Basic Pathology Final

Medicine & Surgery – MedinTo Università degli Studi di Torino
BASIS OF HUMAN
PATHOLOGY
Prof. F. Biasi, P. Gamba,
G. Leonarduzzi
Academic Year 2019/2020

BASIS OF HUMAN PATHOLOGY
CELLULAR ADAPTATIONS TO STRESS
Introduction
General Pathology is the study of a disease, not only of the clinical features but also of the following
aspects:
- etiology (causes);
- pathogenesis: processes that occur during the progression of a disease;
- morphological changes of the cells, tissues, organs during the progression of the disease;
- functional derangements
Cellular responses to stress and noxious stimuli

Homeostasis: any self-regulating process by which biological systems tend to maintain stability
while adjusting to conditions that are optimal for survival. It is achieved by a set of mechanisms that
are put in place by every living being to maintain its composition (glycaemia, calcemia, homeostasis
of the number of blood cells, homeostasis of body temperature, number and size of the cells). [taken
from the slides]
General understanding of cellular adaptations

Normally cells are in a steady homeostatic state which they try to maintain. Once the cell is stressed
it can change its homeostatic state and set a new one, necessary to adapt and overcome different
types of stress. All types of adaptations are reversible, as once the stress disappears the cells can
recover and return to their normal state without any consequence.
How do cells react to stress caused by environmental changes? Fortunately, the cells are a
stationary system and not a static one, meaning they can change their size, phenotype and
proliferative abilities. As a result, they can react to, and survive various environmental stress. They
adapt to the environmental changes by undergoing reversible changes in size, number, phenotype,
metabolic activity, or function of cells. Resulting in new steady states, allowing the cell to survive and
continue to function.
Hence, the adaptation is a positive modification of the cells that enables them to survive various
environmental stress, even though there can be negative side effects to these adaptations. However,
if the stress is too severe the cell dies.
Cells are normally in a state called normoplasia, meaning that the dimension, composition
(glycemia, calcemia), temperature, metabolic activity and number of cells has to remain constant,
this applies to the tissues and organs as well (as they are made up of cells).
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Types of Adaptations
There are different types of adaptations. As an
example, in the picture we can observe a ciliated
epithelium constituted by columnar cells. If
necessary, these cells can become bigger (undergo
hypertrophy) by working harder; or they can begin to
proliferate remaining however of the same size
(undergo hyperplasia). The result of these two
adaptations is the same: the tissue becomes bigger.
If cells have to slow down their activity they can adapt
through atrophy which can mean both decrease of
Figure 1 the size of the cells or a decrease in the number of
cells which are opposite of hypertrophy and
hyperplasia. An atrophic tissue can indicate both a
tissue in which cells are smaller than normal or there
are less cells than normal.
Another type of adaptation is metaplasia in which cells completely change their phenotype, their
morphology. A differentiated cell changes its phenotype and becomes another cell type. In figure n.1
on the left, normal cells of respiratory tract columnar cells of respiratory tract, but in case of a smoker,
they undergo metaplasia and become squamous ones. In normal conditions, the epithelium is
pseudostratified columnar and ciliated, but in case of a smoker it becomes multi-stratified squamous
and the cilia disappear. ALL these adaptations are reversible.
Bizzozzero’s classification of cell types

- Labile: cells endowed with a high proliferative ability. These cells react to an increased work
load by hyperplasia, as they have a higher proliferative ability. E.g. epithelial cells, blood
cells;
- Stable: cells which can proliferate when needed but normally do not proliferate, by remaining
in the G0 state of the cell cycle. E.g. Hepatocytes. If there is a liver damage, the remaining
hepatocytes immediately begin to proliferate to restore the size of the liver;
- Permanent (perennial): cells with no proliferative ability, they cannot proliferate once they
are differentiated meaning that if we lose them, we cannot acquire new ones. E.g. neurons.
In this case, if a higher work load is required, the cells would react by hypertrophy. E.g.
skeletal muscle
Cell injury and cell death
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In normal conditions cells are in a state of homeostasis

(normoplasia) which they try to maintain, but if stress occurs
they can adapt by hypertrophy, hyperplasia, atrophy and
metaplasia in a reversible way. Therefore, once the stress
disappears the cell can recover its original state.
If the cell is unable to adapt to the stress it undergoes injury.

Cell injury does NOT mean that the cell will necessarily die,
this is because cell injury is also a reversible state. Hence, if
the stimulus is not too strong and is transient (short lasting)
the cell can recover, while if the stress is very severe and is
progressive (is repetitive/long lasting) then the injury becomes
irreversible and the cell dies.
There are two different types of cell

death: necrosis and apoptosis.
Usually when stress kills a cell, the
cell dies by necrosis as apoptosis is
programmed cell death.
Cell injury is characterized by:

- swelling of the cell as a
result of the water that enters
and bloats the cell;
- blebs of the cytoplasm/cell membrane, which are visible under the microscope, hence we
can deduce that the cell is suffering;
- cell organelles also swell as a result of the water that enters.
If a cell surpasses the point of non-return, meaning that it cannot return to a healthy cell state via
reparations, the cell will undergo necrosis, hence the cell dies.
Cell death occurs due to the fragmentation of the cell membrane and organelles as a result of the
alteration of the ion pumps. Resulting in the subsequent spillage of cell debris into the local
surroundings, causing an acute inflammatory response.
Environmental changes can cause cell death or cell adaptations depending on their severity.
Hypertrophy
Increase in the size of cells, that results in an increase in the size
of the affected organ. The hypertrophied organ has no new cells,
just larger cells. This occurs in permanent cells which are unable
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to proliferate, hence increase in size to compensate for the increased work load.
This occurs when a cell adapts to an environmental stress by increasing cellular activity. In tissues
containing stable cells both hyperplasia and hypertrophy can occur.
The difference between hypertrophy and pseudo-hypertrophy

In pathology there are a lot of pseudo-hypertrophies and one should not confuse hypertrophy with
pseudo-hypertrophy. Hypertrophy occurs when the cells are really increasing in size, meaning that
their cytoplasm, cell membrane, nucleus, RER, lysosomes and other organelles are actually
increasing in size and number so as to adapt to an increased work load. Whereas, pseudo-
hypertrophy is the accumulation of exogenous compounds (water, blood, lipids, amyloid etc...).
When you have an accumulation of materials that weren’t previously there, you have a degenerative
disease, meaning that inside the cells there are materials which would not normally be there. The
word degenerative doesn’t describe the death of the cell, but instead the accumulation of toxic
compounds inside the cell that eventually leads to cell death. An example is hepatic steatosis.
Hepatic steatosis
A degenerative condition in which the hepatocytes are filled with lipids (mainly triglycerides), thus
making the cells appear larger than normal. However, even though the organ is physically larger it
cannot be classified as a hypertrophic organ as the cytoplasm and organelles are not actually larger
than usual. Hence one cannot call this organ a hypertrophic organ as this is not an adaptation.
The hepatocytes appear as a ring with a stone (“signed ring cells” or in Italian “cellule ad anello con
castone”) because of the following reasons:

- The ring is the cytoplasm which stains more heavily due to the acidic proteins present in it,
and it appears more dense than usual because it is condensed in the periphery;
- The stone of the ring is the nucleus which is also stained;
- The white space in the ring is the potential space that the lipids would normally occupy which
shows no staining as there is nothing to stain. The reason for this is because all lipids are
removed when washed by the alcoholic solvent during preparation.
Physiological Hypertrophies
- Hypertrophy of striated muscle cells in the heart and skeletal muscles in athletes due to
increased functional demand (the heart must work harder).
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You can notice that the left ventricle of the athlete is larger than that of the normal heart, due to the
increased workload demand. Hence the myocytes hypertrophy. This is a reversible state as the
heart can return to the normal size when the athlete stops working out,
as the workload on the myocytes has decreases.
- Hypertrophy of smooth muscle cells of the uterus during

pregnancy due to the stimulation by hormones.
On the right there is a normal uterus and on the left there is a uterus of
a pregnant woman which is much larger. In this case the smooth muscle
cells have hypertrophied in preparation for the series of contractions that
would push the baby out during child birth.
If you were to compare, under a microscope, the

smooth muscle cells of a pregnant uterus to
those of a normal uterus, you would see that the
cells of the pregnant uterus are more spread out
(the nuclei are further apart), while those of a
normal uterus are more compact. Hence, those
of the pregnant uterus are hypertrophied cells.
If you compare two areas of about the same
size and you notice that in one (right side in the
picture) there are less nuclei than the other (left
side), it means that the cells are bigger and
therefore they have hypertrophied.
Pathological Hypertrophies
Pathological hypertrophies can be a result of an increased functional demand or due to the
stimulation by hormones.
- Hypertrophy of the myocardium.
Occurs in hypertensive patients, patients with
valvular lesions and in patients with lung diseases,
due to the increased functional demands as the
heart finds it difficult to pump the blood and therefore
becomes bigger to work better. In this we can’t really
talk about reversibility, it is not really reversible.
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- Compensatory hypertrophy in paired organs.

It can occur following the removal or destruction of one of them as
seen with the kidneys. This is a result of the increased functional
demand placed on the remaining kidney.
- Hypertrophy of endocrine glands on which the adenohypophysis

hormones act on.
Glands become hypertrophic in response to excess hormone
stimulus, which happens frequently when there are adenomas (benign tumors that form in glandular
epithelial) in the adenohypophysis. This results in the overproduction/secretion of pituitary hormones
which results in the increase in size of glands which are hormonally controlled by the pituitary.
For example:
1. thyroid hypertrophy (goiter) for hyperproduction of the thyrotrophic hormone (TSH);
2. Adrenal hypertrophy for adrenocorticotropic hormone hyperproduction (ACTH): Cushing's
disease;
3. Gigantism/acromegaly (hypertrophy/hyperplasia) for hyperproduction of somatotropic
hormone (STH) also known as Growth hormone (GH).
The mechanisms by which cells become hypertrophic

1. Stimulation by growth factors. e.g. IGF1 (Insulin-like Growth Factor 1), when released it
stimulates hypertrophy;
2. Ionic channels. Ionic flows
can stimulate downstream
enzymes which regulate
specific gene expression
which provides an adaptive
response to an increased
demand. E.g. activity of
calcium channels can
stimulate downstream
enzymes (calcineurine) and
produce hypertrophy. The
phosphatase Calcineurine dephosphorylates the transcription factor NFAT therefore
facilitating translocation in the nucleus. Consequently, it increases the transcription of its
target genes which lead to hypertrophy;
3. Other chemical mediators. Depending on the tissue, some molecules (NO, angiotensin II...)
can support the hypertrophic cellular response;
4. Oxygen intake. A functional demand increase to the cell implies an increased intake of
oxygen. Angiogenesis is stimulated (indispensable component of adaptive hypertrophy).
What happens in a hypertrophic cell?

- Increased volume of mitochondria (↑ ATP).
- Increased gene expression. Many signaling pathways activate a large group of transcription
factors. e.g. the phosphatase Calcineurine dephosphorylates the transcription factor NFAT
by facilitating translocation in the nucleus. Consequently, it increases the transcription of its
target genes.
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- Activation of survival pathways (Akt, PI3K, ERK, Nrf2...). The activation of these kinases
leads to specific pathways which are essential for the cells to survive specific types of stress.
- Increased protein synthesis.
- Inhibition of protein degradation via proteasomes and autophagy.
Protein degradation by the proteasome (inhibited in hypertrophic cells!)

The proteasomic complex is
composed of at least 32
different proteins organized in
two 19S subunits (one at the
entrance and another at the exit
of a "barrel" structure). The 20S
subunit in the middle represents the proteolytic center.
The proteins addressed to degradation are "marked" with small chains of ubiquitin molecules (Ub).
They are recognized by one of the 19s subunits, they are degraded within the 20s subunit.
Degradation products are 3-25 amino acids long. Peptides are released through the lower subunit
19s. These peptides can then be further degraded by cytosolic proteases.
Why is ubiquitination important?

In addition to protein degradation, ubiquitination also plays a role
in other cellular processes, depending on the conformation of the
poly-Ub chains:
- gemmation of the cellular membrane;
- internalization of receptors after binding with specific
ligands;
- vesicular transport;
- sorting of proteins within cell compartments;
- gene expression.
E.g. The NFkB inhibitor, called IKB, is degraded after

ubiquitination as it is recognized by the proteasome, allowing the
release of NFkB (a transcription factor) that mediates the
expression of genes that promote cell survival.
Good target for the pharmacological manipulation of tumors.
Activities of proteasomes in given diseases
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Mutation in constituents of the Ubiquitin-Proteasome system may play an important role in the
pathogenesis of certain diseases.
e.g. Parkinson's disease, Alzheimer’s disease
Autophagy (inhibited in hypertrophic cells!)

Senescent organelles and large, denatured protein complexes are targeted for lysosome-driven
degradation by encircling them with a double membrane derived from the ER and marked by LC3
proteins. It is a system of self-digestion that plays a crucial role both in the cellular adaptation and
in the pathogenesis of certain diseases.
The inhibition of autophagy leads to protein aggregate accumulation (Alzheimer's disease).
The cargo degraded by autophagy includes organelles, proteins and protein aggregates, and
constituents of the cytoplasm. Autophagosomes fuse with lysosomes that provide the hydrolytic
enzymes. The breakdown
products of autophagy are
released into the cytoplasm,
where they are recycled into
metabolic and biosynthetic
pathways.
Hyperplasia
Increase in the number of
cells, that results in an
increase in the size of the
affected organ. The hyperplastic organ has become larger due to
the increase in the number of cells which however maintain their
original size.
This occurs when cells need to increase their cellular activity,
hence, enabling them to adapt to an increased workload. In tissues containing stable cells both
hyperplasia and hypertrophy can occur.
Physiological hyperplasia
- Hyperplasia of breast epithelial cells during pregnancy and lactation
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The glands are bigger in the lactating breast as they have undergone hyperplasia as a result of
hormonal stimulation, which resulted in a higher proliferative rate that causes the glands to
increase in size as more cells are present.
Pathological hyperplasia
Pathological hyperplasias can be a result of an
increased functional demand or due to the stimulation by hormones.
- Compensatory hyperplasia for liver regeneration

As seen in partial hepatectomies, in individuals who have a hepatic cirrhosis, hepatocarcinoma or
have donated one lobe/piece of the liver for transplantation, the remaining cells proliferate so as to
restore the liver to its original size. Once the mass of the liver is restored, the proliferation is
terminated.
When a liver is donated from a dead individual, one can

transplant it into several patients, because only a small
piece is sufficient as the hepatocytes can proliferate and
restore the liver. This is because the hepatocytes are
stable cells which normally do not proliferate, but if
needed, they can start to proliferate.
Liver regeneration is composed of two main components:

- Increased proliferation of the remaining hepatocytes;
- Liver regeneration from progenitor cells which originate from the stem cells in the bone
marrow and travel to the liver via the blood. (extra info irrelevant to the course on slides
number 19-20).
The livers’ regenerative abilities have been known since the Ancient Greeks as shown by
Prometheus’s punishment, a Greek legend. Prometheus in fact had been condemned by the Gods
to an eternity of misery for stealing fire and giving it to the human race. His punishment consisted in
being tied to a pole alone on a hill. Not being satisfied, Zeus sent an eagle that every day would rend
his thorax and eat his liver that however would regrow each night in order to condemn him forever.
- Compensatory marrow hyperplasia
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The bone marrow is able to undergo rapid hyperplasia in response to a

deficiency of terminally differentiated blood cells (anemia, profuse
bleeding, hypoxia). The stem cells increase the proliferation rate as a
response to the low number of erythrocytes, therefore more and more
reticulocytes are in the blood flow.
- Lymphocytic hyperplasia in immune defense.

When there is an inflammation, many lymphocytes (neutrophils) arrive on the site of inflammation
because they are phagocytes, and as lots of lymphocytes are needed during an inflammatory
response, they have to proliferate and increase their number. Even though these cells are not
resident cells, this is still an example of hyperplasia in which the cells adapt to the increased work
load by increasing their number.
- Regenerative hyperplasia of the epidermis (wound repair, Psoriasis)

The stratum basale (the lowest layer in the epidermis) is composed of proliferative cells. Normally,
the cells gradually start to become senescent as they reach the upper layers. However, in psoriasis
the cells of the epidermis start to increase the proliferative rate, hence they accumulate causing this
condition of hyperplasia as they proliferate more quickly than they die. This is an adaptation to an
increased functional demand.
- Hyperplasia of the parathyroid glands as a consequence of decreased calcium.
- Gynecomastia due to increased estrogen levels.

The swelling of the breast tissue in boys or men, caused by an
imbalance of the hormones estrogen and testosterone.
- Hyperplasia due to chronic damage.
Atrophy
The reduction in size of an organ or tissue due to a decrease in
cell size and number.
Atrophy results from:
- decreased protein synthesis;
- increased protein degradation;
- apoptosis;
- slowing of cell proliferation.
This is an adaptation to an environmental stress that results from decreased work load in which the
size of the tissue/organ is decreased, hence slowing down their activity.
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This can be obtained in two manners; either hypotrophy (decrease in the size of the cells) or
hypoplasia (decrease in the number of cells).
In the past hypotrophy and/or hypoplasia were used individually to classify this adaptation to a
decreased work load. However, now only the word atrophy is used to classify the decrease in
tissue/organ size, thus one must investigate further and identify whether the tissue has decreased
in size because the cells are undergoing hypotrophy or hypoplasia.
Causes of atrophy:
- Decreased workload (atrophy of disuse).
For example, when an arm or leg is immobilized because of a
fracture in the bone, the single muscle cells shrink.
- Loss of innervation (denervation atrophy).

For example, when a muscle
is no longer innervated,
hence it receives less signals and consequently its work load
is decreased as it is used less often, resulting in denervation
atrophy.
- Diminished blood
supply
Atherosclerosis is a
chronic inflammatory
disease that is
characterized by the presence of atherosclerotic plaques (occurs
in medium and large arteries) that result in the eventual blockage
of the artery which means that the organ which is supplied by that
artery does not receive blood, hence the cells of the organ die due
ischemia.
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This individual had cardiovascular dementia as the loss of

the cognitive functions is in response to brain atrophy that
was caused by the blockage of an artery that supplied it.
This is an adaptation in the sense that the loss of blood flow
to the organ caused the atrophy, however, it is not
reversible.
- Inadequate nutrition
Muscles and organs become atrophic when nutritional
intake is low.
- Loss of endocrine stimulation

- Tissue compression
For example, when you have a tumor, the surrounding tissues can become atrophic, because the
mass applies pressure on the tissues.
Physiologic atrophy caused by a reduced functional demand

- Senile atrophy due to reduced physical activity and hormonal changes
(reduction of muscle fibers).
- Involution of embryonic structures after birth as seen with the cells in

between the fingers that atrophy in order to form the fingers
Physiologic atrophy caused by reduced stimulation by hormones

- Post-partum atrophy of the uterus.
- Reduction of the number of parathyroid cells that produce parathormone
during aging (replaced with adipocytes).
- Testis atrophy during aging (reduction of gonadotropic stimulation).
- Endometrial atrophy during menopause (reduction of estrogen levels)
Metaplasia
The reversible change in which one differentiated cell type changes into another cell type, as a
result of stress that would be better confronted by a different morphology. The change in
differentiation is controlled by the change in gene expression. In all metaplasias there is an
increased risk of developing a benign tumor which can eventually become malignant.
There are many different types of metaplasia and they are named based on the cells that they
become, not the cells they used to be.
Squamous metaplasia in the trachea of smokers
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Normally the trachea is characterized by a ciliated pseudo-stratified columnar epithelium which beats
mucus up and out of the respiratory tract. The mucus traps large particles.
When a person smokes daily, ash and other particles are wafted away by the cilia. However, heat
and toxins in cigarette smoke damage the epithelium causing the cells to need to adapt and undergo
metaplasia. Robust stratified squamous epithelium replaces the respiratory epithelium as it is more
suited to withstand the stress from the heat and toxins in cigarette smoke. The upper layers of the
squamous metaplasia can be keratinized. Consequently, daily smokers have obstructive chronic
bronchopneumonia disease, because they do not have ciliated pseudo-stratified columnar
epithelium which would beat the mucus out of the respiratory tract. This causes them to have to
cough very hard so as to attempt to remove the mucus from the respiratory tract. As mucus remains
stuck in the lungs the accumulation of bacteria in the lungs occurs.
Finally, metaplasia is a pre-cancerosis meaning that these cells have a higher probability of
developing into malignant tumors. If you stop smoking then the cells can return to their normal ciliated
pseudo-stratified columnar state, so you should stop smoking because smoking is not good for you.
This adaptation is reversible: if a smoker stops smoking for 1-2 weeks then the metaplastic cells
should start to return to their ciliated pseudo-stratified columnar state. (We will learn more about this
in lectures to come).
Barrett’s esophagus (Columnar metaplasia)

A condition in which there is a metaplasia in the lower portion of the
esophagus due to a complication of gastroesophageal reflux
disease.
Replacement of the normal stratified squamous epithelium of the
esophagus by simple columnar epithelium with goblet cells, so as
to protect the lining of the lower esophagus by the secretion of
alkaline mucus that neutralizes the stomach acid which is
frequently exposed on the esophageal lining.
Also, in the case of

Barrett’s esophagus, the
metaplasia increases the
risk of tumor development, hence it must be treated.
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Mechanisms that occur in skeletal muscle

hypertrophy and atrophy
In hypertrophy there is an increase in protein synthesis and a
decrease in protein degradation, on the contrary a decrease
in protein synthesis and an increase in protein degradation
take place during atrophy.
Conservation of skeletal muscle mass depends on the
balance between the rates of protein synthesis and protein
degradation.
During prolonged skeletal muscle inactivity, mitochondrial
ROS (Reactive Oxygen Species) are produced. ROS inhibit protein synthesis and increase
proteolysis; the result is the net loss of muscle proteins and thus fiber atrophy.
In this case we are talking about disuse muscle atrophy, which occurs when a muscle is no longer
active as usual (e.g. when an arm or leg is immobilized to recover from a fracture). It has been
observed that an antioxidant treatment (scavengers of free radicals and ROS) is able to inhibit or
delay disuse muscle atrophy.
Role of Satellite Cells in muscle fiber growth

(hypertrophy)
IGF1 (Insulin-like growth factor, one of the main factors
involved in cell hypertrophy) is produced by the myocytes
during intense muscolar activity. IGF1 stimulates the
satellite cells to divide and give rise to two daughter cells,
one of these fuses with the cell membrane of the fiber and
provides a new nucleus to the cell that is used to increase
protein synthesis. When a trained individual stops training
muscle fibers atrophize, get smaller but all the nuclei are
maintained, retraining will thus be a fast process.
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In the case of myocardial hypertrophy, mechanical stretch is detected by mechanical sensors

present on cardiomyocytes’ membranes that can activate signaling pathways which improve
mechanical performance.
Figure 1
CELL DEATH PROCESSES

Two types of cell death have been recognized: necrosis and apoptosis, these differ in their
morphology, mechanisms and role in physiology and pathology.
Necrosis is the accidental cell death (occurs for example when cells are in hypoxia or lack nutrients),
it is a kind of passive death and it is always a pathological process.
Apapoptosis (programmed cell death) is a genetically determined death, it serves many normal
functions and is not necessarily associated with cell injury. However there are pathologies in which
apoptosis is inhibited and others in which it is induced. Whereas normal cells die after a limited
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number of replications, cancer cells lose the ability to undergo apoptosis and are immortal. In
laboratories immortal cell lines obtained from tumors are regularly used as model (e.g. neurons are
taken for neuroblastoma); these cells can’t undergo apoptosis because some genes involved in the
mechanism are mutated and can be frozen and conserved at a temperature of - 80 °C.
Nowadays, one of the fields of research is the understanding of the molecular mechanisms of cell
death and the ability to manipulate cell viability to, for example, induce cancer cell death or inhibit
cell death after a heart attack (myocardial infarction), a cerebral infarction, hepatitis, hepatic
cirrhosis... In those pathologies in which cells die, it could very important to prevent, stop or delay
cell death.
When a stress occurs (e.g. hypoxia, exposure to toxins, lack of nutrients...), if it is not too intense
and its duration not too long, cells can adapt (hypertrophy, hyperplasia, metaplasia, atrophy).
Sometimes cell injury is reversible and if the stress ceases the cell can restore its original state.
When a point of non-return is reached (MTPT opening), cell injury becomes irreversible and the cell
dies, usually by necrosis.
In necrosis, also called colloid osmotic lysis death, the cell breaks, fragmentation of the plasma
membrane and nuclear envelope occurs, the content of the cell is released in the extracellular
environment and an inflammatory reaction is triggered. Necrosis is a passive process that involves
several cells. Cell debris have to be cleaned up.
Cell resistance to stress depends on three factors:
1. Intensity of the stress;
2. Duration of the stress;
3. Grade of susceptibility of the cell, some cells are more resistant, others more delicate. Some
cells are able to activate pro-survival pathways, molecular pathways that if activated protect
cells from stress (PI3K-Akt, MAPK pathway Erk, NRF2, NfKB).
Apoptosis is generally considered physiological, labile cells undergo apoptosis when they become
senescent after a limited number of replications. Apopstosis is an active (requires energy: it involves
the expression of specific genes and thus requires ATP) tightly regulated suicide program in which
cells destined to die activate intrinsic enzymes that degrade the cells’ own nuclear DNA and nuclear
cytoplasmic proteins.
Causes of pathologic cell death (Necrosis or Apoptosis) are:

- Hypoxia;
- Lack of nutrients;
- Exposure to toxins;
- Denaturation of enzymes or inhibition of their activity;
- Denaturation of structural proteins.
Tissues that undergo ischemia (restriction in blood supply) are characterized by necrotic cells and
always undergo inflammation.
Look at figure n.1: on the left there is a necrotic cell while on the right an apoptotic one. In necrotic
suffering cells, ions pumps present on the cell membrane stop working, water gets in and the cells
swell. Blebs on the membrane of cells are index of cell suffering. At a certain point the plasma
membrane, the organelles and the nucleus break down and the leakage of the cellular content
causes inflammation.
On the contrary, apoptotic cells shrink and break up into fragments, called apoptotic bodies, which
contain portions of the cytoplasm and nucleus. The plasma membrane remains intact, but its
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structure is altered in such a way that macrophages can recognize and phagocyte apoptotic bodies
(“eat me signal”: phosphatidylserine, negatively charged and usually restricted to the inner leaflet
of the plasma membrane, is exposed on the outer leaflet and recognized by macrophages). During
apoptosis the content of the cytoplasm is not released, and no inflammatory reaction is triggered.
Another important difference is that while necrosis is a fast passive process, apoptosis requires
time (hours).
Up to a certain point necrosis is reversible, it can regress and cells can recover their functions,
apoptosis on the contrary is irreversible once started even though no morphological changes can
be observed with the microscope.
Necrotic tissue is digested, in the necrotic process every cell membrane (plasma membrane,
organelles membranes and nuclear envelope) breaks down, thus digestive enzymes (contained in
lysosomes) are released. In acute inflammation, granulocytes (mainly neutrophils) secrete lysosomal
enzymes (potent proteases) and phagocyte cellular debris. Hence, in necrotic tissues, many
proteolytic enzymes are present, the ones coming from the dying cells (autolysis) and those from
the granulocytes (etherolysis).
Morphological changes in necrotic cells

- Nuclear changes:
o Pycnosis: the nucleus shrinks (gets smaller) and shows increased basophilia. The
chromatin condenses into a solid, shrunken basophilic mass (along the nuclear
membrane);
o Karyorrhexis: the pycnotic nucleus undergoes fragmentation but remains inside the
cell;
o Karyolysis: the nucleus in the necrotic cell totally disappears because it is dissolved
in the cytoplasm and can be extruded from the cell (after plasma membrane
fragmentation).
- cellular swelling;
- plasma membrane alterations (membrane blebs);
- denaturation and coagulation of cytoplasmic proteins;
- breaking of the organelles;
- dilation of ER;
- swelling and calcification of mitochondria;
- aggregation of cytoskeleton elements;
- intracytoplasmic myelin figures (large, whorled phospholipid masses derived from damaged
cell membranes);
- Cell disintegration;
- Acute inflammation.
There are different types of necrosis:

• Coagulative necrosis;
• Liquefactive necrosis (or colliquative necrosis);
• Caseous necrosis;
• Fat necrosis or steatonecrosis;
• Fibrinoid necrosis;
• Gangrenous necrosis.
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Coagulative Necrosis
It is the most common cell death of tissues that undergo ischemia in all organs except the brain
(liquefactive necrosis). Commonly affected organs are the heart, kidney and spleen. A localized area
of coagulative necrosis is called an infarct. The architecture of dead tissue is characteristic because
it exhibits a firm texture, the tissue appears quite normal and it is preserved for a span of at least
some days, however plasma membranes are not visible anymore. This is because the injury
denatures not only structural proteins (cytoskeleton, ion channels...) but also enzymes (e.g.
proteases) and so the proteolysis of dead cells is blocked. Necrotic cells are anucleated and show
increased eosinophilia in H&E staining.
Ultimately the necrotic cells are removed by
phagocytosis of the cellular debris by
infiltrating leukocytes and by digestion of
the dead cells by the action of lysosomal
enzymes of the leukocytes.
Two examples showed in the pictures:

1. Acute myocardial infarction (on
top);
2. Kidney infarct (on bottom).
The white part in the top picture on the left
is a necrotic area in the left ventricle of the
heart. On the top right, the coagulative
necrotic area in which cells are pinker than the others in shown.
In the bottom right picture depicting a kidney infarct, we can observe that nuclei or necrotic tissue
are disappearing, membranes are broken but the structure of the tissue is quite normal.
Biochemical mechanisms of cell death induced by ischemia

In big arteries, like the coronary artery, ischemia is mainly caused by atherosclerosis, a condition in
which there is an atherosclerotic plaque that develops from the inside of the artery that grows. A
thrombus can then form from an atherosclerotic plaque and block blood flow.
[Coronary artery supplies blood to the heart. Usually in big arteries there is a condition named
atherosclerosis that is characterized by the presence of a plaque that develops from the inside of
the artery wall. When the plaque is broken, a thrombus can be formed over the atherosclerotic
plaque. When there is thrombus over the plaque ischemia occurs so that cells do not receive oxygen
and undergo necrosis. Since there is no oxygen, anaerobic glycolysis is activated, and lactate is
formed, acidosis is determined in these cells and ATP decreases so that consequently because of
ATP depletion, several ion pumps are inactivated or activated e.g. potassium-sodium ATPase pump
doesn’t work so sodium increases inside the cell. There is no ATP so the pumps, whose action is
based on ATP presence, don’t work leading to accumulation of sodium inside the cells; hydrogen
ions have to go out from the cells and so the sodium-hydrogen pumps lead it out. Sodium is higher
in the cell. It must be pushed out, so the sodium-calcium pump is activated, sodium goes out and
calcium gets in. intracellular calcium levels that are higher than normal activate enzymes like
phospholipases which degrade lipids causing membrane damage.]
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- Interruption of blood supply decreases delivery of

oxygen and glucose.
- The metabolism of the cell switches toward
anaerobic glycolysis that leads to an
overproduction of lactate (pyruvate is reduced to
lactate) and thus lactic acidosis (intracellular pH
decreases) and a lack of ATP occur.
- As a consequence, several ion pumps stop
working properly. ATP depletion inactivates the
Na+/K+ pump and activates the Na+/H+ pump.
- Sodium accumulates in the cell and activates the
Na+/Ca2+ pump, this leads to an increase in
intracellular Ca2+. Many cellular functions are
regulated by minimal fluctuations of intracellular
Ca2+ concentration.
- High intracellular calcium level activates
enzymes, included phospholipases (PLA2) that
degrade cellular membrane phospholipids, and
proteases that degrade the cytoskeleton.
- Membrane permeability increases, water comes
in and the cell swells, blebs form and necrosis
occurs.
- Some intracellular calcium also comes from mitochondria and endoplasmic reticulum, whose
membranes get damaged. Mitochondrial membrane is also damaged by lipid peroxidation
due to ROS production favored by low levels of ATP.
- Mitochondrial damage promotes the release of Cytochrome c (Cyt c) to the cytoplasm and
initiates the apoptotic cascade.
20
The non-return point between reversible and irreversible damage is probably represented by the
opening of the MPTP (Mitochondrial Permeability Transition Pore).
These mechanisms have been studied a lot in relation to coagulative necrosis but of course occur
in every kind of necrosis.
Liquefactive Necrosis
Typical of brain ischemia (hypoxia death of cells within the
central nervous system), focal bacterial infection and
occasionally fungal infections (microbes stimulate the
accumulation of leukocytes and the liberation of enzymes from
these cells).
Two mechanisms occur:
1. Autolysis: lysosomal enzymes are released from
necrotic cells. When neurons die, lots of these
enzymes are released in the environment and acid
hydrolysis occurs so the tissue is very digested and
appears liquid; lysis of the tissue because of the
presence of its own lysosomal enzymes.
2. Etherolysis: granulocytes invade the necrotic area
and release potent hydrolases able to digest dead
cells.
The necrotic tissue is a liquid viscous mass and it is frequently creamy yellow because of the
presence of dead leukocytes and it is called pus. The result is an abscess, an inflammatory edema
that accumulates in a cavity formed by liquefactive necrosis in a solid tissue.
21
The colliquative tissue is invaded and removed by macrophages

and replaced by glial (brain) and connective tissue cells.
When looking at a slide of a necrotic tissue it is normal to NOT
recognize any architecture, increased eosinophilia is usually
present. Pink unfocused area with no shape and many violet
points.
Caseous Necrosis
Typically
encountered in foci of tuberculous infection.
It is a mix between coagulative and liquefactive
necrosis, the tissue is not compact but neither liquid.
The area of necrosis has a friable white appearance
(“caseous”: cheese-like). On microscopic examination,
the necrotic area appears as a structureless collection
of fragmented or lysed cells and amorphous granular
debris enclosed within a distinctive inflammatory border.
This appearance is characteristic of a focus of
inflammation known as a granuloma.
The necrotic area in

the center is rounded
by cells that are trying
to phagocyte cellular
debris, in particular
epithelioid
macrophages (named
epithelioid because of their ordered structure), neutrophils and
lymphocytes. All around fibroblasts envelope the granuloma and
secrete extracellular matrix.
Fat Necrosis (Steatonecrosis)

It refers to focal areas of fat destruction, typically resulting from release of activated pancreatic
lipases into the substance of the pancreas and the peritoneal cavity, these events occur during acute
pancreatitis1. In this disorder, pancreatic enzymes leak out of acinar cells and liquefy the membranes
of adipocytes in the surrounding tissues. The released lipases split the triacylglycerides (TAGs)
contained within fat cells. The derived fatty acids combine with calcium to produce calcium soaps
(this process is called fat saponification) which accumulate as amorphous basophilous deposits at
the periphery of the irregular islands of necrotic adipocytes.
Fat necrosis can also occur after a trauma, for example following a breast trauma.
1
The suffix “-itis” always refers to an acute inflammatory process.
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Fibrinoid Necrosis
It is a special form of necrosis usually seen in
immune reactions (chronic inflammatory process)
involving blood vessels (vasculitis). This pattern of
necrosis typically occurs when complexes of antigens
and antibodies are deposited in the walls of arteries.
Deposits of these “immune complexes”, together with
fibrin that has leaked out of vessels, result in a bright
pink and amorphous appearance in H&E stains,
called “fibrinoid” (fibrin-like) by pathologists.
Gangrenous Necrosis
It not a specific pattern of cell death, but the term is
commonly used in clinical practice. It is usually
applied to a limb, generally the lower leg, that has lost
its blood supply and has undergone necrosis (typically coagulative necrosis) involving multiple tissue
planes. When bacterial infection is superimposed there is more liquefactive necrosis because of the
actions of degradative enzymes in the bacteria and the attracted leukocytes (giving rise to so-called
wet gangrene).
Apoptosis
Apoptosis is a pathway of cell death that is induced by a tightly regulated suicide program in
which cells destined to die activate intrinsic enzymes that degrade the cells’ own nuclear DNA and
nuclear and cytoplasmic proteins.
Death by apoptosis is a normal phenomenon that serves to eliminate cells that are no longer needed,
and to maintain a steady number of various cell populations in tissues.
It is important in the following physiological situations:
- The destruction of cells during embryogenesis, for example interdigital cells during hand
morphogenesis undergo apoptosis;
- Involution of hormone-dependent tissues upon hormone withdrawal, such as endometrial
cell breakdown during the menstrual cycle, ovarian follicular atresia in menopause, the
regression of the lactating breast after weaning, and prostatic atrophy after castration;
- Cell loss in proliferating cell populations, such as epithelial cells in intestinal crypts, so as to
maintain a constant number (homeostasis);
- Elimination of potentially harmful self-reactive lymphocytes, either before or after they have
completed their maturation, so as to prevent reactions against one’s own tissues;
- Death of host cells that have served their useful purpose, such as neutrophils in an acute
inflammatory response, and lymphocytes at the end of an immune response. In these
situations, cells undergo apoptosis because they are deprived of necessary survival signals,
such as growth factors.
Apoptosis in pathological conditions can be stimulated or inhibited. Pathological states in which

apoptosis is involved:
- DNA damage. Radiation, cytotoxic anticancer drugs, and hypoxia can damage DNA, either
directly or via production of free radicals. If repair mechanisms cannot cope with the injury,
the cell triggers intrinsic mechanisms that induce apoptosis. In these situations, elimination
of the cell may be a better alternative than risking mutations in the damaged DNA, which may
result in malignant transformation;
23
- Accumulation of misfolded proteins. Improperly folded proteins may arise because of

mutations in the genes encoding these proteins or because of extrinsic factors, such as
damage caused by free radicals. Excessive accumulation of these proteins in the ER leads
to a condition called ER stress, which culminates in apoptotic cell death. Apoptosis caused
by the accumulation of mis-folded proteins has been invoked as the basis of several
degenerative diseases of the central nervous system and other organs;
- Cell death in certain infections, particularly viral infections, in which loss of infected cells is
largely due to apoptosis that may be induced by the virus (as in adenovirus and HIV
infections) or by the host immune response (as in viral hepatitis). An important host response
to viruses consists of cytotoxic T lymphocytes specific for viral proteins, which induce
apoptosis of infected cells in an attempt to eliminate reservoirs of infection. During this
process there can be significant tissue damage;
- Pathologic atrophy in parenchymal organs after duct obstruction, such as occurs in the
pancreas, parotid gland, and kidney.
In previous lectures we saw how cells adapt to different stresses and eventually focused on the
mechanisms leading to the various aspects of cell death. Now, we will go back to the cell function in
general, therefore your biochemical background should help you in recognizing that in the cell
function, phosphorylation and dephosphorylation for instance are key biochemical reactions on
which the cell organizes different programs.
Today, we will see that within the cells there are other key processes other than phosphorylation and
dephosphorylation namely oxidation and reduction of molecules, atoms.
We’ll start talking about oxidant and antioxidant focusing on chemical species, then we’ll introduce
the concept of oxidative stress and that of redox biology.
OXIDANT AND ANTIOXIDANT CHEMICAL SPECIES
These reactions are really vital and imply both a molecule which is losing an electron and is oxidizing
itself and another molecule or atom which on the contrary gains an electron and so becomes in
biochemical terms reduced. A typical redox reaction is for instance the reaction by which
gasoline/petrol is providing energy to the engine, it is an oxidation-reduction reaction. The heptane,
the component of petrol, is reacting with oxygen leading to the production of a number of carbon
dioxide molecules, water and most importantly energy. The energy you get from the oil combustion
is coming from redox reactions. The term “redox” is used when referring to oxidation-reduction
reactions in science/biology. These are vital, and the main example is given by the reaction of the
sugar glucose that in the cell performs glycolysis and then oxidative phosphorylation leading to the
production of water and carbon dioxide with a gain in energy. A molecule of glucose properly cleaved
through an oxidative pathway, first in the glycolysis, then in mitochondria, leads to 36 molecules of
ATP which represent chemical energy.
If you pay attention to this reaction, you will see that the reverse of this reaction is what happens in
plants: they start from water and carbon dioxide, which we are thankful for since they remove carbon
dioxide from the environment and utilize it to produce sugar, nutrients for themselves. There is no
doubt that this kind of redox reaction, reverse and non, is vital for humans and plants.
24
On the right, a list of possible applications of the 1. Oxidation of metals (corrosion, rusting)
redox reaction in industry. Starting from the bottom, 2. Cleaning products removing oxidation
the production of CDs implies a key redox reaction 3. Smelting, to produce a metal from its ore
4. Cathodic protection (galvanized steel)
through which they are able to make a very thin layer 5. Ammonia-based fertilizers (oxidizing
on the surface of the disc. For instance, production of ammonia to produce nitric acid)
cleaning products to remove oxidation of rusting 6. Electroplating (chrome-plated automotive
materials also implies a redox reaction. However, we parts, silver plating cutlery, gold-plated
jewelry)
are interested in what happens in humans, so we’ll 7. Production of compact discs (it depends on
focus now on humans’ metabolism and maybe we’ll a redox reaction, which coats the disc with
also make a few correlations with physiological and a thin layer of metal film)
pathological conditions, therefore on the redox in
pathophysiology.
Figure n.1 is interesting because it provides you with a comprehensive enough view of the main
reactive species acting as key players in
redox processes.
ROS are reactive oxygen species.
Oxygen is the key player of many
biological processes. Before getting into
the analysis of this table, what does
radical or non-radical species mean? In
biochemistry, a free radical is a
Figure 1
molecule or an atom with an unpaired
electron in the external orbital and all the
molecules, just as human beings, like to
be in pairs: if there is an unpaired
electron in the peripheral orbital, the
molecule is in an unstable reactive
condition and tries to let go the
remaining electron or to capture the missing electron from another molecule.
Free radicals therefore are usually pretty reactive molecules, not always though. In the media, it
often happens that they talk about free radicals e.g. sunscreens against free radicals. Public opinion
thinks that free radicals are the “bad guys” in our body which is not always true. Oxygen per se is a
free radical because it has two unpaired electrons in the peripheral orbital but with the same speed,
so that they cannot react; oxygen is therefore a free radical which is NOT reactive. Superoxide anion,
hydrogen peroxide, hydroxyl radical are molecules that we need to gain during inflammation: our
body during inflammation utilizes these radicals to destroy the biological material which has been
phagocyted. These molecules play a key role in the phagocytic processes. Superoxide anion and
hydroxy radical have indeed an unpaired electron, so are they radicals? Yes. Oxidant? Yes. Because
they try to get a stable chemical state and then they remove with strong energy an electron from
another molecule/atom and since they are oxidant, they perform oxidation. Hydrogen peroxide is a
strong oxidant, but theoretically is not a free radical. So, the right definition that comprehensively
considers all these molecules in figure n.1 is not free radical but reactive oxygen species (ROS),
because some have the chemical state of a free radical while others don’t.
These three molecules are key players in physiology during phagocytosis. Under normal
conditions, in all living organisms within the cells there is a basic production of ROS. We are
producing these species but what for? Explained further on.
They are produced during cellular respiration, in particular we have production of superoxide
radical which is converted into hydrogen peroxide which is much more diffusible and performs
different jobs. Peroxisomes are producing a lot of hydrogen peroxide and all together they give rise
25
to other different oxygen species. The message of the lecture is that in our body, in mitochondria
mostly, under normal/basal conditions, we produce a certain amount of ROS.
Which are the sites in a cell where these molecules
are produced under normal conditions?
They are produced in different sites including plasma
membrane, cytoplasm, ER and mitochondria (figure n.2). In
the plasma membrane for instance there is, in every cell, a
very important class of enzymes which is NADPH oxidase
that are able to produce ROS during phagocytosis, that is
one of the main processes of aspecific immune reaction.
There are also lipoxygenases and cyclooxygenases for
the synthesis of prostaglandins and other inflammatory
Figure 2
intermediates that takes place in the plasma membrane.
With the production of eicosanoids, you have a simultaneous generation of some ROS. They are
reactive, so it is better to get rid of them or control them. Within the cell, in the cytosol (the soluble
part of the cytoplasm), there is xanthine oxidase which is an enzyme that is studied when dealing
with ischemia, reperfusion, hypoxia or reoxygenation e.g. during transplantation, the organ to be
transplanted is under hypoxia, then is transplanted in the recipient and the oxygen is restored as
soon as the surgeon de-clamps the surgical field; oxygen is coming back after a period of ischemia
which is a problem because you are reconstituting the system circulation but at the same time you
have a great production of ROS because of the presence of this enzyme. In fact, when you transfer
the organ to be transplanted, you have to put the organ in a solution which is able to preserve it
before transplantation. The key molecule present in any kind of transport solution for to-be-
transplanted-organs is allopurinol which is an inhibitor of xanthine oxidase: in this way, the
transplantation period went from being three hours to twenty hours because you stop the enzyme
which performs reperfusion.
Other important sites of ROS production are the mitochondria because of the enzymes cytochrome
oxidase and NADH dehydrogenase. Finally, another site of production during normal metabolism
is the ER with different cytochrome families: cytochrome P450 and cytochrome b5. We can therefore
understand that there are different sources of production of ROS which is inevitable when performing
physiological processes.
The antioxidant defense
The antioxidant defense is suitably distributed within the
cells (figure n.3), meaning that evolution was able to favor
the production of antioxidants in the right places. If
lipoxygenase and NADPH oxygenase are on the
membrane, then vitamin E, PUFA (polyunsaturated fatty
acids) and β-carotene are located on the same site. In the
peroxisomes, which are the sites of high production of
hydrogen peroxide, there is catalase which cleaves
hydrogen peroxide into water. There is high production of Figure 3
hydrogen peroxide in the peroxisomes which represent the
“accelerator” but then there is the catalase which is able to
modulate the amount of hydrogen peroxide that is present
in a given part and therefore represents the “brake”. Many biological systems are NOT static but can
be modulated: to modulate them you need an accelerator and a brake; in biology no event is static,
but rather dynamic which means that you need something which switches on and something that
switches off.
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Where xanthine oxidase is working, in the cytosol, there are selenium, vitamin C (ascorbate),
glutathione, GPx (glutathione peroxidase) and SOD (superoxide dismutase).
In mitochondria there are selenium, GPx, vitamin E, ubiquinol and SOD.
So, production of ROS occurs in different sites of the cell. ROS are NOT “bad guys”, they could be
useful, but their concentration should be under control since they might be dangerous if they are
free.
Oxidative biochemical stress
When talking about oxidants and antioxidants, we can also talk about an equilibrium within the cells
that is called redox equilibrium. Usually our cells tend to maintain such an equilibrium between
molecules, conditions and parts that are able to stimulate production of excessive amount of ROS
and molecules that are able to quench/reduce such concentration. Prooxidant factors are of many
kinds e.g. mechanism of radiations, UV light and x-ray or γ-radiation, their mechanisms are different,
but they are still all electromagnetic or corpuscular radiations that are effective in generation of ROS.
Going from a limited amount of oxygen in the organ to be transplanted to the reperfusion
phenomenon, so from hypoxia to normoxia, there is an increase or burst in the production of these
species. Our inflammatory reactions are using the advantage of ROS production to kill biological
material, bacteria, microorganisms that are now trying to attack our tissues. On the other side, we
have ubiquinols, highly versatile family of antioxidants.
There is a first barrier represented by enzymes (aforementioned examples: SOD, catalase, GSH-
peroxidase) and in addition there is a second barrier which is composed by “supporters” of these
enzymes which are mainly vitamins: vitamin A, vitamin E, vitamin C which are also endowed with
antioxidant properties. Imagine for instance that one single molecule of vitamin E in our membranes
is able to kill under normal conditions 3000 molecules of PUFA. If you leave a membrane or lipids
on a bench without any defense, it rapidly gets rancid and undergoes oxidation, breaking down; you
feel it because when oxidation breaks down there are many aldehydes. Luckily, this event doesn’t
occur in the membrane because of the presence of vitamin E which, let’s underline it, performs its
function 1 molecule against 3000 at a time, therefore it is pretty efficient. Glutathione and vitamin C,
contrary to vitamin E and vitamin A, are hydro soluble. On the other hand, indeed, vitamin E and A
are compartmentalized in the lipid environment.
Under normal conditions we have an equilibrium between reduction and oxidation reactions but there
are conditions such as inflammation which is pathophysiological, ischemia and reperfusions but also
many drugs, toxins, anesthetics and radiation that are prooxidant. If such prooxidant pressure isn’t
properly counteracted by the defense or if the defenses for some reason are reduced (e.g. people
with low amount of vitamin E, C, A, reduced number of glutathione so with impaired antioxidant
defense), there might be an imbalance, change of the equilibrium.
Oxidative stress is a quite popular term, which has a biochemical not psychological nature and
indicates a condition in which the oxidant reactions are overwhelming the reductive ones. It could
lead to a number of problems. At the level of the plasma membrane, excessive amount of oxidant
species could lead to changes of lipids or the structure of the membrane itself. Remember the
example of the lipids on the bench, the membrane could face the same problem; a stress could occur
because of the ultracellular acute variation in the tension of oxygen. During mitochondrial respiration,
at different ages throughout life, you can have different amounts of ROS produced e.g. in elders, the
amount of superoxide which is leaking out of mitochondria is increasing. One theory of aging is
indeed based on the concept of oxidative stress. General pathologists usually, and we should as
well, try to study causes, mechanism, symptoms but also the process from the very beginning to
promotion, progression and expression of diseases because our main diseases are multifactorial,
multistep processes. It would be silly to focus just on one factor. Aging is a multifactorial process, it
is not a disease, it is the only way to keep on living, but of course it could be different in terms of
quality. During aging you have some oxidative stress. A possible explanation might be that often
27
elderly people do not have a proper nutrition maybe they lack in vitamins, so respiration of
mitochondria is not good.
Free radicals
What can they do when accumulating? They are really reactive, being free radicals or not (hydrogen
peroxide does the same even if it cannot be considered a free radical).
They have different targets which are proteins,
carbohydrates and, within the lipids,
polyunsaturated fatty acids; but let’s not indulge
with this picture that will be discussed again when
examining radiation injuries that are mediated by
reactive species and whose targets are mainly
macromolecules. Eventually you have membrane
changes and death of the cells in case of
membrane lipid degradation, mutations in
translation in case of DNA attack, proteins can be
denaturized, inactivated and in the case of
carbohydrates there are peculiar compounds
called Advanced Glycation End products
(AGEs) which in a way are also increasing during aging in diabetes for instance because of the
reaction of excessive amount of ROS with glucose or other carbohydrates.
The main diseases in humans are chronic diseases, cardiovascular particularly which hold the first
place on the podium, then tumors, chronic pulmonary lung diseases, chronic intestinal diseases but
also early aging, different types of dementia and neurodegenerative diseases. They are
multifactorial, so we are not saying that ROS are the only players: they could contribute to the
pathogens but there are many studies on the role of ROS in Alzheimer disease for instance.
Reactive species and antioxidant defense
When there is an imbalance, what happens? The cell could at least be damaged. Damage of many
cells means that the effect has now spread leading to possible tissue damage, organ damage,
systemic damage and disfunction. Another point you should bear in mind is that in medicine and in
pathology 1+1 NEVER equals 2. So, if you have one pathogenic factor plus another one plus another
one, it doesn’t result in three pathogenic factors but in seven, eight, fifteen, it depends on several
things. In particular, this is true when talking about multifactorial chronic diseases.
The antioxidant system in humans

We are hearing and reading many times about antioxidants. Antioxidants can be of enzymatic or
non-enzymatic type. The key players in the enzymatic defense against the excessive amount of ROS
are superoxide dismutase, catalase and different peroxidases. These antioxidants however also
need some supporters which are glutathione, vitamin C in the cytosol, vitamin E and A in the
plasma membrane and polyphenols (more than 4000 molecules, strong antioxidants) which can be
obtained from the diet and are not only antioxidants since they contain also alcohols.
We concentrate on superoxide dismutase first. The main reactant molecule produced during our
cellular respiration is superoxide which is very important but dangerous if it accumulates. We have
an enzyme inside the mitochondria, which is SOD, that is able to stop an excessive accumulation of
superoxide through the “dismutation” of two molecules of superoxide into hydrogen peroxide. It
performs an important action which however is not sufficient by itself, if the “runner up” catalase isn’t
present since SOD fixes the extra superoxide molecules but tends to produce an excessive amount
of hydrogen peroxide which goes out. Under normal conditions, catalase, which must be localized
in the right place, next to SOD, as soon as hydrogen peroxide is produced, attaches to it and is able
28
to convert hydrogen peroxide into water and oxygen. The coupling of these two enzymes (SOD and
CAT) is very effective, but they must be in the right place (mitochondria and cytosol).
There are many peroxidases (Px) present both in the mitochondria and other organelles and cytosol.
R-OOH are like hydrogen peroxide but are organic peroxides: they are molecules (protein, lipids)
that became radicals in nature because of the attack of free radicals that are unstable. Organic
radicals, since they are unstable, rapidly react with oxygen and give rise to organic hydroperoxide
which is pretty dangerous since it undergoes demolition quickly with production of different
compounds: since these peroxides are starting off as proteins, lipids or nucleic acids, if you ever
break them down, at the end you lose a protein. Peroxides were therefore originally proteins that
became organic hydroperoxides that can be converted by peroxidases into alcohol and water. One
of these peroxidases is obtaining the reducing equivalent NOT from a given compound but directly
from glutathione: glutathione is giving the hydrogen that is necessary to convert the
lipohydroperoxide in alcohol and water thanks to the conversion of glutathione in glutathione disulfide
which is the oxidized form.
Glutathione (GSH) is a tripeptide which is very important in our cell, it has a millimolar concentration,
it’s composed by glutamine, glycine and cysteine (-SH group is very important for the reactions).
Glutathione synthesis occurs through two enzymatic steps:
1. Synthesis of γ-glutamylcysteine;
2. Synthesis of glutathione through the addition of a glycine to glutamylcysteine.
Glutathione disulfide is simply a double molecule with a disulfide bond which is the reversible
oxidized form of this very important molecule.
In our bodies, there are -SH groups (protein thiol or thiol groups) provided by molecules other than
glutathione i.e. proteins, that all together give their contribution to the antioxidant defense to quench
the number of oxidants within the biological fluids e.g. plasma.
Vitamins in general play a key role in the fight against oxidative stress; in fact, we know that
hypervitaminosis and hypovitaminosis have an impact on our cells. Ascorbic acid (vitamin C) is
probably acting in different pathways but it is mainly an antioxidant. It provides electrons to free
radicals e.g. radiation induces a high number of free radicals in the proteins, carbohydrates. These
free radicals need to escape their chemical stage, they need an electron. Ascorbate can move in the
cell easily because it is hydro soluble, while other antioxidants stuck in the membrane (i.e. vitamin E
and A) need to move out; ascorbate is quicker and very generous: it gives electrons easily, because
it can reach the molecules easily, it is a very active and efficient electron donor. By doing this, it
becomes a free radical but there are many reactions and occasions in the body through which it can
go back to its original stage by picking up the missing electron. It works in together with vitamin E.
Uric acid is a molecule that at least in humans cannot be metabolized and so accumulates. It is
produced from purines, from nucleic acid-derived bases, by the enzyme xanthine oxidase that
generates ROS as well. Too much uric acid in the blood causes gout; allopurinol is the drug people
use globally to treat excessive accumulation of urine. Uric acid is not just a waste product but also a
good antioxidant. If evolution lets a defined amount of uric acid in our body is for some reason. It is
a good scavenger of the main ROS, in particular of hydroxy radicals.
Vitamin A is very important for many functions, not only for its very good antioxidant properties but
also for other reasons. Vitamin A and β-carotene are important for the vision, thanks to their structure
and function. A deficiency in vitamin A could imply different problems, but there’s no doubt that the
antioxidant property is one of the majors.
Together with the family of vitamin E, there are the carotenoids among which β-carotene that are
good scavengers of free radicals, in particular like vitamin A and unlike vitamin C, they are lipophilic,
so located within the membranes.
29
Tocopherols (vitamin E) are at least of four types: α, β, γ and δ. The actual form which is more
diffused and available, endowed with the strongest antioxidant effect is α-tocopherol which is
probably the most powerful one, able to keep under control 3000 molecules of PUFA in the
membrane easily.
Vitamin E antioxidant function

These antioxidants are all working together, not
independently. When we focus on a given molecule
or on a given disease, we tend to forget about the
rest, but we must keep an eye on the environment:
pay attention to the whole scenario of a disease,
because it could help. The same stands for
antioxidants in the cell: they are together, they
cooperate. Vitamin E is probably playing the main
role but cooperates with others. For instance, if
there is a lipid oxidation, due to acidity of the fat,
you have peroxy-lipid radical LOO· (the dot stands
for unpaired electron). Peroxy-radical is a lipid, very
reactive in the membrane and needs another
electron to “relax”, the easiest way to get it is to steal
the electron from the neighboring molecules. In this
case, this lipid molecule is not a radical anymore,
but the neighbor has now become a radical, so we
can talk about a chain reaction, which doesn’t stop:
the second radical will do the same job with the third
radical; eventually, in order to stop the chain
reaction, you have a change in the membrane. When
peroxy-radical is attacking the neighboring lipid LH,
this becomes a peroxy that is not dangerous (not a
radical) bringing also a hydrogen equivalent (from
LOO· you get LOOH), but unfortunately you have
another molecule now that becomes a radical
therefore you should stop the deadly pathway.
Tocopherol, in this case, TOH, is giving an electron
easily but becomes tocopheroxy radical which is
not a problem for vitamin C and E since they are
quite stable as radicals (NOT all free radicals are
reactive e.g. oxygen is a free radical). The chain oxidation reaction is stopped because vitamin E
gives up its electron and interacts with ascorbate which gives its own electron so that tocopherol is
restored. What about ascorbate though? It becomes ascorbic radical but how many NADH reactions
are present in the cell? They are everywhere! So, ascorbic radical can get its electron and hydrogen
equivalent back. Glutathione can do the same as ascorbate: it oxidizes itself and reduces
tocopheroxy radical. GSH and ascorbic acid, which are hydrophilic (=moving freely), can search and
find easily the electron that they are missing from the coenzymes i.e. NADH. The fine aspect is that
vitamin A is mimicking vitamin E, but it is within the membrane, in the deep lipophilic environment.
So how could vitamin E cross-talk with vitamin C and glutathione that are in the deep hydrophilic
environment? The tocopheroxy radical is less lipophile and moves to the border between the
membrane and the cytosol, gets closer to the border so that it can cross talk with either GSH or
vitamin C: it can take the electron and becomes vitamin A to move to the original position. Interaction
30
is made possible because of different states of vitamin E and vitamin A. You need vitamin E but also
vitamin C and GSH to make this cross talk, to operate properly you need both.
[He skipped polyphenols and flavonoids]
French paradox
Many years ago, in 1992, an epidemiological study came
out comparing the rate of incidence of CHD (coronary heart
disease) with the daily uptake of fat. The higher uptake of
fat in a given country, the higher incidence of mortality for
CHD. They were expecting that in northern Europe, where
they don’t eat much carbohydrates but mainly proteins and
fats, the occurrence of CHD was relatively higher, and it
was. You can see in different countries a very good
correlation line between daily fat intake and mortality for
cardiovascular diseases. French people that better than us
in underlying important events came out saying that
despite they eat a lot of cheese, they didn’t have a high rate
of death for cardiovascular process but less than expected.
That’s why they talked about French paradox to indicate this “defect”. Italy was of course placed
pretty well but here, in Piedmont, we can also say that we have the “piedmontese paradox” because
we eat a lot of cheese and proteins, we do not exactly follow a Mediterranean diet. The French
paradox awakened the interest of epidemiologists. What was its cause? The wine because it
contains polyphenols and particularly resveratrol. So, we have the French paradox because
French drink a lot of wine and piedmonteses as well. Since that observation, people drew their
attentions on components of the wine but not only also on fruits and vegetables whose compounds
are phenolic compounds that are strong antioxidants, for instance resveratrol is only one of them,
then there are the subclasses of tannins, anthocyanidins, flavanols, isoflavones e.g.
anthocyanidins and flavanols are present in coffee, chocolate and isoflavones (genistein and
daidzein) are present in high amount in soya. They could contribute to the higher or lower antioxidant
compounds of given population or individuals. Not only red wine but also fruit and vegetables are
endowed with a large and efficient variety of antioxidants, which is of important nutritional
consideration. A very important message came out from the French paradox even though it might
be not correct or a bit “amplified”. Of course, biochemists are not epidemiologists, so they focused
on biochemical effects e.g. many polyphenols are good free radicals scavengers. They are relatively
lipophilic, so they try to stay in the membranes and more or less contribute to the modulation of redox
reactions.
Trevor Slater was a pioneer of the evaluation of oxidative damage and break down of lipid
membranes. If you demolish the lipids, eventually the membrane breaks down. He was studying the
pathological conditions. He was the first person who associated the lipid peroxidation (break down
of membranes) with pathogenesis of given diseases e.g. atherosclerosis, alcoholic liver injury, liver
carcinogenesis. He associated lipid membrane oxidation with necrosis and chronic inflammation
reaching a milestone in redox biology.
Hermann Esterbauer moved further and worked on the products deriving from these processes. He
worked on the understanding of the physiology. If oxidative stress is intense, you get irreversible cell
damage but in different pathophysiological conditions like inflammation which is not pathological per
se but could become pathological or in fibrosis, you have a medium intensity of the stress. Under
normal conditions, you NEED some oxidative stress or better redox imbalance in physiology for cell
modulation to occur. Cell modulation by ROS opened a new field of the redox biology whose master
was Mario Umberto Dianzani.
31
Types of cell signaling

We deal with cell signaling when talking about physiology. There are different types of signaling.
Type I signaling involves ligand, receptor and tyrosine kinase. It is a very linear way of signaling
from a given ligand (e.g. hormone) to the nucleus. There is a second type of signaling which is
operated by molecules that we know i.e. glycerol, calcium and so on. The regulation of calcium
homeostasis uses the type II signaling which involves more aspects of the cells. Type III signaling
involves ROS: all the cell is aware of what is going on.
ROS are not simply “bad guys” but they could be physiological molecules important in the modulation
of a number of cellular functions and events because cells are able to cross talk with other cells but
also to generate intracellular messages. Cell signaling is the model for progression from the classic
biochemistry to the modern cellular biology.
In physiology regulated by free radicals, we cannot forget to mention nitric oxide or nitric monoxide.
It is a fantastic molecule important in the vascular system because it is produced by nitric oxide
synthase starting from the amminoacid arginine, producing a radical that is beneficial under normal
conditions. Normally it’s responsible for vasodilation in different systems. People got the Nobel prize
for this discovery and the fourth person whose name is Salvador Moncada discovered a factor called
endothelial dilating factor. The role of the nitric oxide radical is vasodilation. The most practical
pharmacological use of nitric oxide is Viagra. Where is it produced? NO synthase which is the
enzyme that synthesizes NO is present in three types:
- Endothelial NO synthase: constitutive form, produced by endothelial cells which keep
proper caliber in the vessels;
- Neuronal NO synthase: proper of neurons;
- Inducible NO synthase: influent in inflammation and could become a bit more of an
“unpleasant” molecule because during inflammation it meets many molecules of superoxide.
NO per se is good but when it goes together with excessive superoxide it leads to formation
of peroxinitrate (ONOO-) which is dangerous. During long-lasting inflammations, we see the
negative effect of NO, but under normal conditions it is good.
The majority of people consider the free radicals as bad, toxic. We went from free radical toxicology
to the approach of free radicals in pathophysiology and ended with the redox biology. The iconic trio
is comprehensive of Slater (biochemist), Esterbauer (chemist) and Dianzani (general pathologist).
Often biochemists believe they can cure diseases only by curing the cells in the laboratory and forget
they are dealing with humans while medical doctors and general pathologist consider the entire
picture of a given disease: the story, the interaction, the progression and so on e.g. diabetes cases
are different one from the other: one may have complications while another doesn’t, one has to take
three drugs a day while for the other one it is sufficient to eat less bread, therefore we can understand
that diseases are really complex and should be considered in a single individual but also on general
terms.
32
Last week we spoke about cell death, both necrosis and

apoptosis, and about all the mechanisms that underline
these two particular processes. In figure n.1 we have a
summary of these mechanisms. On the left, there is
necrosis which is also called colloid osmotic lysis
because the cell membrane breaks, and all the contents
of the dying cell are released in the environmental space
leading to inflammation. In fact, the necrotic tissues are
characterized by lots of debris derived from pieces of
membrane, of nuclei and all the organelles that are
dispersed in the extracellular space. Therefore, our
organism wants to clean up the debris and possibly also
the causes of cell death, meaning bacteria, viruses,
fungi and so on. Figure 1
Apoptosis, on the right side in the figure, on the contrary
is a programmed cell death: the cells decide on their own to die; to do so they use ATP in order to
activate some signal transduction pathways which lead to apoptosis. In apoptosis the cells are
divided into apoptotic bodies which are rounded by membranes, so there is no dispersion of any
debris in the extracellular space. For this reason, apoptosis NEVER leads to inflammation. During
apoptosis, cells that die completely disappear because there are, in all our tissues, some resident
macrophages that are phagocytes able to recognize the apoptotic bodies, phagocyte and digest
them. We mentioned a lot of examples (lecture n.1) that must be remembered, of both physiological
and pathological apoptosis. On the other hand, necrosis is always pathological.
Mechanisms of apoptosis
There are two main pathways:
- Extrinsic
- Intrinsic
The extrinsic pathways need an extracellular signal to

stimulate the cell to die of apoptosis. While the intrinsic
pathway is activated by the cell itself.
In figure n.2 both pathways are depicted. The extrinsic
pathway, on the left, needs some molecules, that are
released by neighboring cells, like TNF (tumor necrosis
factor) and other stimulating molecules that are
recognized by the receptors located on the cell
membrane. These receptors have an intracellular Figure 2
death domain which binds to the death domain of
another molecule that is present inside the cell that is an
adaptor (in the figure, FADD). The binding between
these two death domains activates some enzymes that
are located in the cytoplasm and are called caspases.
In the extrinsic pathway, caspase 8 is activated and in
turn activates caspase 3, the effector, whose activation
leads to cell death.
The intrinsic pathway, on the right, starts with the release of cytochrome c (Cyt c) by mitochondria.
The increase in concentration of Cyt c in the cytoplasm activates caspase 9 which in turn activates
caspase 3, that is common to both pathways, leading to apoptosis.
33
TNF receptor (figure n.3 in light blue) binds to TNF; the death domain (zig-zag shaped, in green) of
the receptor binds to the death domain of the adaptor (in yellow). Consequently to this binding,
procaspase 8 switches to its active conformation that is caspase 8. At this point, it can activate
some particular types of caspases including caspase 3 that is the caspase involved in the most
cellular processes leading to apoptosis.
Figure 3 Figure 4 The
intrinsic pathway starts with the release of cytochrome c from mitochondria (figure n.4). When these
pores are open (MTPT), Cyt c goes to the cytoplasm and activates caspase 9 that, when apoptosis
starts, is immediately bound to Apaf-1 and cytochrome c to form the apoptosome. Cytochrome c
+ apaf-1 + caspase 9 constitute the apoptosome which activates caspase 3. Afterwards, we have
DNA degradation and all the processes that are characteristic of apoptosis.
Perhaps, for medical doctors-to be, it is not so important to know that Cyt c is bound to Apaf-1 and
caspase 9; but it is very important to know the molecular processes behind cell death (both
apoptosis and necrosis) which are studied a lot in the laboratory. Indeed, researches are trying to
activate apoptosis in tumors, because normally tumor cells are immortal (highlander-l’ultimo
immortale, LOL). Tumor cells don’t die which is the reason why we need to find a way to induce their
death. Finding a way to activate apoptosome formation could be a solution for example, or in general
a way to activate caspase 3 that leads to apoptosis. However, it is useful to know what happens
before caspase 3 activation.
34
Mitochondrial proteins
There are several mitochondrial proteins
which are involved in cell apoptosis. The
names are quite all the same, so it is difficult
to remember all of them, but it doesn’t matter;
the important concept is that they can be
divided into pro-apoptotic and anti-
apoptotic molecules which belong to the
Bcl-2 family. The most famous anti-
apoptotic molecules are Bcl-2, which has the
same name of the entire family, and Bcl-XL. Figure 5
There are several pro-apoptotic proteins among which
Bax and Bak, that bind to these anti-apoptotic molecules, and then all the family of the Bax and Bak
substitutes which are listed in figure n.5.
The anti-apoptotic molecules, so Bcl-2 and Bcl-XL can exist both as a
homodimer i.e. Bcl-XL bound to another Bcl-XL or as a heterodimer
meaning that Bcl-XL can bind either of these two pro-apoptotic molecules Bax
or Bak. In this state, these molecules are anti-apoptotic.
In figure n.6 there is a state in which apoptosis does not happen because Bcl-
XL is bound either Bcl-XL (homodimer) or with Bax/Bak (heterodimer).
If another stimulus arrives signaling the necessity for
Figure 6
apoptosis to occur, Bim, Bik, Bid (figure n.7) and all the
pro-apoptotic molecules which are substitutes of Bax and
Bak take their place and bind to Bcl-XL so that Bax and
Bak are free and can cause the opening of the MPTP
(pores located on the surface of mitochondria) which
leads to cytochrome c release in the cytoplasm that
activates the intrinsic apoptotic pathways [cytochrome c Figure 7
activates Apaf-1 that in turn activates caspase 9; together
they constitute the apoptosome so that caspase 3 is
activated and apoptosis can occur].
The activation of caspase 3 leads to the activation of the
enzymes that degrade DNA (DNAses). If we look at DNA
with the DNA laddering method, we can see (figure n.8)
that the DNA is not fragmentated in the control sample A,
while in sample B DNA is fragmentated due to activation
of DNAses by caspase 3. In this case, you can observe
the laddering due to the different molecular weights on
the gel. Sample C contains necrotic cells, when DNA is
completely degraded.
Alterations in concentration of calcium in the cytoplasm are important in both physiological and
pathological conditions because they can lead for example to necrosis in ischemic tissues
(mentioned in previous lesson). The increase of calcium in the cytoplasm (it can come from ER,
mitochondria or the outside) can also activate apoptosis
Figure 8
because calcium can activate caspase 12, normally
bound to the ER membrane, whose activation leads to activation of caspase 9 which binds to Cyt c
and Apaf-1 forming the apoptosome that activates caspase 3 which in turn activates DNAses so that
apoptosis arises.
35
In all kinds of pathologies,

studying cell death is very
important. Figure n.9
illustrates Alzheimer’s
disease. In pink: neurons,
microglial cells; in light blue:
astrocytes. In this disease
neurons die (oversimplified)
by necrosis because of the
presence in the brain of
amyloid-β-plaques
(represented in black in the
image) which are composed
of a neurotoxic protein called
amyloid-β. These plaques
accumulate outside the
neurons and since they are
made of neurotoxins, cells
die. If cells die by necrosis,
their contents are released in the extracellular spaces and an inflammatory reaction occurs.
Alzheimer’s disease is a neurodegenerative disease, but it is also considered a chronic
inflammation state because necrosis is ALWAYS followed by inflammation. Why is it called
neurodegenerative? Because there is a degeneration: something accumulates into the cells. These
plaques are represented by the black lines outside the neurons in figure n.8 that are neuro-fibrillary
tangles, composed of protein tau (τ) which is useful for microtubules assembly. In Alzheimer’s
disease, tau is hyperphosphorylated, it is strange, which causes the aggregation of tau and
microtubules inside the cells, contributing to their death. She studied the effect of oxidized
cholesterol in Alzheimer’s disease. It is known that hypercholesterolemia is a risk factor for
atherosclerosis, cardiac and brain infarcts, but you might not know that it also leads to Alzheimer’s
disease onset and progression. In particular, they studied the effect of oxidized compounds of
cholesterol. Cholesterol that is present in the peripheral circulation cannot pass through the blood
brain barrier unless it is oxidized. We want to know what 24-hydroxicholesterol and 27-
hydroxicholesterol do to neurons.
On neurons there is a micro-receptor complex composed of CD36, β1 integrin and CD47. It binds
amyloid-β which is present around the neuron causing cell death. We want to know whether
oxysterols (cholesterol oxidation products)
area able to modulate somehow the cell
death caused by amyloid-β. Papers
describing how you can evaluate necrosis
and apoptosis in the cell are linked on slide
49. To evaluate necrosis, you have to
measure the concentration of the
enzymes that are released in the
extracellular space. It is a colloid osmotic
death, so it easy to see whether a necrotic
process is activated or not by measuring
lactate dehydrogenase (LDH) in the
medium. If a lot of LDH is present, it means
that the cells are dying by necrosis. While if
36
you want to evaluate apoptosis, you have to measure the activity of caspase 3: if it is activated,
apoptosis is ongoing; or you can observe the apoptotic bodies through DAPI staining. They have
observed that only 24-hydroxycholesterol was able to induce necrosis because LDH release was
increased after treatment of cells with this oxysterol.
Figure n.9 shows DAPI staining. The fifth square in the bottom line depicts some light blue little
particles that are brighter than the others which are the apoptotic bodies. In these particular
experimental conditions there is apoptosis.
INFLAMMATION
The term inflammation makes you think about a harmful process, but it is not. It is a positive event
that our organism decides to activate to Figure 9
clear up debris, bacteria, viruses, fungi… In
particular, our organism wants to clear up the necrotic debris and also the causes that lead to cell
death. Inflammation may be caused by:
- physical agents: ionizing and ultraviolet radiation, trauma, heat, electric current, cold etc.;
- chemical agents: strong acids and bases, toxic compounds;
- biological agents: bacteria, viruses, fungi, animal and plant toxins, etc.;
All these physical agents cause cell death, therefore in all these cases inflammation is triggered to
eliminate cellular debris. Also, in the case of chemical agents, necrosis occurs, and inflammation is
activated. In case of biological agents, inflammation removes both the causes of cell death and
debris.
The process of inflammation is characterized by the generation of inflammatory mediators,
molecules released by all the cells that play a role in inflammation (endothelial cells, neutrophils,
macrophages, platelets) and also by movement of fluid, cells and proteins from the blood to the
extracellular space.
Characteristics of acute inflammation
- It is predominantly local i.e. you can have inflammation in the liver, in the lung, in the brain,
but it is local;
- It is protective: it helps to eliminate the initial cause of cellular damage (physical, chemical
and biological agents) but also its consequences (necrotic debris). There are some conditions
in which inflammation can become harmful particularly when acute inflammation becomes
chronic, in which case it is obviously dangerous;
- It is activated by non-injured cells. The cells that are damaged do not take part in
inflammation, they die and that’s it, while the healthy ones intervene to protect our organism
by debris and anything else;
- It takes place in the same way regardless of the nature of the damaging agent. We will see
how and what kind of cells intervene in acute inflammatory process, but the mechanism is
ALWAYS the same;
- It is connected with the healing process which can occur by regeneration (in Latin, restitutio
ad integrum). It means that the tissues after an inflammatory process can recover to their
original state, this can happen when the damaged cells are labile cells which can proliferate.
When the damaged cells are perennial ones or when the inflammatory state is too strong,
the healing process occurs by repair (rigenerazione, riparazione) meaning that connective
tissue is created to fill up the space.
When an acute inflammation becomes chronic, so when regeneration or repair do not work, it can
also be harmful establishing a chronic state of inflammation. Inflammation can induce also
hypersensitivity reactions, disfiguring scars called keloids when repair is excessive (we will see
37
also the various types of keloids) and scar retraction and adherence meaning that filaments can
be created when this repair is not performed in the right way.
When an organ or a part of it is affected by acute inflammation, from the terminological point of view
we add the suffix “-itis” to the word. A few examples and “exceptions”: encephalitis (encephalon),
pneumonia (lung), hepatitis (liver), gastritis (stomach), glossitis (tongue), mastitis (breast), stomatitis
(mouth, oral cavity) that occurs often in babies, metritis (uterus).2
Figure 10
Look at figure n. 10. These are the cells and molecules that are important in acute inflammatory and
chronic inflammatory processes. The inflammatory response (in general, acute and chronic) takes
place in the vascularized connective tissue which means that it takes place in a connective tissue
where there are several vessels especially where there are micro-vessels, the microcirculation. All
the cells of the blood are important, some are more important in acute inflammation, some in chronic.
The polymorphonuclear leukocyte (PMN) is the neutrophil, the main protagonist in acute
inflammation. Monocytes are more important in the chronic inflammation because they are the
precursors of macrophages. Actually, they are not really precursors because they are already
differentiated cells. However, under certain stimuli, monocytes adhere to the endothelium, go into
the tissue, and when they become resident cells, they differentiate into macrophages. That’s why
often you can see “monocytes-macrophage system” because macrophages are monocytes that
have further differentiated. Mast cells contain granules containing histamines and are very important
in acute inflammation. Fibroblasts that produce extracellular matrix and proteins are also very
important.
Types of inflammation
We can distinguish between two types of inflammation:
- Acute;
2
Study the conditions mentioned on slide 4, fourth PDF.
38
- Chronic.
Acute inflammation occurs in few minutes, it starts very quickly, it is rapid while chronic is a slow
Figure 11
inflammation that can also take years to develop. Examples of chronic inflammation are:
atherosclerosis, Alzheimer’s disease, tuberculosis, cirrhosis. The protagonists in acute inflammation
are neutrophils, while in chronic inflammation are monocytes, macrophages and lymphocytes if
an immunological response has to be activated. The scheme depicted on figure n.11 gives an
overview of acute and chronic inflammation. When a tissue is damaged it undergoes necrosis,
meaning that a chemical, biological or physical agent acted triggering acute inflammation. This aims
at eliminating the causes and consequences of the damage. It the damaged tissue is constituted by
proliferating cells (labile e.g. epithelial cells), acute inflammation ends through regeneration
(restitutio ad integrum). The tissue is again produced thanks to the ability of the labile cells to
proliferate and create new cells; inflammation disappears. If the damaged cells are non-
proliferating ones (e.g. muscle cells, neurons), the only way to stop the inflammatory response is
through repair: connective tissue is created in that site and a scar is formed. When a scar is formed,
there is loss of function because the tissue is different from the original one i.e. not elastic anymore,
signals cannot be transmitted.
Repair and regeneration are achieved when the stimulus disappears. If the stimulus persists, the
acute inflammation can become chronic, lasting from months to years. If the stimulus is eliminated,
also after moths, the only way that a chronic inflammation can stop is by repair, even if the damaged
cells can proliferate (labile). In chronic inflammation, due to the extended period of time of the
condition, the area of inflammation is too big, therefore it is difficult for the cells to regenerate and
restore the tissue. For instance, in acute hepatitis a big area of the liver is in an inflammatory state,
something has caused cell death. If it is an acute inflammation, hepatocytes, which are stable cells,
when the stimulus disappears (e.g. viruses), begin to proliferate and restore the size of the liver. If
the acute hepatitis becomes chronic hepatitis, lots of hepatocytes die and the remaining ones try to
39
restore the liver architecture but are not able to do it because inflammation persists: they try creating
new hepatic lobules, new central veins, portal triads and so on. New lobules are created but they
are not identical to the original ones and are called “pseudo-lobules” which are not functioning
properly. A liver with a chronic inflammation can progress to cirrhosis and deposition of collagen and
fibrotic processes. In chronic inflammation cells try to regenerate but they are not able to do it, so
they make a mess (“fanno pasticci”). If the stimulus is not eliminated chronic inflammation persists,
even forever.
Inflammation presents specific clinical features, which have been recognized and described by the
Egyptians (as reported in a papyrus dated 3000 b.C.) and then by Celsus in the first century a.C.
The five signs of inflammation are:
1. Heat (calor);
2. Redness (rubor);
3. Swelling (tumor);
4. Pain (dolor);
5. Loss of function (functio laesa).
The first four elements of the inflammation were described by Celsus first, whereas the fifth was
described by Virchow.
Acute inflammation
In acute inflammation there are modification in the vessels, in the microcirculation and also in cellular
events. The two main events in the vessels are vasodilation of the microcirculation and increase
in vascular permeability. Vasodilation leads to changes in the blood flow and in terms of sign of
inflammation, it causes redness of the area (rubor) and presents an increase in temperature
(calor). As regard the increase in vascular permeability, in normal condition endothelial cells are
close to each other, so cells, protein and fluids cannot go out from the vessels in the extracellular
fluid. In case of an inflammatory state, cells do not adhere to one another due to the increase in
permeability that allows the passage of cells, proteins and other plasmatic elements into the
extracellular fluids. This mechanism is used by white blood cells to exit the vessel and reach the
connective tissue where they exert their function which is to clean up the debris. In acute
inflammation, lymphocytes as wells as neutrophils leak out of the vessels into the extracellular space
of the tissues and here they phagocyte bacteria, pathogens and debris. N.B. Neutrophils are
phagocytes like macrophages.
Phases of acute inflammation
1. Vasoconstriction: there is a very fast transient vasoconstriction because the blood has to
arrive in the area where it is needed. This vasoconstriction is comparable to squeezing a
water pump;
40
2. Vasodilation: the vasoconstriction is immediately followed by vasodilation which allows a

faster blood flow to the area. The vasodilation is induced by several mediators, the most
famous is histamine;
3. Active hyperaemia: increased blood flow;
4. Vasoconstriction and hyperaemia bring an increase in the
hydrostatic pressure followed by increased permeability.
Endothelial cells are again, not adhering and therefore cells, lipids
and proteins can reach the extracellular fluids. This results in a
decrease in the colloid osmotic pressure, which is the pressure
given by the concentration of proteins in the vessels. If the colloid
osmotic pressure decreases, then the fluids are stimulated to move
out of the vessels;
5. White blood cells recruitment: vascular permeability is therefore
increased and neutrophils mainly but also leukocytes are recruited
on the site of inflammation because several mediators are
released by the cells involved in inflammation (endothelial cells,
tissues’ cells…). This allows the adherence of the white blood cells
to the endothelium first, then their extravasation into the extracellular tissue where they can
then clean up the debris;
6. Edema: this happens when fluids (reach in proteins) exit the vessels and accumulate
extravascularly. The phagocytes that have reached the extracellular space clean up debris
and pathogens. This leads to the resolution and therefore repair;
7. Resolution.
Q: Is the inflammation caused by an allergic reaction different from an inflammation due to necrosis?
No, all kinds of inflammation share/undergo these phases, what changes are the cells that intervene:
in allergic reaction we have the action of eosinophils and basophils, granulocytes.
Figure 12
Figure n.12 description: in normal condition, in the arterial side the hydrostatic pressure is higher
than the colloidosmotic pressure and therefore we have a net flow of fluids from the vessels to the
extracellular fluids, whereas in the venule side the colloid osmotic pressure is higher and therefore
we have the net flux of fluids into the vessels from the extracellular fluids. Therefore, overall there is
no net flow at the capillaries level. When we have vasodilation, in acute inflammation, in the arterial
side the hydrostatic pressure is increased a lot due to hyperaemia and vasodilation, while the colloid
osmotic pressure is the same, if not reduced because the cells and the proteins have exited the
vessel. Therefore, there is a net flow out of the vessel. It is important to remember that in acute
inflammation, the variation in dilation of the microcirculation (first vasoconstriction and then
vasodilation), leading to an increased amount of blood coming to that area and to increased
41
permeability of the endothelium, causes changes in blood pressures (increased hydrostatic and
decreased colloid osmotic) which as consequence allow the exiting of proteins and fluids from the
vessels to the extracellular environment.
Figure n.13 is an electronic microscope image of endothelial cells,

in normal condition. Normally the endothelial cells are positioned
very close to each other due to the presence of tight junctions. In
inflammation it is visible that there is a big space between the cells.
This is what we mean with increased permeability. It is caused by
different mechanisms, represented in the picture. The most
common is self-contraction of the cytoskeleton of the cell which
causes the breakage of the junctions. Another mechanism is given
by the fact that white blood cells can adhere to the endothelial
cells and release damaging substances in order to separate the Figure 13: endothelium in normal
condition.
cells and be able to extravasate from the vessel.
Pores can appear, and fluids can also exit through pinocytosis.
In the normal tissue, neutrophils and plasma proteins cannot reach the tissue due to the presence
of tight junction.
Figure n.14 is an electron microscopy picture that shows
endothelium in an inflammatory state. It is noticeable the
retraction of the endothelial cells’ cytoskeleton, which is
caused by the histamine produced by mast cells. These cells are
present in the connective tissue and can degranulate releasing
this substance that will in turn cause the contraction of the
endothelial cells, the breakage of the junctions and the leaking of
the vessel content. If the fluid accumulates in the extravascular
area we have an edema. These mechanisms can happen Figure14: endothelium in
inflammation
together.
Edema, exudate and transudate

As we have said before, the extravasation of fluid into the extracellular space from a vessel and its
accumulation is called edema. The fluid, proteins and cells that exit causing the edema which can
be called either exudate or transudate. The difference between the two terms is that the edema
presents with exudate when it is caused by an ongoing inflammatory state, whereas it is called
transudate if it is not caused by inflammatory mechanisms. As we said before, when we have an
inflammatory state, the edema in the tissue is not simply serum, but it is rich in proteins and
42
sometimes cells. When fluids exit the vessels in lack of an inflammatory state, usually there is an
underlying condition which causes blood proteins to be in a decreased concentration compared to
normal. In order to balance the osmotic pressure on the two sides of the endothelium, the fluid tends
to exit the vessel. This fluid forms the transudate. Examples of conditions that cause the transudate
are: hepatic cirrhosis and hepatic carcinoma. In these cases the patient presents with a condition
called ascites, which is the accumulation of fluids in the peritoneal cavity (if we see elderly female
patients which look pregnant here’s why, if we see elderly male patients that look pregnant there’s
no way to know if time was not nice to them or if they also have ascites, good news is they are
definitely not pregnant).
One of the main functions of the liver is to synthetize proteins, when there are problems such as
tumors, cirrhosis, chronic hepatitis and so on, the function of the organ itself is impaired. If proteins
are not produced they are not released in the blood circulation and they cannot exert their colloid
osmotic pressure. Moreover, if the liver presents with carcinoma or fibrotic tissue, the incoming blood
in the portal vein cannot enter the liver easily, increasing the hydrostatic pressure. The high values
of the hydrostatic pressure and low ones of the colloid osmotic pressure result in the leakage of
fluid from the portal vein to the peritoneal cavity. This although, is not a condition determined by
inflammation. In inflammation there is the increased permeability of the vessels which is lacking here.
Recap: Exudate and transudate are different in terms of causes: the former is due to inflammatory
state, the latter is not; they have a composition similar to plasma, except that exudate can present
proteins and cells in it, whereas the transudate is an ultrafiltrate of plasma (is comparable to serum).
Exudate
There are different types of exudate:

1. Serous exudate or serous edema (figure n.15) is an exudate with absence of prominent
cellular response which means that cells are not present or are present in very low quantity.
It has a pale-yellow color and it is similar to serum in composition. It is caused by radiation,
burns which is what causes blisters of the epidermis (in Italian Figure 15: Serous inflammation, low
“flittene”). As shown in the picture the epidermis is separating power view of cross section of a skin
blister showing the epidermis
from the derma and it is possible to notice the dermal papillae. separated from the dermis by a focal
The white part on top is the serous exudate; collection of serous effusion.
2. Fibrinous exudate: contains many proteins, in particular
fibrinogen which is a clotting factor and is the precursor of fibrin. For
instance, it is possible to notice the presence of adherences due to
exudate in a pericarditis (figure n.16). The exudate is not fluid;
3. Catarrhal exudate: it occurs when the damaged tissues are rich in
muciparous cells and it is named following bacterial or viral infections i.e.
rhinitis, pharyngitis…;
Figure 16: fibrinous
pericarditis.
43
4. Hemorrhagic exudate: along leukocytes, white blood cells, proteins and fluid we have
erythrocytes. It is similar to blood, but it is not, because it is NOT a hemorrhagic process but
an inflammatory process;
5. Purulent exudate, also called suppurative inflammation: it is pus.
It is characterized by presence of a lot of neutrophils, necrotic cells
in particular coming from liquefactive necrosis. The fluid is yellowish
and green, it is associated with infection of pyogenic bacteria. This
type of bacteria (examples are staphylococcus, streptococcus...)
cause liquefactive necrosis in infected cells. In this case, the exudate
is always purulent. Figure n.17 shows an example in meningitis.
The purulent exudates have different names:
- empyema for instance is a particular type of inflammatory Figure 17: purulent
edema, implying the presence of purulent exudate in the exudate in patient with
cavities of our body. We can have many types of it bacterial meningitis
depending on the cavity. Pyoperitoneum for example is
the empyema of the peritoneum. Sometimes an acute appendicitis can evolve in
peritonitis which can lead to pyoperitoneum: the peritoneal cavity fills with pus due
to the pyogenic bacteria causing liquefactive necrosis and inflammation. Another
example is the empyema of the articular cavity.
- flemmon: presence of pus along the course of tendons and muscles or under the
skin;
- abscess: is a type of inflammatory edema, specifically a purulent exudate that
accumulates in newly formed cavities arising from the action of neutrophils. It is
shown in figure n.18. The action of neutrophils against pathogens involves the
release of granules and when is required action of a certain entity the enzymes
released can damage and digest our tissue as well as the pathogens, thus
creating a cavity. In other words: purulent exudate accumulated in newly formed
cavities generated by the lithic action of proteases
that accumulate following the destruction of
granulocytes. The exudate can make its way
through the tissue and create a fistula into an
adjacent organ.
Q: Is caseous necrosis a type of abscess? Not

really, liquefactive necrosis is more comparable to
abscess, although caseous necrosis is in between
Figure 18
liquefactive and coagulative. Necrosis in general is connected to chronic inflammation rather than
acute.
How do neutrophils reach the tissue?
We have discussed changes in blood flow and vessels dilation, the increased permeability and
consequently the exudate formation where proteins fluids and cells exit the vessels. So how are the
granulocytes able to reach the tissue? There are three phases to this journey, represented in figure
n.19:
44
1. margination of neutrophils: due to vasodilation and stasis of blood,

neutrophils can remain near the endothelial cells, then they can adhere to
them, roll (adherence and rolling imply a modification in the shape) and form
interactions;
2. transmigration or diapedesis: as shown in the picture, the white blood cell is
undergoing extravasation from the vessel thanks to the increased
permeability. They transmigrate because they are attracted to the site in the
connective tissue thanks to the presence of chemical mediators. Leukocytes
have specific receptors on their membrane which can bind to other receptors
on the endothelial cells and during an inflammatory response these receptors
increase on the surface of neutrophils and endothelial cells as well. This allows
the interaction and adhesion that leads to the extravasation; Figure 19:
3. migration into the tissue thanks to chemotactic stimulus: here the cell margination above
and diapedesis below.
changes its shape to move and follow the chemical trail, it phagocytes the
pathogens or debris. This material is engulfed in a phagosome and afterwards
the lysosome is formed. This presents granules and fuses with the phagosome forming the
phagolysosome. The content of the two compartments meets, so the enzymes of the
lysosome can digest the phagocyted material.
Neutrophils and macrophages are professional phagocytes. This is their main function and
prerogative. There are other cells though that are not professional phagocytes (meaning that it is not
their usual activity) that however can perform it in certain occasions. For instance, the endothelial
cells can in some conditions become phagocytes, as well as fibroblasts, the cells of the connective
tissue.
This is the pathway by which
neutrophils destroy
microbes, mainly through
enzymes that destroy
reactive oxygen species.
NB. There is another version of the slides about acute inflammation (the first part) on Medinto.it
which contains corrections such as spelling of phlegmon, which was previously spelled flemmon.
Recap of past lesson: we spoke about inflammatory reaction, in particular acute inflammation which
occurs when there is death of cells due to microbes, chemical toxins or physical agents, aging and
so on and our body tries to clean up the tissue and remove the debris. There are different phases
during acute inflammation which our body activates. The mechanisms involve both the vascular
system and the cells.
45
First of all, there is a fast and transient vasoconstriction followed by vasodilation that allow an
increased amount of blood to reach the site where it is needed. The increment of blood flow in the
area causes redness (“rubor”) and an increase in temperature (“calor”) which are signs of
inflammation. This is followed by an increase in hydrostatic pressure and vascular permeability,
which means that the endothelial cells are no longer adhering to one another as it happens in normal
conditions because of the presence of tight junctions. Fluids, proteins and cells can therefore pass
through the spaces in between these endothelial cells and reach the neighbouring tissues. We are
speaking about microcirculation, so it is a phenomenon that happens locally. The fact that cells and
proteins exit the vessel and reach the extravascular spaces causes a decrease in colloid osmotic
pressure which in turns causes the fluid to diffuse into the tissue “following” the proteins and cells.
The increased permeability of the micro-vessels drives the cells, and in particular neutrophils, to exit
from the blood circulation and move into the tissue. Figure n.1 shows neutrophils, which are attracted
to the area of the vessel where their action is needed.
Upon their arrival, they adhere to the endothelial cells
because of the presence, on both endothelial cells and
white blood cell, of receptors: they begin to roll against the
vessel wall and form even stronger interaction with the
endothelium until
they stop and
transmigrate into the
tissue. Here is where
the white blood cells
start their work: they
start phagocyting
debris microbes etc.
Neutrophils are
Figure 2
professional
phagocytes, as
macrophages. This
is their principal role and they are therefore the protagonists of acute inflammation. Fluids as well
exit together with proteins causing the sign of “tumor” which is the swelling and can lead to
inflammatory edema.
There are two types of edema: if we speak of inflammatory edema then we will call it exudate.
Transudate on the other hand is a type of edema that is caused by non-inflammatory stimulus.
We will encounter the transudate when we will speak about pathologies of the liver. An example is
ascites. The types of inflammatory edema can be serous, purulent, haemorrhagic and so on.
Interaction between endothelial cell and a
white blood cell. Figure n.2 description: this is the plasma membrane of a
neutrophil. They are professional phagocytes: their role is to eliminate and digest the debris. We will
now be speaking about the mechanisms through which the neutrophils engulf and destroy microbes
and debris.
Neutrophils have specific receptors on their plasma membrane which allow them to recognize the
microbe, subsequently they engulf it through endocytosis. The engulfed vesicle is called
phagosome. After its formation, the phagosome, now in the cytoplasm of the cell, is fused with the
lysosome. The lysosome is a cellular organelle containing enzymes able to digest the microbe. The
fusion leads to the formation of the phagolysosome, where the content of the vesicle comes in
contact with these digestive enzymes that are able to destroy the microbe.
46
Figure 2
There are
conditions in which the classical phagocytosis is not possible. For instance, if
there are too many microbes on the site of inflammation, the neutrophils do not
have time to complete the digestion. They engulf microbes and regurgitate them
back into the tissue continuously, engulfing and regurgitating again and again
while trying to digest them. The mechanism in fact is called regurgitation during
feeding and is depicted in figure n.3. The problem is that by regurgitating the
content of the phagolysosome the enzymes of the lysosome leak in the Figure 3
neighbouring tissue damaging our own cells as well.
This can lead to the formation of a cavity within the tissue, that fills with fluids coming
from the vessel as a consequence of high permeability. The name of this exudate or
edema is abscess (pus inside the newly formed cavity).
In certain cases, the neutrophils are not able to engulf the microbes due to their high
concentration and therefore in attempt to destroy them, they release the lysosomal
enzymes directly in the tissue. The result is about the same mentioned before in the
case of abscess. This phenomenon, illustrated in figure n.4 is called frustrated
phagocytosis. Figure 4
How can granulocytes degrade and kill the microbes?
We have to remember that neutrophils are phagocytes, they present digestive
enzymes in their lysosomes and some of those are involved in the production of ROS (Reactive
Oxygen Species). Examples are: myeloperoxidase, NADPH peroxidase.
The bacterium is opsonized3 which means that little molecules and peptides are bound to the
microbes so that the granulocytes can recognize them thanks to the presence of receptors on their
Figure 5
3
Opsonization is a term that refers to an immune process in which particles such as bacteria are targeted for
destruction by an immune cell known as a phagocyte. The process of opsonization is the means of
identifying the invading particle from the phagocyte.
47
membrane. The bacterium is then engulfed, and this gives rise to the phagosome, which then fuses
with the lysosome forming the phagolysosome. This mechanism is shown in figure n.5.
The enzymes listed in figure n.6 are initially present
on the plasma membrane and, subsequently to the
engulfment, also on the surface of the phagosome
and are responsible for the formation of ROS.
Examples are: NADPH oxidase and superoxide
dismutase, as well as myeloperoxidase and other
proteins responsible for the degradation of
Figure 6
microbes.
Reactive oxygen species can be toxic if they are
present in high concentration and as a consequence to many pathologies or senescence, our tissues
themselves can produce a harmful amount of ROS, but they are also fundamental for the majority of
the signal transduction pathways activated in normal physiological condition such as anti- and pro-
apoptotic signaling cascades. Transient and slight increase of ROS production is necessary to
activate signaling pathways involved in cell survival. They are not to be considered univocally
“enemies”.
The first enzyme activated is NADPH oxidase which is able to reduce oxygen to superoxide anion
(another reactive oxygen species). This enzyme is composed of several subunits, some of which, in
normal condition, are on the cell membrane and others are dispersed in the cytoplasm. When a
stimulus such as the binding and recognition of the microbe by the neutrophil occurs, NADPH
oxidase is activated. Activation happens through the assembly of the different subunits. Those
that were originally in the cytoplasm translocate to the cell membrane where the other subunits are
and come in contact with one another, thus giving rise to an active enzyme. The result of its activation
is the production of superoxide anion. The majority of superoxide anion is transformed into
hydrogen peroxide spontaneously, whereas the rest is transformed into hydrogen peroxide by a
dismutase. Then there are other reactions that can occur and lead to the formation of hydroxyl
radical. The most toxic compound created by these reactions is hypochlorite. Hypochlorite is
formed by the enzymatic action of myeloperoxidase starting from H2O2 in presence of chloride ion.
It is a very powerful compound against microbes.
There are also other antimicrobial pathways that do NOT involve ROS. An example is the reduction
of intracellular pH, as well as the action of lysozyme and lactoferrin which are proteins that degrade
the microbes’ cell structure.
There are several diseases characterized by recurrent bacterial or viral infection due to the fact that
the neutrophils do not carry out their function properly. It happens for example when the NADPH
oxidase or the myeloperoxidase are not present or are present in concentrations that are not
sufficient or for some reason are not functional. There are also conditions in which neutrophils are
not able to interact with endothelial cells because they might lack or present a mutate form of
adhesion molecules such as integrins. Lack of functional integrins prevents neutrophils to adhere
and transmigrate from the vessel into the tissue.
Chediak-Higashi syndrome leads to neutrophils that present defective lysosomal granules and
therefore the neutrophils cannot exert their function properly.
THE MEDIATORS OF INFLAMMATION
We have seen what happens macroscopically during acute inflammation: neutrophils arrive, adhere,
roll, transmigrate, phagocyte. We know that the vessel undergoes vasoconstriction then vasodilation,
the permeability increases etc...
Why do all these processes occur? What are the stimuli that cause the vessel to vasodilate?
48
There are lots of molecule called inflammatory mediators released by all the cell types involved in
the inflammatory reaction: neutrophils, endothelial cells, fibroblasts and so on all produce different
types of mediators. There are two different types of mediators:
1. Cell-derived mediators: they are the mediators produced by the cells and can be:
- Preformed: they are molecules already present in the cells and are secreted only if
needed. Examples are: histamine, which is a mediator contained in granules belonging
to mast cells of the connective tissue; and lysosomal enzymes which are also already
present. The name of the family of compounds to which histamine belongs is vasoactive
ammines;
- Newly synthetized mediators: synthetized only when needed from inactive precursors.
When a stimulus occurs in a macrophage or neutrophil, a signal transduction pathway is
activated leading to the production of a transcription factor and then to the overexpression
of a gene that codifies for the mediators. Then they can exit from the cell and reach their
target. Example is NCP1. Arachidonic acid metabolites, pro- and anti-inflammatory
cytokines, adhesion molecules, ROS […]
2. Plasma-derived mediators: produced mainly in the liver, this is the organ that has the main
function of protein synthesis. In the plasma we have molecules that are in their inactive state
and when they are needed they are activated. They can exit from the blood stream thanks to
the increased permeability and reach their target.
In figure n.7 there is a list of the mediators that we will

discuss about and we will have to remember. In figure n.8
we have a list of the principal effects of the most important
mediators.
Preformed cell-derived mediators

Figure 7 Vasoactive amines
Figure 8
1. Histamine is a preformed cell-derived mediator
present in various cell types but mainly in granules of mast cells. When a stimulus arrives,
49
this mast cell degranulates and through exocytosis releases the content of the granules:
histamine is released in the tissue and immediately goes to bind the surface of the endothelial
cells, which have specific receptors for histamine which are called H receptors.
Upon binding, a signaling cascade is activated which leads to cytoskeleton contraction. This
is one of the mechanisms through which the cells retract breaking the junctions and separate
from one another. Therefore, binding of histamine to the H receptor on the endothelial cell
causes increased permeability and vasodilation. Histamine is responsible for two of the signs
of inflammation: redness and increase in
temperature.
Histamine is one of the first mediators to intervene,
as a preformed mediator, ready to act once
released into the inflammatory centre.
2. Serotonin as well causes the increased vessel
permeability and vasodilation.
Lysosomal enzymes
Another example of cell-derived mediators are the lysosomes enzymes, they are present in the
neutrophils and the monocyte-macrophages lineage deriving cells.
Newly synthetized cell-derived mediators
Arachidonic acid metabolites also called eicosanoids
They derive from a polyunsaturated fatty acid that is arachidonic acid. It is a fatty acid usually
esterified with plasma membrane phospholipids, but under certain stimuli a family of enzymes called
phospholipases (mainly phospholipase A2) are activated and break the bond between the
phospholipid and the arachidonic acid which is released in the cytoplasm. Here it is metabolized by
two enzymes:
- Cyclooxygenase (COX) which produces prostacyclin, thromboxane and
prostaglandins.
- Lipoxygenase (LOX) produces a big family of mediators called leukotrienes.
They cause transient and fast vasoconstriction at the beginning of the inflammatory process.
Arachidonic acid in certain tissues can undergo a non-enzymatic oxidative demolition (lipid
peroxidation) that is independent from these enzymes but is due to ROS. The result is a compound
called aldehyde 4-hydroxynonenal (HNE), which is involved in strong inflammatory processes.
HNE is involved in many chronic inflammatory diseases such as atherosclerosis and fibrosis.
Platelet activating factor
We have the idea that it is a mediator that stimulates the aggregation of platelets, but it is not its only
function. Platelet activating factors comes into play by stimulating the aggregation of platelets in the
final step of the inflammation, such as during the formation of a scar, but it has many other functions:
1. Vasoconstriction;
2. At low concentration they induce vasodilation and increase venular permeability;
3. Chemotactic mediators: means they attract the cells from the blood stream to the
extravascular tissue;
4. They favour leukocyte adhesion to endothelium;
5. They activate phagocytes;
6. Platelet aggregation.
Cytokines
Cytokines they are responsible for the phases of acute inflammation. Cytokines can have different
actions:
50
1. Pro-inflammatory action: interleukin 1 or interleukin 6, TNFα;

2. Anti-inflammatory action: interleukin 10 and interleukin 4 and TGFβ (transforming growth
factor β);
3. Chemotactic activity: monocyte chemotactic protein 1 (MCP1), when their concentration is
high the monocytes are stimulated to adhere to the endothelium and transmigrate into the
tissue, so it has an overall proinflammatory action. They can be produced by a large number
of cells and act not only on monocytes but on other white blood cells too.
Adhesion molecules
Neutrophils can adhere to endothelial cells thanks to the presence of these molecules. These
molecules are not released from the cell surface, but they remain attached to the membrane,
nevertheless they are considered inflammatory mediators. They belong to three main classes:
1. Selectins: expressed in the endothelial cells;
2. ICAM, VCAM, PECAM, also present on the endothelial cells but are involved in different
phases of the leukocyte recruitment:
o ICAM: intracellular cell adhesion molecule;
o VCAM: vascular cell adhesion molecule;
o PECAM: platelet endothelial cell adhesion molecule;
3. Integrins: they are present on the plasma membrane of neutrophils (white blood cells in
general) so when those cells slow down and start rolling and forming stronger adhesions the
integrins are very important because they bind to selectins on the endothelium. The rolling is
allowed by the interaction between ICAM and integrins and then integrins bind VCAM and
this interaction allows transmigration into the tissue. This process is depicted in figure n. 9.
Figure 9
Plasma-derived mediators
Are those mediators that are present in the blood in the inactive form as precursors and are then
transformed into the active form during inflammation. These are:
1. The complement system.
2. Kinins: an example is bradykinin which is involved in vasodilation, increased permeability.
In acute inflammation this family of proteins is transformed from its precursor form of
kininogen into kinin, so they are activated. These complicated mechanisms called the kinin
system as well as the coagulation cascade and the fibrinolytic system are all
interconnecting as it is shown in the figure n.10.
3. Product of coagulation.
During the coagulation cascade the important enzyme is thrombin which leads to the formation of
fibrin from fibrinogen allowing the assembling of the fibrin net and complete homeostasis. The net is
destroyed by plasmin, which is part of the fibrinolytic system. Plasmin is activated by the same
51
enzyme responsible for the production of kinin and for this reason these pathways are
interconnected. Moreover, plasmin activates the complement as well which has importance in the
inflammatory processes. Where does the cascade start though? Who is the responsible for the
activation of all these interconnected pathways? Factor XII, or Hageman factor.
The complement cascade involves little peptides produced that are important in several processes
of acute inflammation such as the recognition of bacteria and opsonization, during which the
complement factor C3A is opsonized to the bacterium and recognised by the neutrophil. The
complement system is also important to activate and recruit leukocytes and destroy microbes.
The products of coagulation are important in the last phases of inflammation.
Acute inflammation evolves leading to:
1. Regeneration or restitutio ad integrum: this means that the tissue goes back to its original
condition of health. In order for this to happen the injurious stimulus must be of moderate
intensity and duration and the tissue itself must be able to actively proliferate and produce
replacement for the cells that have been damaged or killed, so it must be labile.
2. Repair: the initial condition of the tissue cannot be regenerated due to the fact that the stimuli
were either particularly intense (even in labile tissues) or because the tissue is not labile
therefore cannot undergo active proliferation and replace all the injured cells. There is
therefore a production of collagen fibres and of scar tissue as an attempt to fill the empty
space left by the dead cells.
3. Chronic inflammation: if the stimulus persists for a longer period the acute inflammation
degenerates into chronic inflammation which causes the inflammatory signals to remain
active and doesn’t allow a resolution through regeneration but only through repair.
CHRONIC INFLAMMATION
It is important to underline that chronic inflammation is a state that is present in major chronic human
diseases such as atherosclerosis, tumours, multiple sclerosis. This doesn’t mean that these diseases
are necessarily classified as inflammatory diseases (e.g. cancer) but it means that they cause the
development of chronic inflammation whose action is to try and protect the body from the disease
itself. Inflammation is an attempt to protect the body from the disease but while acute inflammation
actually has a protective action, chronic inflammation ends up becoming harmful itself: in case of
many conditions such as cancer, the action of chronic inflammation can promote the progression of
the disease.
Chronic inflammation is an inflammatory state of prolonged duration (weeks or months) and
presents three processes happening simultaneously and constantly:
1. Inflammation: macrophages try to contrast the injury and eliminate the damage;
2. Tissue injury;
3. Repair.
The three elements coexist, which means that while some areas of the tissue are repaired, others
are still being damaged or suffering inflammation. There isn’t a linear progression of the condition as
in acute inflammation.
Chronic inflammation may follow acute inflammation or may start as chronic from the beginning.
Example: atherosclerosis is the process that leads to the occlusion of arteries and vessels due to
the formation of atherosclerotic plaques on the walls of the vessel. This process starts as chronic
when the individual is about 13-14 years of age and entails the accumulation of lipids on the vessels’
walls. Macrophages try to eliminate the lipidic substance through phagocytosis continuously but
cannot keep up with the accumulation and around 50-60 years the individual finds himself with
atherosclerotic plaques. The process starts as chronic.
Chronic inflammation arises from the following settings:
52
1. Persistent infection: there might be microorganisms that are difficult to eradicate, so there
is an acute inflammation developing at first that becomes chronic due to the fact that
macrophages are not able to destroy the microbes,
2. Prolonged exposure to potentially toxic agents: is the case of atherosclerosis. We have
talked about lipids generally, but more precisely the substance accumulating is oxidized LDL
(low-density lipoproteins). Lipids cannot flow freely in the blood because they are
hydrophobic substances and therefore are insoluble in the plasma. They need to be carried
by proteins creating lipoproteins. Digression: if lipids were able to move freely (not bound to
protein carries) in the blood they would gather forming micelle, which are very dangerous
because they could become emboli i.e. aggregates (of various nature: lipidic, solid, gaseous)
that travel through the bloodstream and clog arterial capillaries even far from their site of
origin. Fortunately, lipids can only be transported in the bloodstream by proteic carries which
make the compound hydrophilic and soluble. In the case of atherosclerosis though, the low-
density lipoproteins are oxidized. If inflammation occurs, the endothelium permeability is
increased causing retention of oxidized LDL at the level of the tunica intima of the wall and
the build-up of the LDL even in the lumen of the vessel. The macrophages phagocyte the
LDL and accumulate inside the intima of the artery. The phagocytosis is never enough due
to the continuous accumulation of LDL and eventually results in atherosclerosis and chronic
inflammatory state;
3. Hypersensitivity diseases: such as autoimmune disease where the inappropriate and
excessive activation of immune cells persists.
What are the difference between the acute and chronic inflammation?
1. Acute inflammation is also called angiophlogosis: “angio” indicates processes involving
vessels and microcirculation whereas chronic inflammation is called histophlogosis, where
“histo” implies involvement of the tissue. This means that if acute inflammation regards mainly
what happens to the vessels and the microcirculation, chronic inflammation regards mainly
what happens in the tissue;
2. Infiltration of mononuclear cells, in particular macrophages. These cells derive from a
monocyte lineage. In acute inflammation the main protagonists are neutrophils whereas in
chronic the main protagonists are macrophages;
3. Tissue destruction: it is more extensive then in acute inflammation and the body tries
continuously to repair the damage but is unable to completely repair the tissue and heal.
4. Attempts at healing by:
o Connective tissue replacement of damage tissue: fibroblasts produce
extracellular matrix to substitute the lost cells;
o Angiogenesis: formation of new vessels starting from pre-existing ones. The pre-
existing vessel cells release signals and initiate cascades in order to stimulate the
formation of new vessels that should bring nutrients and oxygen to the area of cell
death and to the newly formed scar tissue in order to allow repair. (NB: In chronic
inflammation we never have regeneration, only repair).
o Fibrosis.
We have mentioned before that the macrophages are the protagonists of chronic inflammation, but
many other cells actually play a role in it: lymphocytes, eosinophils, mast cells that contain histamine,
neutrophils just like in acute inflammation, fibroblasts and plasma cells.
Macrophages.
53
They are very important, as neutrophils they also

contain granules and lysosomes and they are
professional phagocytes deriving from the
monocytic lineage. When they are in the blood they
are still in their undifferentiated form of monocytes,
they act just like the neutrophils: they adhere to the
endothelium, roll, adhere more strongly,
transmigrate and when they arrive to the tissue they
differentiate into macrophages. It is not completely
correct to say that monocytes are precursors of
macrophages, because monocytes are not
precursors: they are cells that are already
differentiated and have their own function in the blood. When they enter the tissue they become
resident cells, so they undergo a further maturation and they activate the transcription of a number
of genes encoding for the production of molecules that monocytes do not produce and become
phagocytes. The functions of macrophages are:
1. The main role of macrophages is to phagocyte both pathogens and microbes as well as
debris, necrotic cells and dead tissue;
2. Initiation the process of tissue repair;
3. They secrete mediators of inflammation such as cytokine TGF (transforming growth factor)
β which stimulates the proliferation of fibroblasts and their production of collagen, important
for scar tissue formation and repair;
4. They interact with macrophages and lymphocytes and reciprocally activate each other.
They display antigens to T lymphocytes and respond to signals from T cells, thus setting up
a feedback loop that is essential for defence against many microbes by cell-mediated
immune responses.
The macrophages accumulate in the tissue during chronic inflammation. why and how does that
happen? There are three mechanisms that determine their accumulation:
1. Continuous recruitment of monocytes from the blood: due to the presence of
chemoattractant and chemotactic substances the monocytes are stimulated to adhere, roll,
adhere more to the vessel wall and then transmigrate into the tissue where they become
macrophages;
2. Proliferation of resident macrophages: there are macrophages that are normally present
in the tissue, they are called resident macrophages and they can be activated and stimulated
to proliferate.
3. Immobilization of macrophages on the inflammatory site.
54
Figure n.10 description: macrophages can derive from the blood because monocytes can exit from
the vessel and differentiate into the tissue, but they can also be already present in the tissue because
they derive from progenitors present in the yolk sac or the foetal liver.
Figure 10
Some resident macrophages are produced during embryogenesis and remain in those organs to
carry out the function of professional phagocytes. Microglia are the cells that are responsible for
cleaning up pathogens, necrosis or debris in the central nervous system. In the liver the cells
responsible of the phagocytosis are the Kupffer cells, whereas in the alveoli of the lung we have the
alveolar macrophages. A macrophage resident in a tissue can be activated directly by microbes or
by molecules produced by leukocytes such as interferon γ and it can become:
1. M1 macrophage which is the classical macrophage and is the pro-inflammatory
macrophage and produces pro-inflammation mediators such as cytokines;
2. M2 macrophage, which is the anti-inflammatory macrophage. They are activated through
the action of certain molecules such as interleukins. It is involved in tissue repair and
suppression of inflammation. This mechanism is studied in certain conditions in order to
stimulate their activation and suppress the inflammatory process as well as slowing down
several diseases by inducing this phenotype. This type of macrophage has been discovered
just recently.
55
Figure n.11 depicts how macrophages communicate with lymphocytes.

Through interleukins (cytokines) the
macrophages stimulate the activation
of the leukocytes and in turn the
leukocytes produce interferon γ which
stimulates the proliferation of
macrophages M1 (the classical
pathway), so the pro-inflammatory
ones.
Figure 11
CHRONIC INFLAMMATION PT.2
Chronic inflammation has an important role in promoting the progression of major chronic diseases,
including tumors, atherosclerosis, Alzheimer’s disease, cirrhosis and so on.
This inflammatory response is a response which attempts to protect our organs from damage,
however in these cases of chronic inflammation, the inflammation becomes dangerous itself, as the
inflammatory state in tumors and other diseases induces the progression of the disease. Chronic
inflammation can last for weeks or months in which inflammation, tissue injury and repair coexist. It
may follow acute inflammation, or it may begin insidiously without any manifestations of a preceding
acute reaction.
For example, atherosclerosis is the process which results in the occlusion of medium to large
arteries, as a plaque forms. This is a chronic inflammation which begins insidiously without any
manifestations of a preceding acute reaction from when we are 12-13 years old. Lipids start to
accumulate in the tunica intima of the artery, which is not normal, hence, our body tries to eliminate
these lipids by acquiring macrophages that phagocyte the lipids, but lipids continue accumulating.
Eventually, when we are 50-60 years old, large plaques form which can lead to ischemia (oxygen
deprivation) of specific tissues (e.g. in the heart, heart attack; in the brain, stroke).
56
Chronic inflammation arises in the following settings:

- Persistent infections by microorganisms that are difficult to eradicate, such as
mycobacteria and certain viruses, fungi, and parasites (Mycobacterium tuberculosis e
Treponema pallidum);
- Prolonged exposure to potentially toxic agents, either exogenous (e.g. particulate silica)
or endogenous (oxidized LDLs in atherosclerosis);
- Hypersensitivity diseases (allergies): chronic inflammation plays an important role in a
group of diseases that are caused by excessive and inappropriate activation of the immune
system, autoimmune diseases (e.g. rheumatoid arthritis).
Chronic inflammation is characterized by:

1. Infiltration with mononuclear cells, which include macrophages, lymphocytes, and plasma
cells;
2. Tissue destruction, induced by the persistent offending agent or by the inflammatory cells;
3. Attempts at healing (repair) by:
- Connective tissue replacement of damaged tissue by increased fibroblast ECM production;
- Angiogenesis (proliferation/formation of small blood vessels from pre-existing vessels);
- Fibrosis.
This is in contrast to acute inflammation which is manifested by vascular changes, edema, and
predominantly neutrophilic infiltration.
Cell types mainly involved in chronic inflammation:

- Monocyte/macrophages: monocytes are circulating cells while macrophages are the
monocytes found in the tissue (differentiated tissue cells);
- Lymphocytes;
- Eosinophils;
- Mast cells;
- Neutrophils;
- Fibroblasts;
- Plasma cells.
Macrophages
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They are the dominant cells in most chronic inflammatory reactions, which contribute to the reaction
by secreting cytokines and growth factors that act on various cells, by destroying foreign invaders
and tissues, and by activating other cells, notably T lymphocytes.
Functions of macrophages include:

- To ingest and eliminate microbes and dead tissues;
- To initiate the process of tissue repair resulting in scar formation and fibrosis;
- To secrete mediators of inflammation, such as cytokines (TNF, IL-1, chemokines, and
others) and eicosanoids. Thus, macrophages are central to the initiation and propagation of
inflammatory reactions;
- To interact with lymphocytes. They display antigens to T lymphocytes and respond to
signals from T cells, thus setting up a feedback loop that is essential for defense against
many microbes by cell-mediated immune responses.
How can macrophages in the inflammatory region during chronic inflammation?

Three mechanisms determine macrophage accumulation:
1. Continuous recruitment of monocytes from the blood by the expression of adhesion
molecules and chemotactic factors;
2. Proliferation of resident macrophages (seen in atherosclerosis) which are already in
there due to derivation from embryonic tissue, therefore they can start proliferating and
get ready to phagocyte the debris;
3. Immobilization of resident macrophages in the inflammatory site.
Maturation of mononuclear phagocytes
In the steady state, some tissue macrophages, including microglia and alveolar macrophages, may
be derived from embryonic precursors and populate the tissues from the beginning, as they do not
originate from monocytes that come from the blood.
The development of macrophages from hematopoietic precursors and monocytes may be more
prominent when tissue macrophages need to be increased or replenished, as after injury and during
inflammation.
Classical and Alternative Macrophage Activation

These three abovementioned mechanisms give rise to a lot of macrophages which however still
need to be activated. Different stimuli activate monocytes/macrophages to develop into two
functionally distinct populations: M1 and M2 macrophages.
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Classically activated (M1) macrophages.

Their activation is triggered by microbial products and pro-inflammatory cytokines, particularly IFN-
γ. They phagocytose and destroy microbes and dead tissues and can potentiate inflammatory
reactions. M1 macrophages are pro-inflammatory macrophages, produce lots of proinflammatory
mediators that are able to kill bacteria, fungi and so on, thanks to the enzymes present in their
lysosomes in the same way as neutrophils do.
Alternatively activated (M2) macrophages

Their activation is induced by interlucin-13, interlucin-4 and other pro-repair cytokines (e.g. TGF-β-
1) that are important in tissue repair and the resolution of inflammation. M2 macrophages are indeed
anti-inflammatory macrophages that are involved in tissue repair and fibrosis.
When one observes a slide of a tissue, which is suspected to be in an acute inflammatory state, and
registers the presence of many macrophages, it doesn’t always mean that acute inflammation is
becoming a chronic one: it could also mean that acute inflammation is in regression, because lots of
macrophages are of the M2 phenotype which produces cytokines involved in tissue repair (the main
one is TGF-β-1, tumor growth factor 1).
In order to distinguish between the two situations, one must observe which types of cytokines are
being released. If the cytokines released are involved in tissue repair, like TGF-β-1 that stimulates
fibroblasts to produce ECM leading to scar formation and repair, then it is the end of the inflammatory
response, in the recovery phase.
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Meanwhile, if the cytokines (e.g. interferon-γ, IFN-γ) released by the lymphocytes present in the
tissue are involved in the microbicidal response and inflammatory response, this result in M1
macrophage activation. In this case, it is a chronic inflammation as the macrophage count in the
inflammation site is high, and the cytokines released are pro-inflammatory.
There are two types of chronic inflammation

1. Non-Granulomatous Inflammation
This is a non-specific inflammation, caused by very virulent
microorganisms that are difficult to eradicate and are responsible
for chronic colitis and bronchitis. The presence of these
microorganisms leads to an increase in macrophages,
lymphocytes, fibrin and so on but nothing else stands out. It can be
due to repeated exposure to irritants (tracheitis and bronchitis for
exposure to cigarette smoke, esophagitis and chronic gastritis due
to excessive production of HCl)
2. Granulomatous Inflammation
It is more specific and is characterized by the presence of granulomas. It is a specific inflammation
because there are different types of granulomas that can form depending on the type of etiological
agent that they are exposed to. However, we mainly observe the tuberculus granulomas (TBC
granuloma) in this course.
Granulomas are concentric and spheric structures, which can be found in tissues when
macrophages are not able to completely degrade the microorganisms or foreign materials which
survive in the phagolysosomes.
When the macrophages try to phagocyte, digest and eliminate a
virus that is too resistant, they surround the foreign material and
differentiate acquiring a new phenotype so that they become
similar to epithelial cells. In this case, they are called epithelioid
cells. If you hear about epithelioid cells, you have to know that
they are macrophages arranged like an epithelium. Sometimes
they fuse into a syncytium with other macrophages to become
stronger, giving thus origin to giant cells.
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These cells can be distinguished as they have large cytoplasms with multiple nuclei, derived from
the fusion of macrophages. So, if you
look at the microscope at a giant cell,
you can see the plasma membrane and
a lot of nuclei in the cytoplasm.
Externally the granuloma can be
surrounded by lymphocytes and
plasma cells (it depends on the greater
or lesser capacity of the causative agent
to induce the immune response).
Fibroblasts and collagen fibers
envelop all the granulomas. One should
not get confused between granulomas,
giant cells and epithelioid cells:
granulomas are made up of many giant
and epithelioid cells. Giant cells are
multiple epithelioid cells fused together.
Moreover, there are two types of giant cells that form granulomas:
- Langhans giant cells: syncytia composed of several macrophages with peripheric nuclei;
- Foreign body giant cells: syncytia with nuclei dispersed in the cytoplasm.
Immune granulomas
Caused by a variety of biological agents capable of
inducing a persistent T cell–mediated immune
response.
Granulomas are produced when the inciting agent is
difficult to eradicate, such as a persistent microbe or a
self antigen. Cell turnover is frequent (high turnover
granulomas). In such responses, macrophages
activate T cells to produce cytokines, such as IL-2,
which activate other T cells, perpetuating the response, and IFN- γ, which
activates the macrophages.
It is not established which macrophage-activating cytokines (IL-4 or IFN-γ) transforms the cells into
epithelioid cells and multinucleate giant cells.
Foreign body granulomas

Caused by relatively inert foreign materials (animal,
vegetable, mineral or obtained by synthesis),
characterized by low solubility. Normally enter the
body by pneumoconiosis (granulomas forming in the
lungs by inhalation of powders) or transcutaneously.
The foreign material can usually be identified in the
center of the granuloma and cell turnover takes place
very slowly (low-turnover granulomas). The epithelioid cells and giant cells surround the foreign
body by adhering to its surface.
It is also characterized by an absence of the T cell–mediated immune responses.
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They are so-called because typically, when there is a granuloma formation because of the presence
of a foreign body or inorganic material, these are the giant cells that you find inside the granuloma.
Granulomas caused by transcutaneous penetration of: splinters of wood, vegetable or fish plugs,
sea urchin, splinters of bullets, textile fibers, talc, oil, paraffin, suture materials, silicone, metals, coal,
etc. …
For instance, that’s the case of ingrown hairs: when pubic hair remains under the skin and a small
spheric granuloma forms under your skin.
Types of conditions caused by pneumoconiosis:

- Anthracosis: induced by the deposition of inhaled carbon particles. The granulomatous
reaction is poor but becomes severe when associated with silicosis or chronic bronchitis;
- Silicosis: serious occupational lung disease (miners inhaling silica particles). Silica
particles are phagocyted by macrophages that are killed because of their toxicity. Numerous
granulomas are formed. They are gradually replaced by connective tissue with formation of
large areas of fibrosis that affect the functionality of the lung;
- Berylliosis: severe pneumoconiosis affecting the workers involved in the beryllium
processing. Granulomas are similar to tubercular ones with little tendency to necrosis;
- Asbestosis: the inhalation of microscopic asbestos mineral fibers suspended in the air
e.g. lung carcinoma.
Classical Granuloma
It has a spherical structure with a necrotic
area in the middle in which the
microorganisms/indigestible product which
killed the cells are located. They are in the
middle of the sphere because multiple
macrophages, which derive from the
circulation or were resident cells, surround the
area by assuming the giant cell/epithelioid
phenotype.
If the causes of inflammation were

microorganisms then there are also many
lymphocytes and plasma cells present,
however when the inflammatory cause is a
foreign body then there are fewer
lymphocytes and plasma cells present.
Fibroblasts surround the different cells that form the granuloma and produce lots of ECM. The ECM
is formed by fibrous proteins (e.g. collagen) which surrounds the entire granuloma (including the
fibroblasts) so as to make it stronger and the absence of vascularization prevents the supply of
oxygen and nutrients to the cells, thus leading to necrosis.
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Many granulomas undergo fibrosis, that however is

NOT limited to the fibrotic envelope of the
granuloma. Instead, fibrosis can also occur inside
the granulomas as sometimes the central necrotic
area can be replaced by fibrous tissue (scarring) or
undergo a massive insoluble calcium deposition
(calcification) or be circumscribed by a connective
tissue reaction (encapsulation).
In some cases, there is a colliquation of the

necrotic material (necrotic material becomes
liquefactive) which, if drained, leaves cavities that
can cause serious complications like in tuberculosis.
Tuberculosis is the prototype of a granulomatous

disease caused by infection and should always be
excluded as the cause when granulomas are
identified.
The etiologic agent is Mycobacterium tuberculosis, present particularly in the lung, but that can also
be found in other organs (intestine, skin, bones). When the necrosis involves a series of neighboring
tubercles, cavities are formed. Healing occurs by scarring, often associated with calcium salts
deposition (calcification).
Today, we will speak about wound healing, the tissue repair. Thus, we will conclude the topic about
acute and chronic inflammation, but today we will focus on tissue repair and fibrosis. As you can
see, the protagonists of tissue repair are macrophages. They differentiate to have a particular
phenotype, the M2 phenotype.
We already know that we have two types of macrophages activation:
1. M1 macrophage: it has a pro-inflammatory role, it cleans up the tissue when there is a tissue
injury. This macrophage is able to phagocyte the debris, the bacteria, the viruses, in fact, it
has a microbicidal action: it is able to kill or digest bacteria. Inside its lysosomes there are
a lot of mediators, such as enzymes that are important for the creation of ROS (reactive
oxygen species) or NOS (nitrogen oxygen species), both are very dangerous for bacteria.
This type of macrophage is also able to produce cytokines and chemokines, pro-
inflammatory molecules able to induce vasodilation and vasoconstriction, to increase vessels
permeability and to attract neutrophils;
2. At the end of the inflammatory process the first type of macrophage acquires another
phenotype, in fact it differentiates, and it’s activated as M2 macrophage. It is able to produce
growth factors and cytokines, which are useful to stimulate, for example, the proliferation
of fibroblasts, cells that produce collagen and other components of the ECM and are
therefore important in tissue repair. Later, it starts to produce other cytokines, similar to the
latest one but with opposite effects, one example is the interleukin-10, an anti-inflammatory
cytokine whose presence stops inflammatory processes).
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WOUND HEALING
There are two types of wound healing depending on the type of tissues that have been injured:
1. If the tissue is formed by labile cells, able to proliferate whenever they want, the process
ends with a complete regeneration of the tissue (restitutio ad integrum). The tissue is
recovered exactly at its original state because when the cells sense the injury, they start
immediately to proliferate and to restore the size, the quality and the function of the original
tissue. This type of process is called regeneration;
2. If the injury is too strong or if the duration is too long, even if the tissue is made by labile cells
it can’t be regenerated, but rather has to deposit extracellular matrix. This type of wound
healing is characteristic of all tissues made of permanent cells. For example, in an injury of
the heart, even if the inflammation is acute, permanent tissue can only be repaired by
connective tissue deposition. This type of healing occurs also when there is the repair of a
chronic inflammation, in which case it is the only possible type.
Cells of labile tissues (labile cells) include:

- Epithelia of the skin, which is a multi-stratified epithelium, from the basal layer, cells
continue to proliferate until they become senescent and they die when they are at the
surface;
- Hematopoietic staminal cells in the bone marrow continuously proliferate when it’s
needed. In fact, when cells of the blood become senescent, they die. Thus, thanks to
the bone marrow, they can be replaced continuously;
- Epithelia of ducts of exocrine organs;
- Cells of the GI tract;
- Cells of the urinary tract
Stable tissues are those tissues composed by cells that don’t replicate, that are quiescent, but that
can start to proliferate if stimulated. In this case, depending on the injury, you can have a complete
restoration of the tissue (regeneration), or a repair by ECM deposition:
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- Hepatocytes in the liver (the classical example);

- Kidney;
- Pancreas;
- Endothelial cells i.e. cells of the blood vessels;
- Fibroblasts;
- Smooth muscle cells.
Permanent tissues include:

- Neurons;
- Myocytes i.e. cardiac muscle cells;
- Skeletal muscles, although satellite cells can provide new nuclei and sometimes
regeneration capability.
However, several studies now, in the research field, have discovered that these cells (permanent
cells) have some capability to replicate. Perhaps, in the future, we will be able to stimulate them to
replicate. Still, in physiology and pathophysiology, the capability of these cells to regenerate tissues
is not sufficient. Thus, when there is a damage of this type of tissue, the healing will be by deposition
of ECM.
Regeneration and repair

In a normal epithelium, cells are labile. When there
is a superficial injury, cells can regenerate, and you
can see in the figure n.2 that the tissue returns
completely identical to the original one. However,
when the injury is severe or includes also the
underlying tissues (for example, speaking about the
skin, the injury concerns also the derma), it is not
possible anymore for the cells to regenerate the
tissue, and so there is repair, deposition of ECM
and, frequently, a scar formation.
Regeneration Figure 2
First type of healing is regeneration. Molecules
important in stimulating the survival cells are the
growth factors, which are released by the
remaining cells.
You are not required to know by heart all the growth factors shown in the tables 1 and 2, but they
are useful for next course like pathophysiology. These growth factors are:
- Epidermal growth factor (EGF);
- Transforming growth factor-α (TGF-α);
- Hepatocyte growth factor (HGF), specific for the liver;
Vascular endothelial growth factor (VEGF): it acts to stimulate vessels to produce
new vessels. It’s a growth factors that is very important during wound healing, in fact,
new vessels have to bring oxygen and nutrients to new tissues that are forming. When
there is a malignant tumor, the problem is that cells don’t stop proliferating and the
tumor continues to grow; this is possible because new vessels are continuously
65
forming inside this mass because VEGF is continuously produced. The formation of
new little vessels starting from existing ones is called angiogenesis;
- Platelet-derived growth factor (PDGF);
- Fibroblast growth factor (FGF);
- Transforming growth factor-β (TGF-β): it stimulates the fibroblasts to proliferate and
to produce ECM and it’s a good anti-inflammatory cytokine;
- Keratinocyte growth factor (KGF).
Table 1 Table 2
Cell cycle in tissue repair
We already studied cell cycle, so we won’t see it in detail here but it’s important to address it because
you will find it mentioned in this course several times e.g. tumors.
Regeneration starts when cells enter the cell cycle and they start proliferating. There are two
important checkpoints: G1/S and G2/M. Stable cells that are quiescent are in the G0 state of the cell
cycle but when growth factors bind to the receptors present on the surface of these stable cells, the
cells feel that they have to enter the cell cycle in G1/S which is a very important passage regulated
by several molecules that are synthesized or activated only when the cell has to proliferate.
For example, cyclins are molecules that bind kinases i.e. enzymes able to phosphorylate the
substrate. Usually a kinase phosphorylates another kinase which in turn phosphorylates another one
until a transcription factor is activated. Then, the transcription factor migrates to the nucleus where
it binds to the DNA and stimulates the expression of genes. In this case, when cyclin dependent
kinase (CDK) binds to the cyclin, CDK is activated and it can phosphorylate a protein that is called
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retinoblastoma protein (RB).

When the cell is quiescent, RB is
normally bound to E2F, a
transcription factor. When the
protein is phosphorylated, E2F
releases RB and translocates to
the nucleus where it binds to
specific regions of the DNA
leading to expression and
synthesis of molecules that are
important for the cell in order to
pass into the S phase. At this
point, the cell can begin to
proliferate.
This is important because we will

speak about retinoblastoma and
how this tumor can occur.
Liver regeneration
A classic example of regeneration is the liver regeneration. We already saw something about the
liver when we spoke about hyperplasia and adaptations: when you have a resection of a part of the
liver (liver hepatectomy), cells that remain alive immediately understand that they have to proliferate
so they enter the cell cycle to restore the initial size of the liver. Restoration of the liver can occur in
two main mechanisms:
1. Proliferation of remaining hepatocytes: main present mechanism, explained above (liver
hepatectomy);
2. Repopulation from progenitor cells: there is a little amount of progenitor cells inside the
liver that in the case of liver resection are activated and differentiate into hepatocytes.
Figure n.4 depicts the different phases of liver regeneration in a microscopic point of view, looking
at the cells, but it is not fundamental to learn the phases’ names.
1. Priming phase: there are cells that are always present in the liver, called Kupffer cells, that
are macrophages. N.B. macrophages can derive from monocytes from the circulation, or they
can be already differentiated in the tissues and have different names depending on the tissue
they are in, in case of the liver, they are called Kupffer cells. They are resident macrophages
which produce interleukin 6 (IL-6) that activates the hepatocytes by increasing on their
surface the levels of several receptors specific for other growth factors;
2. Growth factor phase: other growth factors, produced by the cells present in that area, bind
to the receptors and the cells start to proliferate. If growth factors bind to these receptors, a
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signal transduction pathway is

activated inside the cells so that,
for example, the cells start to
synthesize cyclins or CDKs:
cyclins bind to CDK which
phosphorylates RB leading to E2F
translocation in the nucleus, thus,
cells start to proliferate but not for
a long time. When the size of the
liver is restored, there are stop
signals (are still studied) that bind
to other receptors on the cells
stopping proliferation. These cells
are very important to stimulate
liver regeneration: perhaps they
Figure 4
are M2 macrophages. There is
also the possibility (studied now) of the involvement of staminal cells that can differentiate
into hepatocytes.
Scar formation
Second type of wound healing is the scar
formation. Side note: M2 macrophages are
fundamental so remember this type of
macrophage activation.
Main characteristics of the scar formation process:
1. Angiogenesis: it is the formation of new
blood vessels starting from pre-existing
vessels. This is very important because
when we have scar formation, we lose a
big amount of the tissue, therefore the new
tissue that is forming needs to be
vascularized;
2. Formation of granulation tissue: it’s a
type of transient tissue which takes the
place of the lost tissue and it’s called like
this (granulation) due to its composition. It
has lots of little red points before having
the formation of the scar. In fact, when
pieces of skin are lost, the tissue appears
rich in red points, granules. These
granules are vessels which constitute the
new microcirculation of the tissue. It is a
transient tissue because it is completely
replaced by a scar. It is composed by lots of fibroblasts that have migrated in this area and
started to proliferate and to produce ECM: there is new ECM, lots of fibroblasts and lots of
new vessels. Obviously, there is a lympho-monocyte infiltration: it is an inflammatory state,
therefore there are neutrophils and some lymphocytes. At the end, this type of tissue is
replaced by new connective tissue and there is the connective tissue remodeling;
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3. Production of the fibrous scar: the transient tissue is completely replaced by a scar.
Q: We have seen how growth factors are involved in regeneration, but is there angiogenesis also in
scar formation? Is it still due to the endothelial growth factors?
A: Yes, but in scar formation the new vessels are important because they have to give nutrients to
the fibroblasts that have migrated in the area and are responsible for the deposition of ECM; after
that, when fibroblasts finish to do their job, they die, and the vessels disappear, so it’s a transient
angiogenesis. However, angiogenesis is always stimulated by VEGF.
In the area of injury we have lots of debris, perhaps lots of bacteria and an inflammatory state is
immediately activated: cells that arrive in the area are neutrophils which start to clean up the area
and then, when they have almost finished, monocytes arrive in the tissue and differentiate in
macrophages of the M2 phenotype and stimulate the migration, for example, and the proliferation of
fibroblasts by releasing TGF-β. Fibroblasts start producing ECM; there are lots of fibroblasts in the
granulation tissue but less neutrophils because when they, and also inflammatory cells in general,
finish their job, they decide on their own to die undergoing apoptosis. So, the granulation tissue is
made of some inflammatory cells, lots of fibroblasts and new vessels whose formation starts from
pre-existing vessels. The presence of vessels is necessary because the empty space (cavity) is now
full of cells that have to live and therefore need nutrients, which will be carried through blood. Then,
with the passage of time, cells die through apoptosis (if they died from necrosis, another inflammation
would occur), lots of collagen is produced and then a scar, which is the definitive new tissue, is
formed. Sometimes though, the new tissue doesn’t work very well e.g. it might not be as elastic, if
we speak about skin for instance.
Slides n.14 and 15 are there just to give you an idea about the duration of this process.
Scar formation phases:
1. Inflammatory phase: is typical of the first three days, it’s characterized by pain (dolor),
redness and edema. It takes 0-3 days;
2. After 3 days there is the proliferative phase: it takes from 3 days to a month. The scar is not
present yet but there is a granulation tissue that is a pink tissue characterized by lots of new
vessels, fibroblasts and loose connective tissue which is not the definitive one;
3. Maturation of the scar: it can also take one year to become a definitive scar.
Q: Does the effect of the inflammatory state persist during the phases, for example speaking about
pain, do the patients feel pain even after months from the injury during the maturation phase?
A: If a person feels pain, the inflammatory state is not ended. Sometimes, the infection can persist,
perhaps because neutrophils couldn’t eliminate quickly the bacteria. If infections occur, the
inflammatory phase can be prolonged and so there is a delay of the healing process. Perhaps the
inflammatory and the proliferative phases can also overlap (in Italian, “sovrapposte”). However, there
is a moment when pain disappears and the scar is formed; it depends also on individual
characteristics e.g. someone might need a lot of time to heal and have a definitive scar while others
might take less.
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Angiogenesis
How does it occur?
Quiescent vessels of the microcirculation are made of
endothelial cells and of a basement membrane and
periendothelial cells that are called pericytes if
vessels are very small, while smooth muscle cells
when vessels are bigger. When an angiogenic factor
(e.g. VEGF) arrives, pericytes are separated as
instructed by the growth factor, the basement
membrane is broken in that point, and the endothelial
cells start to proliferate. When endothelial cells meet
another vessel that is generating from another blood
vessel, they fuse together, a cavity is formed, and a
new vessel is created.
Regarding tumor, researchers are looking for a way to
prevent or block angiogenesis. However, in some
cases, angiogenesis is important e.g. in wound
healing. In skin problems like ulcer in elders,
researchers are trying to stimulate angiogenesis. But
in other cases, angiogenesis needs to be blocked e.g.
in tumors. In fact, inside chemotherapy there are molecules whose function is that to inhibit
angiogenesis.
Deposition of connective tissue
70
Talking about the formation of granulation tissue, connective tissue can be formed when fibroblasts
reach the place where ECM is needed (migrate), then, when they are there, they start to proliferate
and producing the ECM proteins. The growth factor that stimulates fibroblasts to produce ECM
proteins is TGF-β, which is an important inflammatory mediator produced by macrophages of the
M2 phenotype. It stimulates fibroblasts migration and proliferation; thus, fibroblasts start to
synthesize collagen fibronectin, and, at the same time, TGF-β is able to inhibit the action of
metalloproteinases which are enzymes that degrade the ECM; thus, the ECM is accumulated
because its synthesis is increased, and, at the same time, its degradation is reduced. TGF-β doesn’t
directly inhibit these enzymes: it stimulates fibroblasts to produce an inhibitor of metalloproteinases
called TIMPs (Tissues Inhibitors of Metalloproteinases).
Figure n.7 depicts fibrosis which is the repair of tissues when there is a pathologic condition. We
will hear about it when we speak about hepatic cirrhosis and a lot of other pathologic conditions.
However, the wound healing we’re describing now is a physiologic process. Fibrosis is characterized
by the deposition of lots of ECM proteins by
fibroblasts: the process is the same but it’s
pathologic. In fibrosis, the injury can’t be repaired
because it is very severe. Macrophages present
in the tissues (in liver, Kupffer cells) produce TGF-
β which stimulates the fibroblasts to produce new
ECM proteins and so on. The result of the process
is the presence, inside the injured tissue, of lots
of ECM proteins that compromise the functionality
of the organ.
For example, in the case of the liver, when there
is a pathologic condition or an injury e.g. chronic
inflammation or chronic hepatitis, lots of
hepatocytes die therefore lots of debris and
bacteria are present. So, the resident cells try to
restore the liver since they are able to, but in this
case, it is not an hepatectomy (surgical procedure
performed by a doctor), but rather a disease.
Thus, they start to proliferate but they don’t do it
in the right manner: weird lobules are created and Figure 7
lots of ECM is produced. In fibrosis, hepatocytes
create a new parenchyma which is completely
disorganized and non-functional.
Remodeling of connective tissue

The first connective tissue produced and present in the granulation tissue is a loose connective
tissue which has to be remodeled in order to create a real scar. The remodeling is achieved thanks
to the action of the matrix metalloproteinases enzymes, produced by macrophages. Remember:
macrophages produce matrix metalloproteinases which degrade the ECM. It is important because
you will find these enzymes when speaking about atherosclerotic plaques which are found in
medium and large arteries. These plaques grow until they occlude the vessels, therefore they cause
heart ischemia or stroke. Atherosclerotic plaques have big a fibrous cap made by proteins of the
ECM matrix. There are plaques in which lots of metalloproteinases are present. In these plaques,
the ECM is degraded thus making them prone to rupture because the fibrous cap is damaged by
these metalloproteinases. There are other plaques in which there are less enzymes, perhaps
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because there are less macrophages. In this case, the fibrous cap is stronger, thus it’s difficult for it
to break. These enzymes can be inhibited by inhibitors of metalloproteinases.
Figure n.8 regards a granulation tissue. The red

part is composed of new vessels, so it is the
angiogenesis which gives this look to the tissue.
The white part is where ECM has been deposited.
It’s also an edematous tissue because the
inflammatory phase (immediately before
granulation) quite always has an exudation from
the vessels to the tissue: edema among the cells.
Then there are lots of fibroblasts and inflammatory
cells. However, after the phase of the granulation
tissue, in a few months, the transient tissue
disappears and a scar appears. The scar is characterized by more deposition of ECM because,
Figure as
8
some fibroblasts die, the remaining ones acquire
new phenotypes that are more able to synthetize
ECM leading to its accumulation. The edema is
reabsorbed, drained and also the inflammatory
cells die of apoptosis. The scar is pink when it’s
newly formed then, in some years, it becomes
white.
Healing of skin wounds

Until now, we have been describing general wound
healings, also of tissues that are inside our body.
Healing of skin wound is a specific example of wound
healing.
Two types of skin wounds healing:
- Healing by first intention: when there is a
clean, non-infected wound, made by
surgical incision. A wound characterized by
a non-loss of tissue. There are surgical
sutures to close the parts of the wound
together. In this case, there are different
phases:
1. Inflammation;
2. Proliferation of epithelial
and other kinds of cells;
3. Maturation of the connective
tissue scar.
However, at the end, you can have a very thin scar or
sometimes also nothing because if the cells proliferate
well and the incision is very thin, you can have the
complete restorage, so regeneration of the tissue.
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- Healing by second intention: it happens when there is the

loss of a lot of tissues. In this case, abundant granulation tissue
is created. Sometimes, you have wound contraction meaning
that fibroblasts, that are a lot in the granulation tissue, can acquire
a particular phenotype and can be called myofibroblasts cells
(“myo-” like muscle cells) so fibroblasts with some characteristics
of muscle cells e.g. they have actin filaments that only muscle
cells have. They can differentiate and contract the wound to try
to make the remaining, living epithelium parts closer to each
other.
Abnormalities in tissue repair

These can be of several kinds:
1. Ulceration;
2. Wound dehiscence;
These two are due to problems in the formation of granulation
tissue or in the formation of the scar. They are wounds that always remain open, don’t repair and
can be infected and inflamed since the scar does not form.
In the other cases, the scar is produce too much: there’s an excessive production of granulation
tissue which leads to prominent scar and to:
3. Hypertrophic scars;
4. Keloids;
5. Exuberant granulation: presence of a lot of granulation tissue;
6. Contraction: exaggeration of contraction i.e. fibroblasts differentiate into myofibroblasts that
contract the wound it’s physiologic but, in some cases, there might be an exaggeration of contraction,
even after surgical incision and sutures.
Pressure ulcer of the skin: it can occur when an old person or someone who has difficult in moving
is forced to stay in bed for a long time. There is a loss of tissue, a large gap between the edges of
the tissue can be formed. With the right medicines, you can have restoration of the tissue, but the
layer of epidermal re-epithelization is very thin, in fact, contraction of the wound healing is very
difficult to repair.
73
Keloids: it’s the excess of collagen deposition e.g. when the scar is formed too much; it’s genetically
determined e.g. it’s very typical in African people. It cannot occur when a scar develops in the stern.
In the microscope we can observe that this is not skin but rather a lot of ECM, which is stained in
pink. The correct formation of the scar depends on the characteristic of the person not on the surgical
procedure which was performed.
DISEASE CAUSED BY RADIATION TRANSFER

Introduction
74
The topics of the lesson concern disease caused by radiation transfer, with a brief introduction about
radiation and the description of non-ionizing radiation. The topic will be concluded during the next
lesson (where the topic will be ionizing radiation) since she probably won’t be able to conclude it in
this lesson. The slides for the lessons can be found on Campusnet, although since we prefer the
Moodle system, they will try to upload them also there.
Radiation
What is radiation? Radiation is energy that travels in the form of waves or high-speed particles. The
energy is transferred from one point to another of the matter with a speed equal to or less than that
of light in free space.
We have two types of radiation with two different origins:
- Natural radiation, also called basic radioactivity, which is the result of spontaneous
changes of energy within an atom;
- Artificial radiation, which is produced by men for scientific, industrial or war purposes.
However, we are also exposed to environmental artificial radiation, for example the radiation
produced by tv, tv screen, radio waves, microwaves, etc.
Natural radiation
In this group we have several subtypes of radiations, namely cosmic radiation, terrestrial radiation
and radiation from living beings.
With cosmic radiation we mean first of all the sun, then protons, helium ions and other elements,
and some electrons. This natural radiation (cosmic) reaches the earth and, while going through the
atmosphere, it interacts with different atoms present in the air, producing the emission of secondary
radiations, like electrons, mesons or gamma-rays (γ-rays).
Another type of natural radiations are the terrestrial radiations, which derive from the disintegration
of radioactive substances present in the earth crust. For example, we can find radium, uranium,
thorium and other radioactive molecules. So, we are also exposed to this natural radiation.
Then we have radiation from living beings. This is emitted from radioactive isotopes that are
present in the living beings, but the amount of this radiation is really low, and it doesn’t cause any
pathological effect, since it is a really low portion of the basic radioactivity.
Effects of basic radioactivity
We are exposed to this every day.
Basic radioactivity can have physiological effects, for example it is necessary for the survival of
various species (we need the sun, it is important for us), it can perform important functions like the
photosynthesis processes of the plants and it is important for the vision of animals. These are, let’s
say, the positive effects of the natural radiation.
However, basic radioactivity can also have negative effects: it can be one of the risks for cancer
(we have to keep in mind that radiation of natural origin must be added to the artificial ones, so we
are exposed to the synergetic action of these types of radiation); therefore, because of the synergetic
action between artificial radiation and an excessive exposure to basic radioactivity, we have an
increase in the risk of cancer development. Some people are more exposed than others, for example
the workers of metalliferous minerals mines or people who manipulate some radioactive compounds
(most exposed to this basic radioactivity).
TYPES OF RADIATION
75
Electromagnetic and corpuscular radiation

Natural and artificial radiation are then further
divided into two groups, constituting two types
of radiation, electromagnetic and corpuscular
(or particulate) radiation.
The first type, the electromagnetic radiation,
is characterised by energy in the form of
photons with 0 mass that propagate with a
speed equal to that of light.
The second type, the corpuscular or
particulate radiation, has an energy in the
form of a mass of various entities and a speed
that is lower than that of light. They are
constituents of atoms and their nuclei.
Concerning electromagnetic radiations, it
has already been said that they have 0 mass
and that their energy corresponds to their
frequency. In this group we can find radio
waves, micro waves, infra-red light, visible
light, ultraviolet (UV) rays (the most important in this group), X-rays and γ-rays.
Instead, concerning corpuscular radiation, it has been said that they have variable mass and that
their energy corresponds to their speed. In this group we have β-rays, that are electrons, α particles,
protons, neutrons and mesons.
Ionizing and non-ionizing radiation
Depending on the energy and the biological
effects induced by radiations we can divide
them (the electromagnetic and the
corpuscular ones) into two other classes,
non-ionizing and ionizing radiation.
All the non-ionizing radiations are
electromagnetic radiations; in this group we have radio waves, tv waves, micro waves, infrared
waves (or light), visible rays and UV rays (all included in the group of electromagnetic radiation).
Most of electromagnetic radiation are non-ionizing radiation. Concerning their activity, the radiation
with less activity are radio waves but the activity increases going up to UV rays.
The second group is ionizing radiation. In this group we have the corpuscular radiation plus X-rays
and γ-rays (which are in the electromagnetic radiation group). The ionizing radiations are all
corpuscular plus two types of electromagnetic radiations, X-rays and γ-rays. The activity is low
concerning accelerated protons and neutrons and it is increased considering X-rays or α-, β- and γ-
rays.
76
Figure n.3 shows the electromagnetic spectrum where all the radiations are divided in non-ionizing
and ionizing radiations. Just to repeat, in the group of non-ionizing radiation you have tv waves, radio
waves, microwaves, infrared light, visible light and UV rays. The wavelength increases in this
direction [left], so radio waves have a larger wavelength compared to UV rays and the frequency
increases in this
direction [right].
Concerning ionising
radiation, you have
X-rays and γ-, β- and
α-rays.
The wavelength is
very short, the
frequency is higher
and, of course, also
the energy will be
higher. In fact,
energy increases
when the
radiations have a
shorter Figure 3
wavelength.
The meaning of energy is the power of penetration of this radiation into a biological tissue. The
biological effects exerted by this radiation depend on dose, on the amount of radiation that can
be absorbed by the target cells (or tissue) and on the nature and size of the irradiated sensitive
target.
So, energy is equal capacity of penetration of
the radiation. Different types of radiations have
a different capacity to penetrate into the tissue or
target cells. For instance, you see that α particles
can be stopped by a single sheet of paper or skin
(so they cannot penetrate into the body); β
particles, which are electrons, are stopped by an
aluminium plate or by the body; X-rays and γ-
rays, that have energy as photons, are stopped
by lead (in fact, people working in the X-ray
department in the hospital are protected because they stay in a special place and, especially young
women, they have to use some lead stuff to protect some organs, to prevent penetration of X-rays);
neutrons can pass the lead but are stopped by concrete.
To sum up, we have natural and artificial radiation, which are divided into electromagnetic and
corpuscular radiation and then, depending on their energy and their effect, they are divided into
ionizing and non-ionizing radiation. Non-ionizing radiations are all electromagnetic.
Non-ionizing radiations
Non-ionizing radiations have an energy lower than 10eV. They are all electromagnetic radiations,
such as radio and tv waves. Of course, with radio we include also mobile phones, which are
dangerous, representing one of the main oncogenic risks, especially for brain cancer. When we use
our cell phone, there is an emission of radio waves; if you use it for a few seconds a day it’s ok but
if you use it for hours and hours, of course you are exposed to these radio waves for a very long
77
time and, as said before, biological effects for radiation depend on dose, size but also exposition to
the radiation. Other non-ionizing waves are micro waves, infrared light, visible light and UV rays.
The energy of this radiation is directly proportional to the frequency and it is inversely proportional
to the wavelength: in figure n.3 we clearly see that if the frequency increases also the energy
increases, if the wavelength increases the energy and the frequency decrease.
When these radiations have a collision with the matter, for example if the radiation hits one of the
electrons, the energy released by the radiations causes the atom excitation. The atom starts to be
excited and the electron moves from a more internal
orbital (with a lesser energy), jumping to an outermost one
(second or third one in figure n.5) with a bigger/highest
energy. But the electron stays inside the atom, it is not
expelled/removed (this happens in ionizing radiation,
when there is the expulsion of the electron). In this case, it
stays in the more external orbital because it has received
more energy. Following excitation, the atom becomes
reactive. Figure 5
A quick explanation about different types of non-ionizing
radiation is provided but then the focus will be mostly on UV radiation, which is the
most dangerous among this group.
Radio waves and microwaves
They are not dangerous if the use of radio, tv, mobile phones or microwaves, is a normal use (i.e. in
a short time and correctly). Accidental exposure to intense source of microwaves can cause damage
or injury. For example, the effects caused by microwave exposure can be of thermal type (for
example in overheating) and eye injuries, such as cataract (the crystallin of the eye will be damaged
and the subject won’t see properly), while for radio waves the principal damaging effect of excessive
exposure is an increment of brain tumours and leukaemias (the phone is one of the main risk of
brain cancer concerning radio waves).
Infrared radiation
In this case the most exposed people are those working with glass (glass blowers), for instance if
you go to Venice one of the principal souvenirs is this type of glass, and the metal workers. Infrared
radiation can also be produced by nuclear weapons and in this case the people can show serious
burns and sometimes also immediate death. They can cause also a thermal effect, in which case
the severity of the injury depends on the dose, on the distance and the duration of exposure; although
this is true for any kind of radiation: if I stay in the sun for half an hour, it is fine, if I stay in the sun
every day for eight hours, of course the pathological effects of the UV radiation increase, so I have
a major risk to develop for example cancer in the skin; also eye injury (cataract) can be caused.
Radiation in the visible light
This type of radiation is present in all the situations where an intense light is produced, so in eclipses,
nuclear explosions or lasers. We are exposed to an intense light. Also in this case, radiation can
have both beneficial effects and pathological effects. As for the beneficial effects, bright light is
important for plant photosynthesis processes but also for animal vision. Visible light can also be
used to degrade bilirubin, a substance derived from the degradation of heme which gives a yellow
colour to the skin, typically visible in some babies born with yellow skin due to an accumulation of
bilirubin; it is sufficient to expose them to a fluorescent blue light and the bilirubin is degraded, proving
that this radiation can be useful in the medical practice. Moreover, radiation can be used for
therapeutic purposes, for example in lasers, and to induce molecular evaporation. But this
radiation can also cause pathological effects, for example it can cause retinal lesions, in particular
rod and cone cells damage and it can stimulate the retinal pigmented epithelial cells to transform
78
light energy into thermal energy, which can cause protein denaturation resulting in temporary or
permanent loss of the visual function, since there is damage concerning the eye.
Ultraviolet light
The most dangerous non-ionizing radiation is the UV rays. They have a wavelength between 100
and 400 nm and they are the most dangerous because they have the highest energy among the
other non-ionizing radiations due to their position on the electromagnetic spectrum (see figure n.3):
they have a shorter wavelength, a higher frequency and so a higher energy. They are the most
dangerous also because they have the highest capacity to penetrate and to reach the target cell and
they are more active (more penetrating and more active), so they cause more pathological effects
compared to the others.
The most dangerous UV are UVA and UVB. We have three kinds of UV rays (A, B and C):
- UVC have a wavelength between 100 and 280 nm. They are the less dangerous and do not
cause any pathological effects because they are stopped by the ozone layer, so they do not
reach our body, being stopped in the atmosphere;
- The most dangerous, UVA and UVB,
have a wavelength between 280 and
400nm, and between these two types of UV,
the more dangerous are the UVB ones
because they are more absorbed by living
organisms. They both pass the
membrane, UVB and UVA can cross the
ozone layer, reach the body and
penetrate into the epidermis and papillary dermis.
Effects of UV irradiation exerted on the skin are based on the dose of radiation absorbed, on the
exposure time and on the period and place. The natural radiations are of course the UV rays
produced by the sun and the intensity of this radiation depends on altitude, geographical coordinates,
season and weather condition. For example, today we have clouds, so the intensity of the natural
radiation produced by the sun is lower than when it is a totally sunshine day. But of course, also
altitude and season influence intensity, for example on high mountains it is stronger and during the
summer it is stronger: the intensity can change. Concerning artificial UV sources, we can talk
about lamps, lasers and welding equipment.
The biological effects of UV on the skin depend on dose, duration, exposition etc. but we can
distinguish two types of effects:
- Direct effect: this happens when there is a direct energy absorption, preferentially by
proteins and nucleic acids (DNA). There is a direct damage of proteins, DNA and lipids (but
especially proteins and nucleic acids);
- Indirect effect: this happens when UV action is amplified by the presence in the skin of
fluorescent sensitising substances. These substances can be either of endogenous nature
(called porphyrins, which cause diseases called porphyrias) or of exogenous nature
(different classes of drugs). In this case we have a photodynamic action and so an indirect
effect. During this reaction many kinds of free radicals are formed.
79
As already said, the UVC is not dangerous

because it cannot cross the ozone layer, while the
UV that can reach our body and penetrate the
epidermis and papillary dermis are UVB and
UVA. They can cause an oxidative effect, so they
can induce the production of free radicals
(there is an oxidative stress condition). UVB can be easily

absorbed by the DNA and they can have a carcinogenic
effect. They are responsible for different skin tumours. The
sun, as all the other radiations, can have beneficial/positive
effects, for example it can promote/induce the production
of vitamin D (there is a stimulation of endogenous vitamin
D production in the skin), it can warm up (we feel hot when we are in the sun, we can feel the heat),
it can have a psychological effect, in moderate doses it can also help in certain skin
conditions/diseases and it can also have a beneficial effect on some autoimmune diseases, for
example multiple sclerosis, because it can alter the cell-mediated immune response and modulate
the systemic immune responses. Moreover, UV can be used to sterilise materials and
environments because some microorganisms are sensitive to UV rays, so they are used to sterilise
some instruments, some rooms etc because they can destroy many kinds of microorganisms.
However, an excessive exposition to the sun can have pathological effects. First of all, it is the major
cause of skin cancer (so it is the major oncological risk for skin cancer) and UV light is the main
cause of skin aging, it accelerates it, and it is a factor in the development of aging spots.
Tugba’s question: Is the allergy of sun-sensible people due to excessive exposure to UV light as
well? For example, dermatitis that develops especially during the summer because of the UV, can it
be classified as pathological?
Prof’s answer: Pathological effects, yes, because there is dermatitis for excessive exposition to the
sun. If it’s a normal inflammatory symptomatology, for example if I go to the sea side and my skin is
white and I don’t use protective cream and I stay for eight hours in the sun, I will have some
inflammatory reaction (erythema, blisters, necrosis, etc); if it’s a disease, especially dermatitis, a
disease of the skin, that can be amplified when people are exposed to UV rays, in this case the sun
can increase this pathological effect.
80
Of course, the pathological effects come out

when I stay in the sun for a long time every day
(i.e. following a repeated and extended exposure
to UV radiation) and if I stay in the sun without any
protection I immediately have some
inflammatory reactions on my skin,
such as erythema, blisters,
necrosis and desquamation, and
after a while I can have
hyperkeratosis and
hyperpigmentation (like aging
spots) because there is an
increased melanogenesis (which is the production

of melanin); I can also have injuries to the eye, for
instance conjunctivitis, cornea ulceration, retinal
lesions and cataract, although also regarding the
eye, only the UVB and the UVA are dangerous for
the vision (because UVC are stopped by the ozone
layer). The UVB can damage the crystalline lens,
resulting in the development of a cataract and it can
damage the cornea, while the UVA can reach the
retina and provoke other injuries.
Late effects on the skin comprise skin aging, although the most dangerous effects of UV rays are of
course skin tumours. UVB and UVA can penetrate into the dermis and into the epidermis and so
they can cause in a first step inflammation, an allergic reaction, oxidative stress and, in the end, they
can induce the development of skin cancers.
UV rays can have two different effects, a direct and an indirect one.
Talking about the direct effects, UV rays can directly damage nucleic acids, in particular DNA,
proteins and also lipids. Concerning DNA damage, they can directly break DNA and, as a
consequence, there is the formation of abnormal dimers: DNA is formed by four nitrogen bases,
adenine, guanine, cytosine and thymine, and the complementary coupling in the DNA double helix
is between adenine-thymine and guanine-cytosine; in this case, however, there is the formation of a
T-T dimer, a mutation, or also of a thymine-cytosine (T-C) dimer, both induced by UV (these are
the two main problems that can occur in the DNA), which are at the base of mutations. Usually there
is a mechanism that is able to repair some of these damages to DNA, but sometimes these repair
mechanisms don’t work properly or are damaged, so the mutations stay and accumulate in the cell
causing a neoplastic transformation. The principal cancers induced by UV rays are the basal cell
epithelioma, the spinocellular epithelioma and the melanoma), which are all malignant skin
cancers (we will talk about these skin cancers when we will talk about cancer in the next lessons).
UV can also damage proteins, provoking oxidation of the thiol group and intra- and inter-molecular
structural rearrangements, causing protein denaturation. They can also damage membrane lipids
and cause/provoke/induce lipid peroxidation, in particular of the so called PUFA (polyunsaturated
fatty acids). As a consequence, there are: free radicals production (which equals in oxidative stress),
ruptures of the membrane and cell death. These are the direct effects of UV rays on proteins, nucleic
acids and lipids.
81
Then there are also indirect effects. In this case the indirect effects of UV are mediated by
exogenous photodynamic substances. During these reactions there is a high production of
reactive oxygen species (ROS) and other types of free radicals. These indirect biological effects of
UV are induced/provoked by several photodynamic substances, such as:
- Dye colorants, for example eosin and methylene blue;
- Tar derivatives, for example acridine;
- Furocoumarins, which are derivatives of specific plants, for example bergamottin, that is
present in the peel of bergamot fruits and grape fruits; these are all substances which have
these photodynamic properties;
- Phytodermatitis, for example, because there are some vegetables and plants that make
certain people’s skin more sensitive to the effects of UV lights, such as the fennel, the dill,
the lime, the lemon, the figs;
- Some skin products like lavender, lime, sandalwood;
- Some drugs, since there is a big number of drugs that can induce skin sensibility to the sun
and to UV rays; there are a lot, both systemic photosensitizing drugs and topic
photosensitising drugs, used as therapeutic drugs, like anti-infectives, anti-inflammatories,
oncologic, immunosuppressant drugs, oral contraceptives, psychotropic, etc. (there are
many drugs for systemic or topic use that can stimulate the sun effects).
Then there are endogenous photodynamic substances that can provoke indirect effects of UV
rays. These substances are called porphyrins and the diseases that occur when they accumulate
in the skin or in some organs are called porphyrias.
Porphyrias
Porphyrias can be either a hereditary or acquired diseases. They are caused by an altered heme
biosynthesis, which involves a great number of enzymes; it starts from glycine plus succinyl CoA
and through the specific enzyme δ-aminolaevulinic acid, there is a process made of many steps with
many enzymes involved to produce the heme. The biosynthesis of heme happens mainly in the liver
and in the bone marrow; if one of the enzymes involved is lacking or inhibited, we can have the
accumulation of intermediate metabolites of porphyrins in the liver, in the skin or in the bone
marrow. Regarding the hereditary porphyrias, we have two main types: hepatic and erythropoietic
porphyria.
The hepatic porphyrias are mainly of autosomal dominant transmission and they are often
associated to precipitating factors also called “triggers”. They are caused by the accumulation of
porphyrins but the pathological effects can be amplified/stimulated by specific triggers: these include
the sun (specifically natural UV rays), drugs, smoking, alcohol, stress, pregnancies, oral
contraceptives, low calories and hypoglycidal diets, dehydration, infections, but also some foods (like
cabbage, broccoli, garlic, chocolate, tomatoes, some spices, etc) and some chemical solvents.
82
In the synthesis of heme, as

already said, starting from
glycine and succinyl CoA
thanks to different types of
enzymes, at the end we
obtain the heme. When one of
these enzymes is inhibited or there
is a lack of this particular
enzyme (the reduction of one of
these enzymes) we have
different types of porphyrias; for
instance, in the case of
inhibition or deficit of δ-
aminolaevulinic acid
dehydratase we have this type
of porphyria [i.e. ALA-dehydratase-deficiency porphyria]; if we have a deficiency of porphobilinogen
deaminase (that is important for the conversion of porphobilinogen to uroporphyrinogen I), we will
have acute intermittent porphyria (one of the most common porphyrias).
To sum up, there are different types of porphyrias and each type is linked to a specific enzyme.
On the table (figure n.13),
different types of porphyrias
are reported, specifying
whether if they are of
autosomal dominant
transmission or not, which are
the affected organs, which is
the site of altered metabolism
(especially the liver but also
the bone marrow) and which
are the clinical symptoms; in
fact, symptomatology can
differ: there can be some
neurological problems, or
some skin problems and there
Figure 13
are both acute and non-acute forms of porphyrias.
Today we talk about two of the most frequent and severe types of porphyrias.
Acute intermittent porphyria
This type of porphyria is caused by a deficit of hepatic porphobilinogen deaminase activity, which
means the organ involved is the liver.
It is an asymptomatic disease in 90% of the cases, only 10% of the defect carriers show clinical
symptoms. The main clinical symptoms are:
- colonic abdominal pain characterised by vomit, nausea and constipation or diarrhoea;
- some neurological problems, such as paralysis;
- some psychiatric disorders, for example anxiety, agitation, insomnia, depression;
- convulsions.
Affected people don’t show skin problems since they don’t have a photosensitive skin.
83
It is possible to recognise that people are

affected by acute intermittent porphyria because
they have a special colour of urine: they have
dark red urine (also said Porto wine), due to the
presence of metabolites that accumulate in the
urine. So, how can you do a diagnosis of these
people? You find in their dark red urine a large
amount of porphobilinogen during the acute attacks; it is difficult to recognise that people are affected
by acute intermittent porphyria when they don’t have an attack because the urines are normal, and
metabolites cannot be found in the urine.
The crisis is after puberty and is due to the action of exogenous agents (triggers or precipitating
factors), such as the consumption of steroid hormones, barbiturates, sulphonamides, fasting, stress,
disease infections, surgical procedures, or excess of alcohol. These people are normal when they
don’t have a crisis, which can occur after the consumption of these triggers.
Therapy. There are different treatments; during severe attacks one of the therapies consists in the
administration of heme intravenously and in this case the symptoms can be reduced in a few days,
even though it is important to start this therapy during the early steps/stages to avoid or delay nerve
damage (because, as already said, in acute intermittent porphyria there is damage of the
neurological system). To reduce the abdominal pain, it is possible to use narcotic analgesics, also
useful to reduce nausea and vomit, and some other drugs to reduce insomnia. The therapy for
convulsive attacks is problematic because you reduce the convulsions, but the drugs can be triggers
for porphyria (so it is better not to use this treatment).
Porphyria cutanea tarda
Another type of frequent porphyria is the so-called porphyria cutanea tarda. This one is due to a
deficit of uroporphyrinogen decarboxylase, that converts uroporphyrinogen III into
coproporphyrinogen III.
This exists in both hereditary and acquired form and is caused by a decreased activity of the enzyme
uroporphyrinogen hepatic decarboxylase.
Also in this case, the crisis are induced by several triggers, for example alcohol, oestrogens, drugs,
iron, hydrocarbons, etc.
It is possible to recognise that people are affected by this type of
porphyria because there are some clinical symptoms;
affected individuals have troubles with the skin (in contrast with
the previous type of porphyria, in which patients don’t have any
skin problem but only neurological problems), such as
blisters, bullous formations and fragility of the skin that is
more exposed to the sun (i.e. face, hands, etc) which is due to
accumulation of porphyrins in the liver. The accumulation of
these metabolites in the liver is responsible for hepatic injury, so
these people show hepatic siderosis
for accumulation of iron, a chronic
hepatic damage that can evolve in
hepatic cirrhosis. Then, these
metabolites can reach the skin
through the systemic circulation and
induce skin problems. Affected peoples often show also hirsutism4
4
Excessive body hair in men and women on parts of the body where hair is normally absent or minimal, such as on the
chin, chest, face or body.
84
but they usually do not have neurological problems or abdominal pain (in acute intermittent porphyria
they have mainly neurological problems, in this case they have mainly liver and skin problems, they
don’t have neurological or psychiatric disorders or abdominal pain).
Acquired porphyrias
Concerning acquired porphyrias, one of the main triggers is alcohol.
Acquired porphyrias can be associated with other problems, for example with hepatitis, HIV
infections, hepatocellular carcinoma and with lead poisoning, all of which act as triggers/precipitating
factors.
For example, it has been discovered that a long time ago in Turkey they used a
special fungicide added to treat wheat. A lot of children that were fed with this
wheat later showed this type of porphyria (porphyria cutanea tarda) because of
the inhibition of this enzyme. In particular, they were affected by hypertrichosis5.
Another trigger can be lead poisoning, which can induce the inhibition of
ferrochelatase and ALA dehydratase. Even in this case, children showed
development defects, decrease in IQ, hyperactivity and insomnia while the
adults showed abdominal pain and mental confusion.
Regarding the therapy, the best thing is to protect these people from excessive
exposition to UV rays. In cases where there is a big liver damage, another solution is the liver
transplantation. Another therapy can be the administration of heme to reduce the clinical
symptoms and it is better that these people reduce the consumption of all the triggers seen
before, so it is better if they don’t drink, if they don’t use specific drugs, if they have a diet not so rich
in proteins and carbohydrates. Moreover, also the administration of chloroquine can reduce the
symptoms in about three months.
Xeroderma Pigmentosum (XP)
It is a rare genetic disease inherited in an autosomal recessive pattern which can occur after
exposition to UV rays. It is characterized by the inability to repair DNA damage which occurs after
excessive exposition to UV rays. [That is: extreme sensitivity to ultraviolet radiation because of the
inability to repair certain DNA photoproducts]. It involves both sexes and all ethnic groups (incidence
1:250,000).
DNA damage which consists in the formation of pyrimidine dimers
(especially thymine homodimers), which represent a mutation that is
readily repaired by the DNA repair mechanisms that can be activated
to remove DNA damage; among them, there is the Nucleotide
Excision Repair (NER) mechanism.
NER is able to repair different bulky lesions that distort the double helix
of the DNA which include the various pyrimidine dimers caused by
sunlight and the covalent bonds that can form between DNA bases
and large hydrocarbons (such as the carcinogen benzopyrene).
In this pathway, a large multienzyme complex scans the DNA for a
distortion in the double helix, rather than for a specific base change.
Once it finds a lesion, it cleaves the phosphodiester backbone of the
abnormal strand on both sides of the distortion (position 5’ and 3’), and
a DNA helicase peels away the single-strand oligonucleotide
containing the lesion. The large gap produced in the DNA helix is then
repaired by DNA polymerase and DNA ligase.
5
Excessive hair growth over and above the normal for the age, sex and race of an individual.
85
Taken from slides: GADD45 protein (Growth Arrest and DNA Damage) is one of the most important
component of the DNA damage sensing mechanisms. It activates NER and interacts with other
proteins (p53, p21, Cdc2, MEKK4, PCNA), it stops the cell cycle until the lesion is repaired and thus
protects against cancer development.
Once the lesion is recognized by this repair mechanism, the damaged tract of DNA is removed from
position 5’ to 3’; not only the area containing the mutation is removed but also the nucleotides nearby
are, just to be sure to remove the entire damaged area. At this point, there is the synthesis of the
right new tract of the removed region that will be inserted in the DNA.
All these repair mechanisms involve a different number of enzymes (e.g. DNA polymerase and DNA
ligase) which must work properly. In the case of NED, there are 8 genes that encode for proteins
involved in these mechanism; it is sufficient that even just one of these genes does not work properly
or has a mutation, for the repair mechanism not to be able to work properly and remove DNA
damage. This is what happens in patients suffering from Xeroderma pigmentosum: in affected
people, some of these genes (at least one) are mutated so that the DNA repair mechanism doesn’t
work anymore; they cannot remove damaged pieces of DNA.
Taken from slides: Xeroderma pigmentosum is caused by mutated genes involved in repairing
damaged DNA, mainly NER-related genes. Researchers have identified at least eight inherited
forms of XP due to eight different mutated genes (XPA, XPB, XPC, XPD, XPE, XPF, XPG, XPV).
The proteins produced from these genes play a variety of roles: recognize DNA damage (XPC
complexed with HR23B, XPE, XPA), unwind regions of DNA where damage has occurred (XPB,
XPD, helicase activities, subunit of TFIIH), excise the abnormal sections (XPF, XPG), and replace
the damaged areas with correct DNA (XPV, POLH). The major features of xeroderma pigmentosum
result from a buildup of unrepaired DNA damage.
Normally, we are exposed to UV rays (sun-exposure) but luckily, we have this DNA repair system
that because of the presence of different genes (e.g. gene encoding for DNA polymerase or ligase)
enables us to repair all the damage that appears on our DNA. In case these genes are mutated, the
repair mechanism cannot work properly.
As mentioned before, NER consists of 8 genes encoding for specific enzymes and it’s important that
all of them work properly, otherwise we cannot have reparation of DNA damage. For instance, a
person who has mutations on these genes (meaning no production of the right enzymes to repair
damage), when exposed to UV rays (sunlight), might be affected by XP. It is better for people who
are aware of their condition to avoid exposure to sunlight.
Clinical symptoms of Xeroderma Pigmentosum (people who have severe mutations of this
repair system):
- Severe burns when exposed even to minimal doses of solar radiation. This often occurs in
infancy at the first exposure to sunlight;
- Development of many freckles (accumulation of melanin produced by melanocytes) at an
early age;
- Development at young age of multiple cutaneous malignant tumors: basal cell
carcinomas, spinocellular carcinomas and melanomas; due to growth of tumors, they might
have deturpation on the face;
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- Eyes are very sensitive to sunlight; indeed, they must wear sunglasses at all times to protect
the eyes otherwise might easily be affected by recurrent conjunctivitis6 in which retina and
crystalline might be damaged;
- Blister formation on the skin at minimal exposure to the sun because it is very sensitive
(inflammatory response);
- Spider angiomas (visible vessels on the skin’s surface resembling a spider web - “spider
web blood vessels”), the so-called couperose skin (chronic skin condition caused by
weakening of blood vessels) in all the areas that are most exposed to the sunlight;
- Alterations of the skin which is rich in cysteine (-SH) (the stratum lucidum and stratum
granulosum, alteration in transcription of myelin and keratin because of altered TFIIH that is
involved in NER, but also in transcription of several protein-coding genes) that result in dry
or squamous skin;
- Ichthyosis: squamous skin (xeroderma), irregular dark patches (pigmentosum);
- Brittle hair and nails;
- Sometimes also neurological signs can appear because they can be due to altered
myelinization, these include: hearing loss, poor coordination, difficulty in walking, loss of
intellectual function, difficulty in swallowing and talking, and seizures.
Diagnosis
Usually, these clinical symptoms appear already during the first years of life, in the early childhood
(age 1-2). The main indication for a diagnosis is the appearance of severe burns after a minimal
exposition to UV light.
The diagnosis is based on clinical data, such as lesions to skin (e.g. blisters) and eyes (e.g.
conjunctivitis) and neurological manifestations (data concerning the NS), and also on the family
history since XP is a rare genetic disease so there is an autosomal recessive transmission pattern
(there might be people in the family affected by this rare disease). You can evaluate also the mutation
on this DNA repair system (NER): collect biological samples (e.g. skin, blood) and further investigate
if there are mutations affecting genes involved in the repair mechanism, so that you have the proof
that the repair mechanism is not working anymore.
Affected individuals are at high risk of developing skin tumors, mainly basal cell and spinocellular
carcinomas (the risk is 1000-2000 times higher than in normal subjects). Skin tumors develop early,
at an average age of 10 years, and many patients die at early age because of skin cancer (<40% of
affected subjects survive beyond 20 years). If you know you have XP, you should avoid exposing
yourself to sunlight to protect your skin and your eyes. In fact, with an early diagnosis, if severe
neurological symptoms are not shown (less severe types) and precautions are taken to avoid UV
exposure, a normal lifespan is possible; otherwise, it can be a severe and dangerous, even if rare,
disease.
IONIZING RADIATIONS
Any type of particle or electromagnetic wave that carries enough energy (>10 eV) to knock electrons
from atoms or molecules, thereby ionizing them.
Radiations with an energy higher than 10 eV.
6
It is an inflammation process. Remember: “-itis”!
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In this group we have all the corpuscular radiations (with high kinetic energy) and two
electromagnetic radiations e.g. X-rays and gamma-rays (which have high frequency, low wavelength
meaning they have high energy).
Corpuscular radiations propagate energy by means of atomic and
sub-atomic particles supplied with high kinetic energy (velocity):
- α particles: composed of two protons and two neutrons
bound together into a particle identical to a Helim-4 nucleus;
- β particles: high-energy electrons or positrons emitted during
radioactive β decay;
- Protons;
- Neutrons.
Electromagnetic radiations propagate energy in the form of photons with a speed equal to that of
light and no mass, only electromagnetic waves with very high frequencies (short wavelength) (E=hf)
carry enough energy to be ionizing:
- X-rays;
- γ-rays.
Last time, while talking about non-ionizing radiations, we said that the electron will absorb energy
but will not be removed from the atom, it will jump to a higher orbit instead. In this case when the
collision of radiation with the matter occurs, energy is released causing expulsion of the electron
from the atom. It is able to cross all the orbits and be removed from the atom becoming a reactive
positive ion through a process called primary ionization. The electron that is removed can then
leave its energy to other electrons becoming a negative ion through a process called secondary
ionization. The first electron receives a lot of energy, passes through all the orbits and then goes
outside the atom where it passes its energy to other electrons therefore going from being a positive
to a negative ion.
Recap: When these high energy radiations collide with the matter, electrons are knock out from their
orbits and the atom becomes a positive ion. This event is called primary ionization.
Released electrons lose energy as they interact with other atoms and when all the energy acquired
during ionization has been yielded, the electron is absorbed by and atom that becomes a negative
ion. This is the secondary ionization.
Penetration
Penetration capacity of ionizing radiation in the tissues varies according to the nature and energy
of the radiation. The capacity to penetrate in a tissue is inversely proportional to the wavelength i.e.
when the wavelength decreases, the frequency increases and so does the energy which means that
the radiation has more capacity to penetrate the tissues.
X-rays and γ-rays penetrate deeply into the tissue because they have more energy, while α and β
particles are absorbed in the most superficial layers (i.e. don’t have a great capacity to penetrate the
tissues).
Radiation units
To measure the radioactive material, different radiation units are used:
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- Ci (Curie) or Bq (Bequerel) is used to express

the number of disintegrations per second of
radioisotope in a radioactive material (decays).
It can measure the amount of radiations emitted
by a source, radioactive material;
- Gy (Gray) is used in medical practice to
express the energy the target tissue per unit of
mass (1 J of radiation absorbed by 1 kg of
mass) (J/kg).
- Sv (Sievert) is the unit of radiation absorption
that takes into account the relative biological
effectiveness of ionizing radiation, since each
form of such radiation has slightly different
effect on living tissue. 1 Sv is generally defined
as the amount of radiation roughly equivalent in biological effectiveness to 1 Gy of γ-radiation.
Ionizing radiations, as all radiations, are present in the environment, but individuals can be also
exposed to artificial radiations. Ionizing radiations are largely used in diagnostic imaging. Damage is
prevented by shielding with lead-based materials through which radiations cannot pass.
Ionizing radiation is a double-edged sword: it
is widely used in medical practice (cancer
treatment, e.g. radiotherapy, diagnostic
imaging, therapeutic and diagnostic
radioisotopes), but can cause adverse short-
term and long-term effects such as fibrosis
(late pathological effect due to chronic
inflammatory response), accumulation of
mutation (mutagenesis) that can lead to
carcinogenesis and teratogenesis
(congenital defects due to abnormal embryo
development, when a pregnant woman
receives high doses of radiation so that the
body will be born with defects).
Ionizing radiations exert two types of actions

on target molecules:
- Direct action: energy is directly transferred on the cellular constituents (DNA, proteins,
lipids), which are damaged by the radiation; when the energy is transferred on these
molecules there is an oxidative damage, many free radicals are produced and as a
consequence the structure and the function of DNA, proteins and lipids are damaged.
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Description of the image in

which the damage on
constituents are described:
Free radicals are
responsible for different
types of damage on nucleic
acids, but the result occurs
as mutations, cell death
although, sometimes, if the
repair mechanisms are
working properly, there can
be damage repair.
Concerning proteins, the
production of a great
amount of free radicals is
responsible for the
oxidation of sulfhydryl groups and for intra- and intermolecular structural rearrangements,
whose final result is the denaturation of the protein. Concerning lipids, free radicals are
responsible for the peroxidation of PUFA, which causes the breaking of the membranes and
so the consequence is, also in this case, cellular death. (These free radicals cause oxidative
damages to other cellular structures - oxidative stress status - and the production of other
radicals - chains reaction-.);
- Indirect action: the energy is transferred from the radiation or the expelled electrons to
water7 that undergoes ionization - water radiolysis - and free radical formation. This
overproduction of ROS leads to an oxidative stress and thus cell damage (both in the nucleus
and the cytoplasm). All the cells can be damaged by the production of free radicals, whether
they are proliferating or not. If the radiation affects the cytoplasm, there can be damage
concerning the endoplasmic reticulum, mitochondria and
lysosomes (which will again lead to cellular death).
Greater oxidative damage is produced in the presence of oxygen that
increases the effects of the radiation (radical formation from oxygen
ionization). Poorly vascularized tissues with low oxygenation, such as the
center or rapidly growing tumors, are generally less sensitive to radiation
therapy than nonhypoxic tissues.
Rapidly dividing cells are more vulnerable to injury than are quiescent
cells. Except at extremely high doses that impair DNA transcription,
irradiation does not kill nondividing cells, such as neurons and muscle
cells. In dividing cells DNA damage is detected by sensors that produce
signals leading to the upregulation of p53, the “guardian of the genome”.
p53 is a transcription factor that in turn upregulates the expression of
genes that initially lead to cell cycle arrest and, if the DNA damage is too
great to be repaired or repair mechanisms are aberrant, that of genes that
cause cell death through apoptosis.
Therefore, tissues with a high rate of cell proliferation, such as gonads, bone marrow, lymphoid
tissue, and the mucosa of the gastrointestinal tract, are extremely vulnerable to radiation, and the
injury is manifested early after exposure. All these pathological effects on cellular constituents
7
Our body is made for 70-80% of water, which when is hit by radiation is ionized (water ionization). About 80% of the
radiant energy is absorbed by the water which is main constituent of the cells (about 70%).
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depend on the dose of the radiation: massive doses can immediately directly kill the cells (all kinds,
proliferating and non); low doses, instead, have as principal effect the water ionization, with the
production of great amounts of free radicals, which especially alter proliferating cells, resulting
eventually in DNA damage (e.g. T-T dimer formation) and protein denaturation; however, these
effects will cause the death of the cell for apoptosis, not
necrosis.
The effect of ionizing radiations is thus dose-
dependent: low doses cause DNA damage (free radical
production through water ionization/radiolysis) in
proliferating cells that will undergo apoptosis, while large
doses will transfer energy directly to macromolecules
and will kill both proliferating cells and quiescent cells
mainly through necrosis. Sometimes, if DNA repair
mechanisms work properly, DNA can be repaired and so
tissue reconstitution is achieved, but if they don’t work
properly (for instance, if there are some mutations in the
genes encoding for specific enzymes of the
mechanism), there will be a failed or aberrant repair and,
as a consequence, cell death or carcinogenesis due to
accumulation of mutations, but also, as already said,
teratogenesis (if the fetus receives radiations, it will not
develop properly and malformations will be present).
It has been reported that the administration of 1 Gy

(Gray) of ionizing radiation produces about 150,000
ionizing events in the nucleus but only about 1000 DNA
double strand breaks could be detected. This big
discrepancy is mainly due to DNA repair mechanisms:
as already said, one of them is NER but there are also
other two important repair mechanisms: Non-
Homologous End Joining (NHEJ) and Homologous
Recombination (HR). It is also important to have a
system that prevents the formation of free radicals; this
is possible thanks to the action of different enzymatic
systems, for example the superoxide dismutase (SOD),
the catalase and the peroxidase, the presence of
compounds rich in sulfhydryl groups, like glutathione
(GSH), and the right amount of natural antioxidants,
such as vitamin A, C and E.
The pathological effects of ionizing radiation depend on:

- Dose of absorbed radiation;
- Duration of the exposure;
- Cell sensitivity (radio sensibility).
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As we already know, there are three classes of cells in our body: labile cells, stable cells and
quiescent/perennial (or permanent, nds8) cells. Labile cells are, for instance, the epithelial cells or
the blood cells; stable cells are, for example, the liver cells; perennial cells are, for example, the
neurons. Labile cells are the most sensitive to this kind of radiation, because they proliferate very
quickly: this is why ionizing radiation can be used for therapeutic purposes, for instance in oncologic
patients (all neoplastic cells behave as labile proliferating cells, even those in brain tumors).
Therefore, fast dividing labile cells are the most radiosensible. Neoplastic cells, independently from
their tissue of origin, proliferate quickly and can be considered labile, this is why radiotherapy is used
to kill cancer cells.
Here various cell types are listed grouped according to radio sensitivity in descending order:
- Lymphocytes, spermatogonia, ovarian follicular epithelium, oocytes, proliferative bone marrow
cells;
- Epithelial cells (gastrointestinal, epidermis, etc.);
- Parenchymal, connective and endothelial cells;
- Muscle, bone, and nerve cells.
Pathogenic effects of ionizing radiations

The pathogenic of ionizing radiation on the body can be divided in early and late effects: some of
them will appear immediately (hours or days), some will appear after a while (in months or years).
Of course, these effects will depend on the dose and on the cell sensibility.
Early somatic effects. If I am exposed to very high doses of radiation, for example between 6-10
Gy, death occurs within a few minutes or hours from exposure due to a fall in blood pressure
(Neurovascular syndrome), heart failure and brain hemorrhaging (this is the so-called death by rays
or par radiation).
If I am exposed to low doses of radiation, for example lower than 6Gy, I will have serious damage to
the mucosae (because they are epithelial cells, highly proliferative), for instance those in the GI tract,
resulting in severe diarrhea, nausea, intestinal bleeding, hair loss (after exposure higher than 2Gy)
but no immediate death (although it can occur in a few days for loss of water in the case in which
there is no recovery from vomit and diarrhea). Then, in two weeks, the damage to bone marrow stem
cells becomes detectable: less production of red cells and white cells results in leukopenia, anemia
and thrombocytopenia (Hematopoietic syndrome); these conditions make the patients more prone
8
Nds: nota dello sbobinatore
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to infections (both viral and bacterial) which cause the death of 50% of the patients (their immune
system doesn’t work properly).
Late somatic effects arise slowly after repeated exposures to low doses of ionizing radiations
(Chronic Radiation Syndrome):
- Radiodermatitis: inflammatory lesions on the skin; often appears after weeks of radiotherapy in
oncologic patients in the area where radiotherapy is done. There is the formation of erythema,
necrosis, etc. of various severity, ranging from erythema to necrosis with the formation of ulcers
that hardly ever heal, occurs in cancer patients undergoing radiotherapy;
- Tumor development, with a prevalence of epithelial tumors, leukemias and thyroid tumors;
- Suppression of gametogenesis and consequent sterility;
- Destruction of gastrointestinal tract and ulcers formations;
If mutagenesis occurs in germ cells, the mutation will be transmitted to the offspring.
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It is important to underline all the different types of tumors that

can develop as a consequence of ionizing radiation:
- #1 Leukemia (following Chernobyl and Fukushima disaster, but
also after Hiroshima, the prevalence of leukemia increased
enormously);
- #2 Thyroid Cancer;
- #3 Brain Cancer.
Also other tissues can be affected, for instance there can be Lung
Cancer, Breast Cancer, Hepatic Angiosarcoma, Skin Cancers
(mainly basal cell or spinocellular carcinomas and melanomas)
and Osteosarcoma.
Radiation during pregnancy

It is important for women to know not only to avoid exposure to
radiation, but also not to swallow or breath in radioactive
materials, because those can reach the fetus through the blood
stream.
Exposure to radiations can be dangerous for the developing fetus especially during the period that
goes from the 2nd week to the 18th week. The effects of radiations change considerably depending
on the timing of irradiation:
- Pre-implantation Phase (first 9 days from fertilization): death of the embryo or no consequence
and subsequent normal development; there can be immediate death of the embryo or nothing at
all (so there is a normal development with normal subsequent events);
- Morphogenetic Phase (from 9th day to the end of the 2nd month): during this period the cells of
the embryo are highly sensitive to radiations (max radio sensitivity) and irradiation can result in
various organ malformations (teratogenesis);
- Fetal Phase (from the beginning of the 3rd month until
the end of pregnancy): in this period the organ
malformation risk decreases but the embryo is more
susceptible to neurological problems, for example mental
retardation, due to incomplete or abnormal encephalic
development (peak at weeks 8-15).
Irradiation from the third week of gestation increases the risk
of developing leukemia in the post- natal period: especially
after nuclear disasters like Hiroshima, Chernobyl and
Fukushima, a lot of babies were affected by leukemia.
CHEMICAL DETERMINANTS OF DISEASE
This is one of the different lessons that you will have dealing
with environmental factors that induce different types of
diseases. There are many different types of environmental
factors that can induce specific diseases: physical (previously discussed in Leonarduzzi’s lessons),
chemical (today’s topic) and biological (dealt with in the microbiology course). Next week we will
start with a series of lessons about the big topic which is cancer. (she’s particular involved in these
lessons).
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Now we will discuss environmental factors related

to changes in the pH in our body.
Some of the slides related to the balance between
bases and acids are topic of another colleague’s
lecture in physiology, but we will also discuss them
here to stress their importance.
The mechanism of action of chemicals are different and the body wants to maintain the equilibrium
between acids and bases within our body/itself. This type of process, which is called homeostasis,
can be disrupted through four routes:
- pH modification;
- solvent action;
- protein denaturing agents;
- selective inhibition through biochemical functions (related specially to poisons e.g. drugs and
chemicals in general
The chemical buffer systems of our body are very important as they maintain an equilibrium
between the acids and bases in our body. The buffer systems that maintain chemical homeostasis
in the body are largely discussed in physiology, however, here we discuss the pathological aspects
of these systems when they are chemically altered.
pH modification (acid-base imbalance)

The most important way to maintain this balance is through buffer systems that can be related to
an immediate or quick or slow response: There are immediate, quick and slow systems that regulate
the homeostasis of the pH of the body:
- the immediate response is related to the buffer systems of the salts in the body in which
they combine acids and bases in order to maintain very low variations of pH levels in the
body;
- another type of system that can balance the acids-bases equilibrium is the respiratory
system which constitutes a quick response because it occurs in few minutes in our body
through ventilation and regulates the concentration of CO2 (carbon dioxide) in the body;
- the renal system is important because we also need to remove excess of acids or basis;
it is a slow response.
Among the different important types of buffer systems related to the salts, the bicarbonate buffer
system is the most powerful extracellular buffer system in the body, it is an open system which is
regulated both by breathing and renal system: they regulate the concentration of carbon dioxide
(CO2) and that of bicarbonate ions (HCO3-) respectively.
The Bicarbonate Buffer System

The system involves HCO3- (bicarbonate ions), CO2 and also H2CO3 (carbonic acid) and it is
activated when we introduce high quantities of acids e.g. chloride acid (HCl). The introduction of a
high quantity of HCl induces the release of hydrogen ions (H+) whose excess is balanced by the
availability of bicarbonate ions and the final formation, according to this reaction, of water and carbon
dioxide.
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- This reaction can be reversed, changed (directed) towards a basic or an acidic environment:
When a strong acid, such as HCl, is added, the H+ is released and buffered by HCO3-
(bicarbonate ions) and H2CO3 (carbonic acid) is formed, so the reaction shifts to the right;
- When a strong base, such as sodium hydroxide (NaOH) is added, free OH- (the -OH comes
from the NaOH dissociation) is combined with H2CO3 (carbonic acid) to produce HCO3-
(bicarbonate ions), so the reaction shifts to the left.
The concentration of these two important acids or bases can change the equilibrium of this reaction.
Depending on the concentration of bicarbonate ions or carbon dioxide, we can induce a low pH or a
high pH:
- HCO3- increase results in an increased pH which is called alkalosis;
- CO2 increase leads to a decreased pH which is called acidosis.
A HCO3- (bicarbonate ion) increase will result in an increased pH, which leads the acid-base balance
to alkalosis, while a [CO2] increase will result in the decrease of the pH which leads the acid-base
balance to acidosis.
So, generally there’s an equilibrium between acids and bases (HCO3- and CO2 concentrations), but
if we introduce a large quantity of base, we have alkalosis, while if we introduce acids, we have
acidosis.
How does our body maintain this equilibrium between acids and bases?
Our body tries to maintain this equilibrium through the alkaline reserve. The alkaline reserve, for
example, in the blood is defined as the concentration of bicarbonate ions in the plasma
corresponding to the quantity of H+ ions that can be fixed in the blood by our erythrocytes, without
any big pH variations. (plasma [HCO3-] about 25 mEq/l).
If there is a decrease of the alkaline reserve, we have acidosis and if there is an increase of the
alkaline reserves, we have alkalosis.
Acidosis can be induced by an excess of acids, but the acids can be volatile or fixed. On the other
hand, alkalosis is due to the decrease in the concentration of acids in our tissues. Alkaline reserve
is associated to the rate and depth of our breathing.
If there is an increase or decrease concerning fixed acids we have metabolic acidosis or alkalosis,
but if these changes are related to volatile acids we have respiratory acidosis or alkalosis.
So, we have two types of both acidosis and alkalosis depending on the type of acids and the route
by which these different types of acids are buffered in our body:
- Metabolic acidosis or alkalosis;
- Respiratory acidosis or alkalosis.
The alkaline reserve is also defined as the quantity of carbon dioxide in terms of cubic centimeters
(cm3) that can be fixed by our body, specifically by 100 ml of blood at 0°C at 760 mm pressure
(normal values of alkaline reserve is 70 cm3).
Buffering systems in the body

We have different types of biological systems in our body (e.g. cells, tissues, blood…). The way in
which one biological system can cross-talk to other ones is important. The cross-talk between the
buffer systems amplifies the buffer effect.
In the cells: normally, the slow reaction that converts CO2 into H2CO3 (carbonic acid) occurs into the
water and keeps it in the cytosol. However, an increased diffusion of carbonic acids from the cells
(tissues) toward the external or interstitial fluids can induce different changes (can be slow or very
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quick) of CO2 into the external environment of the cells. This type of diffusion9 leads to the formation
of the concentration gradient (in Italian, “gradiente di concentrazione”).
In the blood: CO2 dissociates, as we have seen in the aforementioned reaction, into hydrogen ions
and carbonate ions, but when it dissociates, it can
perform different activities:
- for example, related to breathing depending on
the quantity;
- it can be fixed by proteins of the plasma (8%);
- it can be linked to amino acids where it remains
unchanged (27%) as the CO2 is acidic and the
amino group is basic (formation of hydrogen
bonds/dipole-dipole interactions);
- it can be transformed directly into bicarbonate
through action of carbonic anhydrase enzyme
(65%);
- it can then quickly dissociate again according
to the reaction that we have seen before
causing the availability of free hydrogen ions
which can be linked to hemoglobin; therefore, there are H+ ions linked to hemoglobin but the
HCO3- is free: there is an increase of base form of this ion which diffuses in the plasma.
In the lung:
- Hb (Hemoglobin) is oxidized and is less able to retain the quantity of H+ ions, thus the excess
H+ ions that are not linked can react with HCO3- producing again the H2CO3 that dissociates
into CO2 and H2O;
- CO2 is very diffusible in the alveolar air and is exhaled.
In this case, the excesses of H+ ions and carbon dioxide are removed through breathing.
Alkalosis
Metabolic Alkalosis (↑HCO3- ): it is due to the increase of HCO3- and can occur also for example
during therapies e.g. patients that suffer from gastritis are administered a big quantity of bases, in
particular high quantities of citrate or sodium bicarbonate. In this case we have an increase of
bicarbonate ions or is due to an excessive loss of acids (in particular HCl) e.g. in gastric diseases it
can be due to repeated vomit.
Respiratory Alkalosis (↓CO2): when we have a decrease in carbon dioxide due to pulmonary
hyperventilation; in fact, we extrude a lot of carbon dioxide, CO2 moves from blood to alveolar air
faster than that produced in the tissues by an increased oxygen supply, in this case if we breath very
quickly (fast extrusion of CO2) which can cause the decrease in concentration of CO2 in the blood
with alkaline reserve increase.
Another factor which can be involved in respiratory alkalosis can be the alteration of some cations
(cationic metabolism). In particular, the decreased dissociation of Ca2+ salts induces a low
concentration of these salts in this tissue, particularly in muscles where it causes hyper-excitability
of peripheral neurons and subsequent muscle tetany.
9
She literally said “this type of diffusion is called “concentration gradient”” but I think she means that the diffusion of
carbon dioxide toward the interstitial fluids and blood leads to the formation of the concentration gradient which is a
concept that we saw in depth during the physiology course.
97
Compensation response
There are two different compensatory methods to try to maintain this pH.
The one related to the respiratory events is hypoventilation: there is a lower respiratory induction
by the respiratory centers and there is a decrease in extrusion of carbon dioxide and consequently
an increase in amount of concentration of CO2 in the tissue. [from slides: respiratory center is
depressed, ventilation is reduced and the consequent increase in pCO2 brings pH close to normal
values.]
Renal compensation is related to reabsorption of bicarbonate ions by renal tubules which induces
the decrease elimination of H+ ions leading to an increase in pH.
[From slides: less tubular reabsorption of bicarbonates (decreasing the base concentration) and
decreasing elimination of H+ (increasing the acid concentration) with subsequent pH decrease which
returns the pH close to its normal values.]
Acidosis
Metabolic acidosis (↓ HCO3- ): is due to a decrease in HCO3- ions or can be on the other hand due
to an excessive incoming of bases in the body due to exogenous intoxication by chemicals e.g.
intoxication by Ammonium chloride (NH4Cl), which can be split into urea NH3, H+, Cl-.
Respiratory acidosis (↑ CO2): there is an increase in carbon dioxide which is related to the
ventilation of the lung when, for example, there is hypoventilation, the quantity of CO2 extruded by
our body is lower than normal. The high concentration of CO2 cannot be fixed in the blood (too much)
thus diminishing alkaline reserve.
Compensation response
Similarly to what we have seen for metabolic and respiratory alkalosis, it can be in this case
hyperventilation or increased renal reabsorption of bicarbonates and H+ ions elimination with
subsequent pH increase which returns the pH close to its normal values.
What happens in the cells when intoxication occurs depends on if the intoxication goes slowly or
very quickly; for example, with the introduction of the acids it is important to take into consideration
the quantity and the type of acid: if it is chemically strong or not. So, depending on the type of acids,
if the decrease of pH is weak/slow in the cells, the cells undergo autolytic processes, while if the pH
decrease is very strong/quick, we not only observe autolytic processes but also that some cells are
destroyed by protein denaturation, dehydration and induction of the so-called coagulative necrosis.
Coagulative necrosis is different from colliquative (liquefactive) necrosis for its features, but also in
the agents which trigger it (causes). If I have an ischemia it can be cardiac or cerebral, however, in
the brain coagulative necrosis cannot occur but rather liquefactive takes place. This is due to the
type of tissue and the type of cells which constitute it. Usually tissues with high concentration of
proteins undergo coagulative necrosis because proteins are able to denature: for example,
coagulative necrosis occurs in muscles (e.g. cardiac, parenchymal and many other tissues of our
body which have high quantities of proteins). But why is it named “coagulative”? In Italian, the term
“coagulative” corresponds exactly to “denaturation”, they are synonyms (coagulazione =
denaturazione) which is why it was named coagulative in the past: it is has nothing to do with the
coagulation of the blood, it is only because the words are synonyms in Italian.
Colliquative necrosis on the other hand is present in tissue which have less proteins and have high
concentration/quantities of water. In this way, lysosomes and hydrolytic enzymes can be activated
by high quantities of water: they are activated in tissues with a lower pH with high quantity of water.
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For example, if we have a weak basic intoxication, we can see the swelling (sort of colliquative
necrosis) of subcellular elements. You must remember that necrosis is also considered to be an
incidental type of cell death because it is due to an environmental factor which targets the cell from
the outside. The first structure involved in cell death caused by necrosis is the external membrane
and then, coming inside, it targets the other membranes and finally the nucleus (from outside to
inside).
If we have a weak pH change, we can have swelling of cellular membranes, in particular
mitochondrial. If we have a strong increase of pH with bases, we have colliquative necrosis
depending on the type of tissues and hydrolysis of particular proteins (i.e. keratin and collagen –
scleroproteins - which are also important in the scar formation process), in the cells but also in the
tissue causing therefore damage of interstitial environment (tissue outside the cells). We can talk
about other factors that cause the etiology of changes in this equilibrium:
- Environmental: acids or bases;
- Chemicals that can target cells or tissues e.g. solvents.
Solvent action
Solvents are chemicals which can destroy by lysis all the structures of the cell and tissue (can induce
lysis). They are normally divided into two types according to their mode of action and composition:
- Lipid solvents;
- Hydrophilic solvents.
Lipid solvents
Chloroform (CHCl3), carbon tetrachloride (CCl4), acetone physically destroy the membrane. If you
have used acetone, you have seen that the epidermis becomes whiter and whiter due to the modality
of the solvent action that induces the de-epithelization because they lyse. They cause the lipid bilayer
of the cell membranes to dissolve as they dissolve in lipid solutions. This is because their structure
is maintained as a result of their hydrophilic and hydroponic properties interacting in an aqueous
environment, hence, if you introduce lipid solvents into the environment the phospholipids flip and
this causes them to dissolve.
Hydrophilic solvents
These types of chemicals mainly include salts which can induce two types of situations: hypotonic
solutions or hypertonic solutions.
Example: red blood cells can live for some period of time in a buffered solution which retains a normal
6.5-7 pH range. However, if we insert these cells in a hypertonic (solution with water and a high
concentration of salts), the cells will attempt to balance the osmotic gradient and the pH by releasing
water from the cell to balance the concentration of salts so as to reach an equilibrium between inside
and outside. In this way water moves out of the cells which shrink and die by plasmolysis.
On the other hand, if we insert these cells in a hypotonic (solution with water and a low concentration
of salts), the cells will attempt to balance the osmotic gradient and the pH by taking in water from the
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environment to balance the concentration of salts so as to reach an equilibrium between inside and
outside. In this way the water comes in inducing swelling of the cells which eventually burst and die
by osmolysis which in this case is called osmotic shock.
Protein denaturation
This type of chemical damage to an organism can be due to several different chemicals that are
protein denaturing agents, including:
- Acid-Base imbalance which can result in pH alterations which cause the proteins to be in
an environment with a pH which is not their optimal pH, hence, resulting in their denaturation
due to conformational change which causes their inactivation;
- Presence of different types of ions, in particular metallic ions (Cu, Zn, Cd, Fe, Ni, Co,
Al) which can react very quickly with –COOH groups of the amino acids/proteins, which
causes conformational changes to the protein that causes it to lose its functional capabilities,
hence, denaturing it;
- Salts (CaCl2, KI, thiocyanate, LiCl2) can cause denaturation because charge balance is
important for protein stability, because a protein uses charged residues to fold and stay
folded. Severe salt concentration variations in solution can mask charges and eliminate or
inhibit those interactions, potentially exposing internal hydrophobic regions and reducing
protein solubility which could cause protein denaturation.
Another consequence is that water-soluble proteins have concentric "shells" of semi-ordered
water molecules arranged around them, in much the same way that dissolved salts have
associated water molecules making them soluble. If you add too much salt, the waters in the
protein solvation shells are stripped out to dissolve the salt, precipitating the protein out of
solution;
- Several organic substances that act as cationic detergents. Among these, the main
detergents with cationic action are urea and guanidine at high concentrations, they have a
quaternary ammonium as the polar end, which is positively charged and can react with many
molecules in our body, proteins being one of them can be denatured. Tween 80 is also a
nonionic surfactant and emulsifier used in foods and cosmetics and known by its chemical
name polysorbate 80. Tween is a detergent that we use at home to wash everything and in
fact, the law decided to set a particular concentration to avoid active detergent damage.
Selective inhibition of biochemical functions by toxins or poisons.

The other big category that can induce chemical damage are xenobiotics and toxins in general.
Intoxication can be induced by different types of biochemical lesions which vary depending on the
target of the particular chemical involved in the process e.g. metabolic targets such as mitochondria,
citrate cycle and so on. We can measure the damaging activity of these poisons using two definitions
of activity of the chemicals:
- The minimum lethal dose which is the minimal dose able to kill 100% of the animals of a
certain species, that have the same sex, age and weight, in a given time. Total destruction
of the animals;
- The median lethal dose (LD50, half-dose) is the minimum dose required to kill 50% of
treated animals of a certain species, sex, age and weight, in a given time.
These definitions are important during experimental approach related to use of some drugs,
pharmaceutical and chemicals.
Different toxins can have two types of effects:
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- Instant effect: induces acute toxicity (e.g. KCN) because these chemicals can directly link
with important metabolic mechanisms of our cells, hence alter their normal functional role
which leads to an immediate toxic reaction.
For example, potassium cyanide is known to have a very strong and quick action because it
is linked directly to the mitochondrial respiratory chain, it links there and therefore causes a
change in oxidative phosphorylation: oxidation and phosphorylation, normally coupled in
mitochondrial function, are split, so that mitochondria continue to breath, assume oxygen,
but the energy which increases due to oxygen respiration is not linked to the phosphorylation
molecule i.e. ATP, ADP and AMP. There is the dissociation between mitochondrial oxidative
action and phosphorylation (i.e. uncoupling of respiratory chain). This molecule is very strong
and used for homicidal purposes;
- Delayed effect: all the types of chemical that need to have a metabolic transformation (not
direct toxins) but must be metabolized in our body, their derivatives can cause damage, so
they need a metabolic transformation (which is way they are called “delayed” = they need to
be transformed); also, they must target a tissue and induce in this way a chronic toxic action.
Toxic molecule classifications

Depending on their nature, toxic molecules can be classified as:
- Organic toxins: can find their way into our bodies when we unknowingly absorb them from
known and unknown substances that slowly or sometimes rapidly poison us. Blood tests are
the best way to check for organic toxicity and volatile compounds. When it comes to organic
toxins, there is not a lot of specific treatment. The first step is to identify and remove the
offending agent. After removing the offending agent, the treatment continues with the
detoxification of the liver. For liver support and cleansing, abundant Vitamin C is absolutely
crucial. Vitamin C helps the liver deal much better with toxins taken in from our environment.
There are not many toxins that can’t be neutralized by vitamin C;
- Inorganic toxins: include single elements like lead, arsenic, mercury, cadmium, and others.
These toxins, otherwise known as heavy metals, can be very hazardous to the body,
especially since they accumulate over time, migrating toward deeper and deeper tissues and
eventually affecting the brain and nervous system, attaching to enzymes and blocking
metabolism in different ways. A useful but sometimes not very reliable test for heavy metals
is hair analysis, it can lead to further testing. Hair analysis gives us clues as to where to look
for something and treat it;
- Natural as they derive from vegetables (phytotoxins) or animals (zootoxins);
- Synthetic.
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Route of entry of a toxin

They can also be classified according to their route of entry because the same substance can induce
changes in LD50, or of symptoms and the latent period (barriers). The main routes of entry can be:
- Ingestion (KCN, drugs, household chemicals);
- Inhalation (CO);
- Absorption (Hg, Pb);
- Injection: related to injection there are different ways or injections:
o Intradermal;
o Subcutaneous;
o Intramuscular;
o Intravenous;
o intraperitoneal.
Mutations induced by chemical mutagens (carcinogens)

Another important action of some chemicals, especially related to cancer development, is their ability
to induce modifications, in particular of three types.
Incorporation into DNA of another base instead of a normal base:

Thymine is substituted by 5-bromouracil, thus adenine-5-bromouracil is formed, adenine is replaced
by guanine. Hence, at the first replication, adenine that normally is paired with thymine, is replaced
by guanine which is paired with cytosine, so we have G-C instead of A-T.
Chemical base modification:

Modification by induction of deamination, the removal of -NH2 (the amino group) is a chemical base
modification that alters the pairing. There can be two types of deamination.
Nitrous acid (HNO2) mutagen action by deamination (removal of NH2 group) thus altering their base
pair:
a. adenine into hypoxanthine which has more affinity for cytosine (so, during the first
replication of DNA hypoxanthine pairs with cytosine); at the second replication
cytosine will pair with guanine G-C will be formed;
b. Cytosine into uracil, which has affinity for adenine, so in the second replication the A-
T will be formed instead of G-C because G-C pair has been replaced by A-T.
Alkylation:
Chemical mutagens act by giving alkyl groups to the bases such as ethyl-methanesulfonate and
ethyl-ethanesulfonate, which induce methylation at the 7th position of guanine and, to a lesser extent,
adenine. In this way, the linkage with ribose is broken. This is an important mutation related to some
chemicals that are considered to be carcinogens. We have many carcinogens, so many chemicals
able to induce cancer.
Non-specific defense against toxins to maintain alkaline reserve

- Vomiting and diarrhea;
- Panniculus adiposus (fatty layer of the subcutaneous tissues);
- Contact with proteins that are considered not-noble (we have “proteine nobili” but also other
kinds of proteins);
- Buffer systems;
- Antioxidants (see pathology lecture n.3);
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- Metabolic changes: molecule break down;

- Reaction with other substances that form “complexes” without toxicity.
Tolerance [taken from slides]

Increased tolerability to a toxic compound due to repeated use with low doses of the same toxic that
induces:
- Increased synthesis of detoxifying enzymes;
- Specific antibodies production.
Xenobiotic metabolism
Xenobiotic metabolism consists therefore in the deactivation and the excretion of xenobiotics and
happens mostly in the liver. Excretion routes are urine, feces, breath, and sweat. It can be divided
into two phases:
- Drug metabolizing phase I reactions (or non-synthetic reactions): xenobiotics can be
metabolized to nontoxic metabolites and eliminated from the body (detoxification);
- Protective synthesis phase II reactions: xenobiotic metabolism may also result in the
formation of a reactive metabolite that is toxic to cellular components. If repair is not effective,
short- and long-term effects develop.
In particular, related to metabolic changes and reactions with other substances, we have two routes
to metabolize our xenobiotics, two series of enzymes:
- Activating enzymes: catalyze phase I reactions
which include hydrolysis, reduction and oxidation.
Xenobiotics can be metabolized by these enzymes
and turned into a derivative which can be further
metabolized by the enzymes of phase two
reactions;
- Detoxifying enzymes: catalyze phase II
reactions which include glucuronidation,
sulfation, methylation and conjugation. These
enzymes further metabolize the derivative that
was produced through phase I enzymes’
action, thus transforming the primary
metabolite into a secondary metabolite which
can be eliminated through normal process of
elimination (urine, feces, breath and sweat) so
that they have no damaging action.
A xenobiotic can be metabolized through phase I

reactions in a non-toxic metabolite, but it can also be metabolized in a more reactive metabolite
which, depending on quantity and concentration of the substance (xenobiotic), cannot be
counteracted by phase II reactions and can therefore induce toxicity.
Let’s take cancer as an example. There are some chemicals present in the environment of the body
among which cyclic hydrocarbons which are not cancerous. They are introduced in our body through
breathing or injections and then metabolized in our tissues, in particular in the lungs and in the liver,
by phase I enzymes. However, they are metabolized into a derivative which is diol epoxide or
epoxide, which has an alkalotic effect/function. So, this metabolite is directly active toward DNA
damage: induces a very quick mutation of DNA.
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Another example is related to adenine that is converted by phase I reactions but also by phase II
reactions so, it can be eliminated normally. However, we have some bacteria that can reverse these
reactions to make available the deactivated compound that becomes activated again.
The main enzyme and most important one for metabolism and phase I reactions is cytochrome
P450 which is present in the drug metabolizing system found in our microsomes. Normally we tend
to think that this system is present only in the liver (high concentration there), but actually all our
tissues have it.
Another example can be the tissue-specific presence (specifically present in kidney, liver and lung)
of rhodanase enzyme which is able to transform cyanide into thiocyanate, thus inducing the
formation of a non-dangerous metabolite.
Protective synthesis, phase II enzymes consist of different types of reactions:

- Conjugation with different acids:
o amino acids: lysine, cysteine, glutamine in particular;
o sulfuric acids: phenols;
o glucuronic acids: phenols or aromatic compounds.
In this case (protective synthesis), the enzymes need either of these three types of
compounds as a substrate. The acids are transferred to the carboxy terminal groove to make
an ester bond;
- Acetylation: acetyl CoA with amino groups
- Methylation with compounds that contain the nitric groove.
There are four important chemical agents able to induce damage: potassium cyanide, carbon
monoxide, lead and mercury.
Potassium cyanide (KCN) which is present in different compounds; as explained before, is the
inhibition of mitochondrial oxidative enzymes of the respiratory chain, so it blocks oxidative
phosphorylation inducing the so-called histotoxic anoxia because there is oxygen, but it is not used
for phosphorylation. It can have either acute or chronic action. Acute toxicity can occur with either
high doses causing immediate death (1-15 minutes) or low doses with different symptoms (anxiety,
tachycardia, etc. …) especially related to psychologic effects but at the end many of these conditions,
including alteration of breathing and activity of the heart, can induce acidosis (imbalance in the pH)
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leading to a coma followed by convulsions and death by respiratory arrest. There is an antidote for
potassium cyanide which is methylene blue that is able to transform the haemoglobin into
methaemoglobin which has the capability to capture the cyanide ions before they inactivate
cytochrome oxidase enzymes of the respiratory chain.
Carbon monoxide (CO) can be found both indoors and outdoors e.g. environmental and
professional exposure: smoke, car exhausts, industries and so on. The action of mechanism consists
in directly binding haemoglobin, thus inducing the formation of carboxyhemoglobin (HbCO). The
toxicity depends on the percentage of carboxyhemoglobin formed. Also, in this case, psychic
alterations and alteration of breathing, tachycardia, hypothermia and so on occur. Chronic
intoxication presents itself with nervous disturbances and changes in electrocardiogram (EKG).
Depending on percentage of HbCO, we can have only headache and dizziness or go towards coma
and die. There are also after-effects of the intoxication related especially to induction of motor
changes (e.g. amnesic syndromes, paralysis, cortical blindness and so on) which can be observed
through computer tomography (CT scan) or magnetic resonance imagining (MRI). In this case, MRI
shows a brain atrophy which determines the percentage of CO intoxication.
Lead (Pb): it can be found in different types of industries

(professional exposure) and objects which we use
normally such as food, colored glass, ceramics (non-
professional exposure). Its mechanism of action is the
Ca2+ mimetic effect, in particular it inhibits two enzymes
which do not induce the right biosynthesis of the heme
group; these are δ-aminolevulinic acid dehydratase and
ferrochelatase which are not able to link to proto-
porphyrins, so hemoglobin is not built in the proper way.
Also in this case, nerve disorders occur and there’s a
mechanistic link related to the hyperactivation of a
calcium-dependent enzyme inducing protein kinase C with
spontaneous induction of neurotransmitters in the pre-
synaptic axons [from slides: hyperactivation of Pb-
dependent protein kinase C (an enzyme that is calcium-
dependent) with the induction of spontaneous release of
neurotransmitters in presynaptic axon terminals]. In this
case, acute toxicity in children is very important because
there is a serious encephalopathy and high mental
retardation while in adults we can have either severe (convulsion, coma, high impairment of
reproductive organs related to reduced sperm-count and fertility, pregnancy and so) or mild (learning
and behavioral disorders, hyperactivity, irritability and so on) form. Chronic toxicity leads to anemia
due to Ca2+ mimetic effect with decrease of biosynthesis of haemoglobin, there is an increase of the
ratio between the concentration of Hb and erythrocytes count ([Hb]/RBCs is <). There’s joint pain
related to paralysis in particular in the late stages of the disease and other types of hepatic or
parenchymal diseases in general. There is however a therapy: anticonvulsants, mannitol, which
is a quick anti-inflammatory compound, and cortisol are administered. Chelating agents are those
molecules able to link directly lead to avoid its action are administered as well, among them there
are the EDTA (ethylenediaminetetraacetic acid) or the succìmer which is a succinic acid
penicillamine (for prolonged treatment). In general, the important thing is to chelate (to link) as soon
as possible the poison.
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Mercury (Hg): we can be exposed to it at work or have an increase in Hg levels caused by the type
of diet that we conduct, the so-called bioenhancement: higher quantities in particular of a type of
mercury which is the methylmercury, normally present in the sea and therefore eaten by fish. We
have very strong disease such as Minamata disease in which the corporation reversed, discard
tons of mercury in the liver inducing terrible distress of liver in affected subjects; there are also other
important diseases, in fact this is only one of the four poisoning that affected the Japanese (the Itai-
itai disease in 1912 and the Yokkaichi asthma in 1955). There was another one in Ontario (Ontario
Minamata disease) where there was a company that dumped chemicals in the Wabigoon river (so-
called “Grassy Narrows First Nation”)
ENVIRONMENTAL PATHOLOGY
Environmental pathology is the study of all extrinsic pathogens (out of our bodies) which constitute
risk factors for our health. They can be constantly present in the environment (e.g. high or low
temperature if the temperature is too high or too low it can cause pathologies, high or low pressure
(barometric/atmospheric) if it is too high or too low some pathologies may occur, radiations that were
discussed with professor Leonarduzzi etc. …) or accidental exposure to electricity.
Industrially produced pathogens can accumulate in the environment in case of their non-disposal.
Burns
Burns occur when there is a transfer of thermal energy, they are injuries caused by the transfer of
thermal energy from a heat source to delimited district of our organism. You can have a burn when
the temperature of the affected skin reaches 40-45°C. The severity of burns depends on a series of
factors:
- the extent of the area that is burnt;
- the depth of the lesion: there are lesions that involve only the superficial area of skin and
other that go in depth involving dermis, hypodermis and sometimes also muscles and
tendons;
- the percentage of body surface area that is affected: sometimes a non-serious burn but very
extended (involves a big surface of our body) can be very dangerous;
- presence of internal injuries that can result from inhalation of toxic fumes (fire);
- amount of transferred heat;
- temperature reached by the tissue, a burn occurs when the tissue reaches a T of 40-45°
which however is different from 50°C, 60°C, and so on;
- duration of contact with heat source: the injury caused by contact with a medium/low heat
source but for a long time can be as bad as the injury produced by a very hot heat source
that was in contact with the area for a short time;
- nature of tissues: some tissues are very resistance e.g. epithelium with abundant stratum
corneum (palm of our hands, callus palmar), while others are very delicate;
- the promptness of the therapy.
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Figure n.2 gives you an idea of how the skin is

organized. The superficial layer of the skin is the
epidermis, then there is the dermis which is vascularized
while the epidermis is not, and then the hypodermis is in
depth.
There are four different degrees of burns:
- First degree burn: involves the epidermis only;
- Second degree burn: goes down in the dermis
and can reach the papillary dermis or go more in
depth but in general when dermis is involved we
speak about second degree burn;
- Third degree burn: involves also the hypodermis
below;
- Fourth degree burn: is very severe.
Classification of burns
First and second-degree burns are also called partial thickness burns and are characterized by
congestion and pain but not always necrosis: sometimes you have an acute inflammation in these
cases, but not always cell death.
Third and fourth-degree burns are called full thickness burns and are characterized by
carbonization of both epidermis and dermis, which means that there is death of the cells (necrosis).
Every time you have necrosis, an inflammatory reaction is activated.
First degree burns
A first-degree burn is characterized by
inflammation, pain, redness because of the
dermal hyperaemia10 and erythema due to
hyperaemia of the microcirculation of the
dermis. Skin is intact but erythematous, dry,
hypersensitive and sometimes there is a
slight swelling because there is an
inflammatory reaction and sometimes an
exudate (inflammatory edema) goes out
from the vessels giving rise a slight swelling. Even a first-degree burn, if affecting 80% of the body
surface, can cause a state of shock.
Second degree burns

Second-degree burns are characterized
sometimes, not always, by necrosis of the
epidermis and by the presence of
subepidermal vesicles which are full of
serous exudate that does not contain cells nor
proteins, it is clear; these bubbles are called
phlyctena (“flittene” in Italian) and are located
between epidermis and dermis.
10
When we have an acute inflammatory process, we have hyperaemia because the blood comes to the site of injury.
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There are two types of second degree burns:

- IIA or superficial dermal burns: superficial or papillary dermis;
- IIB or deep dermal burns: affect deep or reticular dermis.
Figure 1a In figure 1a Figure 1b

we can observe a
superficial dermal burn, while in figure 1b
we can observe a deep dermal burn. Here, we see that the blister ruptures
so that the underlying dermis is exposed.
Third and fourth degree burns
Third and fourth-degree burns are full thickness burns and, in this case, there is carbonization
which means that a big number of cells die; there is necrosis that is usually of the coagulative kind,
and the tissue can be white or blackish, it is blood-less because all the vessels have been destroyed
by carbonization. Tissues are dry, contracted, rough, hard and there is skin numbness because
also the nerves have been destroyed. Very often nerves, tendons, muscles and bones are destroyed
in these types of burns.
Inflammation rapidly
develops in adjacent vital
tissues because it is carried
on by vital cells not dead
ones: when there is a lot of
debris in a tissue because
cells have died, the
remaining cells that are
alive begin to produce
cytokines, chemokines and so on in order to induce the inflammatory response.
A number of numbers…
Burns that affect more than 50% of the body are potentially fatal, but in general we can speak about
localized or extended burns depending on the percentage of affected body surface:
- Localized: in case of less than 20% of affected body surface in adults or less than 15% in
children;
- Extended: in case of more than 20% of affected body surface in adults and more than 15%
in children.
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Medical doctors use the “rule of nines” which is a tool necessary to know
what is the total body surface area (TBSA) that has been injured. For
example, a doctor can say that in a given person there is a certain
percentage of body surface which is burn. How? Thanks to this rule. In
adults each of these parts corresponds more or less to 9% of the body
total surface i.e. the head is 9%, the arms are 9% each, the anterior
superior and inferior trunk are 9% each and the same goes for the
posterior part, the front surface of the legs is 9% and so is the dorsal one
all multiplied by two, and the remaining 1% is for the genital region.
If one arm and one leg are completely burnt, you can say that 18% of the
body surface is burnt which means that it is a localized burn.
This is very important to find the therapy, because if the body surface is
injured for more than 50% of its total, it is very likely that a hypovolemic
shock will occur, so you have to properly treat the patient.
In children the situation is different because they are not as proportionate
e.g. the head is bigger compared to ours. Head and neck are 19%, arms
are 9% each, 9% for upper and lower anterior trunk respectively and same
for the posterior portions, but the legs are shorter so they only account for
13% for the front and 13% for the back. However, the total is still 99% plus
1% for the genital region.
Healing of cutaneous burns

We have seen how the healing process occurs in general. In case of
cutaneous burns, if they are partial thickness burns (1st and 2nd degree)
and they are not serious, there is no necrosis of the tissue so it’s easy to
have spontaneously restitutio ad integrum, the resolution; but if they are
serious, they take more time to repair but they do it perfectly without a
scar formation because in these types of burns only the epidermis is injured (no vessels, easier to
repair).
Full thickness burns on the other hand are deeper, so they are more difficult to repair, and a scar is
always created; sometimes a re-epithelization might be necessary:
- Artificial skin can be produced in vitro by culturing keratinocytes and then put on the surface
of the burn
- Auto-transplantation of skin taken from other regions of the body.
Why are burns so dangerous, particularly If they involve more than 20% of the body?
They can give a shock. If they involve more than 50% of the TBSA, they are fatal, but when they
involve more than 20% of TBSA, they can cause a hypovolemic shock which means that the
volume of blood is less than physiological level, because during burns the endothelial cells of the
vascular vessels are damaged so lots of fluids go out from the vessels and so there can be a shock.
So, we have in order: fluid loss, reduction of blood volume (“hypovolemic” because “hypo” means
less), reduced cardiac output, reduced pressure, unbalanced electrolytes and obviously if there is
and hypovolemic shock and blood volume is reduced, also oxygen is reduced in blood so that tissues
receive less oxygen. You can have some complications among which sepsis because in burns the
skin, which has the function of protecting our body from microorganism, is damaged, so it’s important
to remove the debris immediately to allow phagocytes to reach the infection site (i.e. neutrophils
which are the main cells involved in acute inflammation) and promote wound repair because if the
skin is infected, the wound is not produced in the right way.
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Low temperatures
We have seen pathologies due to high temperatures, but they can also be due to low temperatures
There are two important terms to remember:
- Frostbite or freezing (they are not synonyms): local phenomenon with local effects (in
Italian, congelamento) when a single area of our body is exposed to very low temperatures;
- Hypothermia: general phenomenon with system effects (in Italian, assideramento or
ipotermia) when the entire body is exposed to low temperatures.
Frostbite is the opposite of burns, but also in this case we have four degrees of frostbite.
Classification of cold injury according to severity
- First degree frostbite: erythema, edema, and sometimes hyperaemia,
just like the erythema of UV exposure for instance;
- Second degree frostbite: characterized by vesicles that are similar to
those that appear during second and third-degree burns, they are
blisters called also in this case phlyctena;
- Third and Fourth-degree frostbite: extended necrosis of the tissues.
Factors that increase the risk of frostbite:

- Behavioral; alcohol, smoke can increase the probability of having a
frostbite;
- Physiological;
- Mechanical: if you go skiing with three pairs of socks on or if the boots are too tight, the
correct circulation is not allowed, frostbite can occur; rings on fingers, immobility (e.g. ski lift).
You can read about treatment and prevention on the slides e.g. wear mittens instead of gloves (in
Italian, moffole).
The
case
report
on
slide
n.24 was about a medical doctor who went on a mountain and got an important frostbite on his hand
(pictures above). The extremities of the fingers are not necrotic, but the blood is not arriving anymore
there, so the circulation is not right. Treatment was carried out with Iloprost which is a prostacyclin11
analog. Since prostacyclin is responsible for vasodilation, Iloprost as well is responsible for
vasodilation of the vessels, allowing blood to arrive to the tissue that do not receive oxygen anymore.
11
We mentioned prostacyclin when we spoke about chemical mediators of inflammation, it derives from the
arachidonic acid metabolism.
110
Hypothermia is the general condition, in which we are all exposed to very low temperatures, not
just a part of the body. Occurs when two mechanisms of production and dispersion of heat do not
work correctly:
- Thermogenesis (production of heat) doesn’t work;
- Thermodispersion (dispersion of heat) works too much.
When hypothermia is present, the temperature rapidly decreases below 37°C which is the
physiological temperature of the body, leading to slowing of biological functions until the circulatory
function is suppressed. Death occurs when the temperature of our body reaches 20-25°C. When a
body reaches 35-32°C there is first an increase in ventilation speed (tachypnea), depth of breath
(hyperpnea), vasoconstriction to maintain the heat inside our body, then tachycardia, hypertension
and so on. When the temperature is 32-29°C there is reduction of respiration, heart rate, blood
pressure, renal function: all the biological functions of our body are slowed down in order not to
spend energy and maintain the heat inside. When the temperature reaches 29° or less degrees there
is the vasomotory paralysis (incapacity to react, move or think). Below the 25°C there is the
apparent death, but they are not because death occurs at lower temperatures. Therapy consists in
very slow heating of the individual.
Effects of atmospheric pressure fluctuations
There are two types of pathologies due to variations of atmospheric pressure:
- Hypobaropathy: due to a reduced atmospheric/barometric pressure, occurs when you reach
high altitudes;
- Hypobaropathy: when atmospheric/barometric pressure is increased, in English is mostly
called “decompression sickness”, occurs when divers go deep in the sea/lake.
Hypobaropathy or altitude sickness is characterized by a decrease in barometric pressure. At the

sea level (0 meters of altitude) barometric pressure is equal to 760 mm Hg, it decreases with altitude
(it is inversely proportional to altitude). For example, at 3000 m we have already a pressure of 525,
at 6000 m a pressure of 349 and so on. The partial pressure of oxygen at alveolar level that we can
tolerate is 67 mm Hg that is present when altitude is about 3000-4000 m.
The decrease in barometric pressure causes a decrease in the partial pressure of the gases that are
present in the air, but the composition of the gases remains the unchanged. The problem is not
nitrogen because it is the most abundant among all the gases present in the atmosphere, but the
problem in hypobaropathies is only oxygen, because increasing the altitude oxygen is reduced and
the partial pressure of oxygen at alveolar level is reduced as well. This situation characterized by
oxygen saturation of hemoglobin (because in the blood oxygen is bound to Hb) is called hypobaric
hypoxia which in Italian is “ipossia ipossica”: “ipossia” is the reduced level of oxygen in the blood
(hypoxia) while “hypobaric” - due to reduced barometric pressure - is “ipossica” meaning due to
reduced oxygen in atmosphere. It is characterized by tiredness, headache, nausea, mental
obnubilation or euphoria. These symptoms can evolve in coma and death, so they can be very
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serious.
Slm = sul livello del mare meaning ASL = at sea level. The table shows altitude at ASL, the
barometric pressure, the partial pressure of oxygen at alveolar level and the O2 saturation (binding
of oxygen with hemoglobin). We can tolerate to reach 3000 m altitude where oxygen saturation is
90% which is not perfect but still a good saturation. Going beyond 3000 m of altitude can be
dangerous because oxygen decreases in our blood and therefore in our tissues as well. When
altitude is 4000 more or less (depends on personal characteristics), a homeostatic reaction of our
organism is triggered: we start hyperventilating (increase the frequency and depth of our breath),
contemporarily there is tachycardia (faster heart rate) to give more oxygen to the tissues.
Hyperventilation is positive event because it allows oxygen to come in and reach the tissues to there
is an increase in alveolar and arterial pressure of oxygen and saturation increases as well. However,
in parallel to oxygen assumption there is carbon dioxide elimination because hyperventilation puts
oxygen in and carbon dioxide out but in an excessive manner. The consequence of elimination of
CO2 is respiratory alkalosis meaning that pH is increased in the blood. In physiological conditions,
the reaction water + carbon dioxide, when in equilibrium, yields bicarbonate and hydrogen ions. But
if CO2 is eliminated too much, its concentration is too low, thus bringing the reaction toward left;
therefore, to create CO2, we transform bicarbonate and hydrogen ions into CO2 and we know that a
reduced concentration of hydrogen ions in blood is responsible for an increase of pH, the respiratory
alkalosis which inhibits respiratory centers triggering
hypoventilation.
Why is hypobaric hypoxia dangerous? It can lead to a
series of events by creating a condition of respiratory
alkalosis because of the hyperventilation that occurs.
The persistence of this condition of hypoxia stimulates
erythropoiesis (production of new RBCs starting from
the precursors present in the bone marrow). In
response to hypoxia, the kidneys are stimulated to
produce erythropoietin which binds to receptors on
precursors of RBCs in bone marrow so that they being
to erythropoietin and start differentiating in reticulocytes and so on. Why do erythrocytes need to be
more numerous? Because if we have more erythrocytes, we can bind more oxygen, and if there isn’t
much oxygen, it helps it to reach the tissues. This is an example of hyperplasia because there is the
production of more cells to overcome the lack of oxygen.
Peruvian Andes
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There are villages located at 4000-4300, whose inhabitants have very low levels of oxygen in their
alveoli, but they are acclimatized meaning that they have particular characteristics which allow the
oxygen to reach their tissues more easily e.g. short stature, big/wide “barrel” chest (pulmonary
functions are very well-developed so they can push air into their lungs more easily), cardiac
hypertrophy (adaptation), greater extension of capillary beds and others. However, there can be
issues because the hematocrit is very high (volume occupied by blood cells), blood viscosity is very
high (lots of RBCs in the blood) which can lead to chronic mountain sickness.
We can have voluntary acclimatization by remaining for days or weeks at higher altitudes to what
we are used.
This condition of hypobaric hypoxia can be prevented
by breathing pure oxygen. Climbers often breath pure
oxygen while climbing very high altitudes: usually
nitrogen constitutes 4/5 of the air, but in this case, they
are occupied by oxygen. The graph shows the
difference between a climber breathing pure oxygen
while climbing (green line) and another who does not
(red line). In the first case, oxygen saturation remains at
90-98% until 12000 m, while in the other case oxygen
saturation is sufficient until 3000 m but then goes down
very rapidly.
Until 4000 m there are no particular issues but above
that altitude we start hyperventilating, so respiratory
alkalosis occurs, and we can have a series of
neuropsychic disorders, also at rest e.g. errors of
calculation, orientation so that it might be difficult to get
home. Over 6000 m, we have a “critical zone”: we have little time until we die.
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Neurological consequences of altitudes are shown in the picture on the right, progressively we have:
altered night vision, dizziness (issues in equilibrium) or tingling, loss of consciousness and acute
decompression.
Effects of high altitude/reduced barometric pressure
- Systemic edema: it is called systemic because it is not localized in one site of our body, but
is rather diffused in the body; however, it is more probable to occur at the level of face, hands,
feet;
- Retinal hemorrhage: it is reversible condition, which is solved when you get down the
mountain;
- Acute mountain sickness (“mal di montagna”): it is very common in our mountains that is
not feeling well while climbing or even simply walking; most common symptoms are
headache, dizziness (you have to sit down), fatigue, loss of appetite, and, under stress, so if
you try to walk or climb, you might have memory deficits (neurocognitive functions), nausea,
vomiting, problem in hearing, shortness of breath, sweating, etc. … The prophylaxis consists
in the use of acetazolamide for carbonic anhydrase12 inhibition; the use of these inhibitors
before or during ascent may prevent respiratory alkalosis which may occur, but the most
active way to stop this acute mountain sickness is to descent (go home). If descent is not
possible, oxygen may be administered by portable hyperbaric chambers. Hyperbaric
12
Carbonic anhydrase is the enzyme responsible for transfer of CO2 out of the blood.
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means that the barometric pressure is increased inside the chamber and pure oxygen is
administered. Acute mountain sickness can be serious or not, it must be recognized because
it might lead to high-altitude cerebral or pulmonary edema which are very dangerous and
might lead to death;
- Chronic mountain sickness (Monge’s disease – named after the person who discovered
the characteristics and potential pathologies which may occur in inhabitants of villages
located at high altitudes): it is due to increase in blood viscosity, when partial tension of O2 is
reduced, erythropoietin is produced by kidneys and we have hyperplasia of erythrocytes. It
can afflict also people who live at high altitudes and are fully acclimatized but can still suffer
from this pathology. Characteristics are similar to those of the acute mountain sickness.
Remember that when altitude increases, barometric pressure decreases, but also other
environmental features change: temperature decreases (hypothermia), UV radiation
increases because of decreased cloudiness and water vapor.
Exposure to high environmental pressures (barometric)

These pathologies are called in Italian “iperbaropatie” while in English they are most commonly
called “decompression sickness” rather than hyperbaropathies.
The pressure increases as the atmospheric pressure is added to the mass of water: when you go in
depth in water (e.g. sea, lake…), the atmospheric pressure is summed to the pressure of the water
that is over your head, so the pressure increases more and more. It is also called “diver’s or caisson
disease” (in Italian, “malattia dei cassoni”).
Two physical laws of gases:
- Boyle’s law: the pressure and the volume of a gas have an inverse relationship meaning
that if the barometric pressure increases while you are going in depth in water, the volume
occupied by the gas decreases which is why at 1 atm ASL the gas occupies 1000 cc, while
it is 500cc when the pressure is doubled (see scheme on the right).
This is why the organs collapse while we are reaching the bottom:
lungs collapse, become smaller and compressed because the gases
inside them occupy less volume and also their functional activity is
compromised. For this reason, gases mixtures at a pressure higher
than the barometric pressure are given inside the tanks (bombole).
In this way, if the gases inside the tanks are at a higher pressure, our lungs don’t collapse as
we go down;
- Enry’s law: the amount of dissolved gases is proportional to its partial pressure in gaseous
phase which means that increasing the barometric pressure, the solubility of the gases
increase. While we are going down, barometric pressure increases, and the gases are
completely dissolved in water, they become liquid. The problem in this case is not O2 because
it is fixed to Hb, the problem is nitrogen because, while atmospheric pressure increase, it
dissolves completely in our blood (but not only). When we are in depth, the nitrogen is liquid,
dissolved in blood, but when you have to ascend again to the surface, you have to do it
slowly, by steps because the barometric pressure is
reduced, and the dissolved nitrogen becomes again
gaseous forming little bubbles which are spontaneously
expelled through respiration. The problem is when you
ascend too quickly because the dissolved nitrogen
becomes gaseous altogether forming big bubbles which
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represent emboli13 (gas embolism). An embolus is very dangerous because it might reach a
vessel that is too small for it to pass, it can block the blood flow leading to ischemic death of
the tissue.
In solution in the body ASL, 1 liter of nitrogen is dissolved in the body, not only in liquids, but also in
lipids (i.e. in adipose tissue, plasma membranes of our cells are composed of a double layer of
phospholipids). When you reach 10/30 m much more nitrogen is dissolved in the body. Over 35 m
in depth, it can be dangerous because its solubility in lipids increases very much; you can have a
condition called “martini effect” because you can feel drunk due to these neurological alterations
(feeling of intoxication, reduction of work capacity, mild confusion and so on). These symptoms go
on until you get nitrogen narcosis: people can fall asleep due to high solubility of nitrogen at these
levels in the plasma membranes of neurons; all the nitrogen dissolve in neurons’ plasma membranes
alters the capacity of neurons to communicate with each other causing this condition.
How can gas emboli be formed in our blood: pressure increases, nitrogen is completely dissolved in
blood; when you go up again, nitrogen, if you ascent too quickly, forms big bubbles which can also
be formed due to pulmonary barotrauma that occurs because, for the gas law, when P increases,
the V occupied by gas decreases, so the lungs collapse, and when the surface is reached, they
become again big rupturing the alveolar capillaries so that bubbles can go into the blood from the
lungs.
Symptoms of decompression sickness include narcosis, pain (in joints, knees, elbows…),
constitutional symptoms (headache, lightheadedness, inappropriate fatigue, nausea, vomiting,
anorexia…), muscular problems like cramps, spams, pulmonary manifestations like dyspnea and
cough.
Cutis marmorata might also be a symptom which is however not very
common), it is a skin rush due to decompression illness. Therapy could be to
go again in depth after you have reached the surface too quickly so that the
nitrogen dissolves again and then to come back up slowly. This solution is
called “in water recompression” and is suggested when you don’t have a
hyperbaric chamber nearby. On the other hand, if you have this chamber you
must go inside where there is a new immersion that is mimicked. In this case,
the pressure is increased in the chamber, bubbles in blood are dissolved again,
the pressure is reduced slowly and in this way the nitrogen becomes again
gaseous forming tiny little bubbles that are spontaneously expelled with
respiration.
ONCOLOGY INTRODUCTION. METAPLASIA,

DYSPLASIA, NEOPLASIA
CELL ADAPTATION
There are some adaptations that occur before tumours
develop; some types of cellular adaptations have already
been seen in the course, like hypertrophy, hyperplasia and
hypotrophy (or atrophy): they are related to
microenvironment modifications that can induce cell death
or, if they are of medium intensity, a sort of adaptation to
reach a new homeostasis of the tissues or of the cells.
13
Whenever you have something, either solid, liquid or gaseous, in the blood which shouldn’t be there, you are talking
about an embolus
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Besides the ones mentioned above, there are other two types of adaptations, dysplasia and
metaplasia, which are important in the development of cancer. The causes of dysplasia are not well
known: they can be different, like environmental factors, genetic factors, etc. Metaplasia is due to
some environmental causes that induce functional changes in our tissues (the so-called cell
phenotype modification).
Metaplasia
The definition of metaplasia is: “a mature tissue, that is a specialised tissue, is transformed in another
type of specialised tissue”: there is a change from a type of tissue with a specific function (well
differentiated, mature) to another type of mature, well-differentiated tissue with other specific
functions.
Most types of metaplasia are due to environmental factors and chemical compounds, such as
the chemicals that are in the smoke of cigarettes. There are several examples of metaplasia in our
body:
1) Squamous metaplasia of the respiratory epithelium: the
pseudostratified ciliated columnar epithelium in the
respiratory tract is lost in terms of functions and morphology
and is replaced by another type of epithelium which is similar
to the epithelium present in the trachea (stratified
squamous)14; the hot air that we introduce when we smoke
can induce a sort of different function related to the
pulmonary and bronchial epithelium which is normally able to
secrete. In this case, it changes its morphology and functional
activity to be similar to the other epithelium of the upper part
of the trachea whose function is related to the contact with
the environmental antigen, so the functions are different;
2) Squamous metaplasia of bile duct: this type of metaplasia
is related, for instance, to the bile duct stones in the Figure 1
gallbladder, where the columnar secretory epithelium
changes into stratified squamous epithelium because of a
physical disturbance of the mucosa due to the stones that are in the bile duct;
3) Barrett’s oesophagus (or Barrett’s metaplasia): it is normally found in patients suffering
from gastroesophageal reflux disease; the stratified squamous mucosa of the oesophagus is
replaced by a columnar epithelium similar to the one found in the stomach, which normally
has goblet cells, able to secrete mucus. The normal epithelium is replaced by a metaplastic
tissue that can also
develop other types of
morphological and
functional changes, like
dysplasia and cancer.
Barrett’s oesophagus is
considered a disease that
can develop cancer in
large quantities of cases:
many patients that suffer
from Barrett’s Figure 2
14
She said: “the normal stratified squamous epithelium in the respiratory tract is lost in terms of functions and
morphology and is replaced by another type of epithelium which is similar to the epithelium present in the trachea” but
it is wrong because at first the epithelium is columnar, not stratified squamous.
117
oesophagus can develop cancer at some point in their life. Figure n.2 is a visual description
of Barrett’s oesophagus: normally, between the stomach and the oesophagus, we have two
types of junctions, the gastroesophageal junction (GEJ) and the squamocolumnar
junction (SCJ); the GEJ is the region that divides the squamous mucosa of the oesophagus
from the columnar mucosa of the stomach: normally, in the healthy subject, the GEJ overlaps
with the SCJ (forming a line that is also known as Z line or border between the gastric and
oesophageal mucosa). In the case of Barrett’s oesophagus, however, the squamocolumnar
junction is increased and is seen into the oesophagus, so there is a part between the GEJ
and the new SCJ in which there is columnar epithelium that normally is not there. This
columnar epithelium is found in gastroesophageal patients in which there is reflux of HCl,
causing a condition of acidosis, and so the last part of the oesophagus must defend itself
from the attack of the acid. The columnar epithelium is normally composed by three types of
cells:
a. Fundus type: cells similar to the fundus of the stomach;
b. Junctional type: cells similar to cardiac epithelium (i.e. of the
cardias); Figure 3
c. Intestinal type: cells similar to cell types of
the intestine with mucus secretion (goblet
cells). Goblet cells are the cells that most
frequently degenerate into malignancy
(dysplasia and tumours) in the oesophagus
of Barrett. Figure 4
As it can be clearly seen in
figure n.3, goblet cells are
also called chalice cells
because of their shape;
they are among (she said
“within”) the other epithelial
cells.
In figure n.4 there is a normal epithelium of

the oesophagus on the left and on the right
there is columnar metaplasia (like in the
stomach). Here in particular there are also
goblet cells in the part with columnar metaplasia in comparison with the other part, which is normal
oesophageal squamous epithelium. Figure n.5 is the picture of a slide of tissue taken from a patient
affected by gastroesophageal reflux.
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To sum up, metaplasia is the change from one

mature tissue type to another differentiated
Figure 5
mature tissue type (an important concept to
understand the development of cancer).
Metaplasia can be reversible: if we change
the conditions that induce it, the epithelium
can change again to a well differentiated,
normal specialised tissue.
When the factors that induce metaplasia
continue for a long time, metaplasia can be
replaced by another definition/condition which
is dysplasia.
Dysplasia
Dysplasia literally means “disordered growth”. In contrast with metaplasia, in which there is a
change to a different type of tissue but the growth is normal and ordered, dysplasia is disordered in
the growth, shape and orientation of the cells into the tissue.
The term dysplasia can be used in two types of conditions: with the definition of an abnormal growth
or also referred, more strictly, to abnormal growth due to changes of the proliferative process.
The changes can include differentiation, cell shape and orientation and also involve the type of
tissue/cells that show dysplasia.
Dysplasia can also be considered a synonym of advanced metaplasia. We can have some
environmental factors or genetic factors that induce metaplasia (a kind of adaptation of our tissues
and cells to a new condition) but the tissue continues to function. However, if the factors continue to
attack this kind of epithelium, the cells cannot recover their function and structure, so they undergo
dysplasia. In advance stages, dysplasia is irreversible and becomes a cancer.
Uterine cervix dysplasia
The uterine cervix dysplasia is the main example of dysplasia.
Figure 6 Figure 7
Disordered growth can be confined
to the epithelium, like in the part on the right in figure n.6, and at a higher magnification (figure n.7)
we can also see differences in the cells compared to the normal cervix tissue. In the part of
dysplastic15 growth there are different shapes and orientations of the cells; normally they are oriented
in the same direction (left part of figure n.6), but on the right of both figures n.6 and 7 we have
different types of cells with a disordered orientation; these cells have also different shapes, like round
cells, etc. The dark part is the nucleus, which has very big dimensions: the ratio between the nuclei
and the cytoplasm of the cells is increased in the case of dysplasia.
15
Dysplastic or dysplasic is the same
119
Based on the morphological changes of the cells of the cervical epithelium, some time ago doctor
Papanicolaou, a doctor from Greece, applied a staining to state the cells of the cervix epithelium,
creating what we now call the pap-test. This test is a cytologic test: it examines the volume and
shape of the cells, the ratio between the nucleus and the cytoplasm, etc.
The pap-test is divided in five classes:
- Class I is related with normal cells in structure, volume, size and so on (the staining is
normal);
- Class II is when we see some atypical changes but that are absolutely NOT related to cancer;
it could be an inflammation or a kind of infection, like HPV, so something that is not of great
concern;
- Class III is an intermediate dysplasia: now we can talk about dysplasia and we can think
about the possibility that there is a cancer. There are two types of tumours16, benign and
malignant;
- Class IV is a severe dysplasia, so it is irreversible. This type of severe dysplasia is indicative
of malignant tumour;
- Class V is related to a malignant tumour with a very high number of changes in dimension,
orientation and such of these cells. In this case we can think about the presence of a
malignant tumour of the epithelium (carcinoma17) that can also spread out from the site of
origin in the body.
CIN 1-2 and CIN 3 is another type of classification of the possibility of the dysplasia of the cervix
epithelium.
In uterine cervix dysplasia, disordered growth can be confined to the epithelium. If the carcinoma is
confined to the epithelium it is said to be in situ; this type of carcinoma has all the features of a
malignant cancer, but it is still confined to the epithelium, so we can see dysplastic changes which
are marked and involve the full thickness of the epithelium but they don’t penetrate the basal
membrane. Most of the surgically resected tumours are carcinomas in situ because the surgeon can
remove all the volume of the tumour.
We can also have other histological classifications related to the dysplasia; for instance, the CIN
(Cervical Intraepithelial Dysplasia) refer to a classification
Figure 8 Basal membrane
Figure 9
called Richart grading, which divides the dysplasia only in three degrees (see figure n.9):
- Early or low grade of dysplasia (CIN I);
- Moderate grade of dysplasia (CIN II);
16
Not cancer, tumour is a better name because there is some misunderstanding about the word “cancer”.
17
The malignant tumour of epithelia is called carcinoma; the term cancer means normally malignant, but it is
very generic.
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- High grade of dysplasia (CIN III), there is carcinoma in situ (still into the thickness of the
epithelium); in situ carcinoma can also undergo to metastasis (spreading).
There is also another classification, SIL (Squamous Intraepithelial Lesions), not very known but
simple and important for doctors and technicians who read the histological specimens because the
classification in only divided in two classes:
- SIL I: low-grade lesion, in which there are changes due to inflammation and infection (e.g.
HPV), which correspond to CIN I and degree I and II of the pap-test;
- SIL II: moderate and high-grade lesions, CIN II or CIN III.
This classification only allows us to understand if it is cancer or not and, if we see it is a high-grade
lesion, eventually go on and see other types of parameters.
Precancerous conditions
A precancerous condition (precancerosis) is a tumoral lesion that can progress towards the
malignant form in a statistically established number of cases; Barrett’s oesophagus is a
precancerous lesion because in a statistical number of cases the patients can develop tumours.
It is important to understand that this is a statistical concept18: epidemiologic data told us that this
type of disease or changes in tissue morphology can lead to cancer development.
Also metaplasia, if prolonged for a long time so that it can become dysplasia, can be a precancerous
condition; for instance, smokers can develop a very aggressive pulmonary tumour called
microcitoma, squamous metaplasia in chronic cholecystitis can induce carcinomas (squamous or
spinocellular cancer), leucoplakia can develop into the same type of squamous cancer, uterine cervix
dysplasia can develop into different types of cancers: if they are related to the epithelium they are
squamous, if related also to fibrotic tissues they can become fibromas or fibrosarcomas, depending
on the type of cells involved, and finally Barrett’s oesophagus
can develop into an epithelial cancer.
Therefore, dysplasia like a phenomenon may be a
precancerosis, but also a disease like Barrett’s oesophagus
can be a precancerosis because it can undergo first
metaplasia then dysplasia and develop cancer.
Figure 10 There are different organs where the precancerous condition
can precede a malignant tumour, the main ones being: uterus,
oesophagus and stomach, the lung and the bronchial epithelium, liver and pancreas.
In some organs it is very difficult that a benign tumour can develop into a malignant tumour, while in
other tissues we can see a change in phenotype from benign to malignant, like for instance in the
colon-rectum, the breast, the bladder and the prostate. The colon-rectum is very important because
it is one of the human tumours that develop in a very long time e.g. intestinal dysplasia to become
cancer can also have latent times of 10 years; normally the different phases of the progression of a
cancer can be studied in experimental animals (simpler than in humans), but we can observe this
type of progression in humans in some types of cancers (like colorectal cancer) because the Δ (delta)
time from the presence of moderate dysplasia to the development of cancer is very long.
Neoplasia
Also called tumour although it is not absolutely equal (tumour means formation of a mass). It is the
third type of adaptation19 (the first one is metaplasia and the second one is dysplasia).
18
The teacher hinted that if asked what a precancerosis is at the exam, we should answer that it is a statistical concept,
not that it is a dysplasia
19
However, neoplasia is not mentioned in the scheme about cell adaptation on slide number 1 of the pdf
121
The name comes from the Latin “new formation” (neo=new). Indeed, neoplasia is characterised by
an autonomous growth without any purposes (afinalistico in Italian) which is also progressive
(either slow or very quick depending on the type of tumour).
Morphological aspects
Regarding the morphological aspects of the cells in the neoplasia, the important feature that
characterises the malignant phenotype of these cells is the dedifferentiation, an alteration in the
maturation of these cells. Normally, after the morphogenesis, we have specialised tissues with
differentiated and mature cells; when we have a tumour, these cells become dedifferentiated: they
appear to go back in their specialisation. The malignancy of the tumour depends on the grade of this
dedifferentiation; the features of the dedifferentiation are related to some aspects of the cells:
- Pleiomorphism (different dimensions, shapes and volumes of the cells);
- Abnormal nuclear morphology (e.g. a bigger nuclei);
- Number of mitosis the nucleus has;
- Loss of cell polarity (e.g. in uterine cervix dysplasia).
Therefore, in some tests like PAP-test, we can see cell-shape alterations and heterogenicity of
cell dimensions with increased or decreased volumes, all of which contribute to an extreme disorder
(since neoplasia can also be considered a severe dysplasia).
Concerning the nucleus and nucleoli, in the nucleus we can see increased nucleus/cytoplasm
ratio, increased number of nuclei (we could also see cells with 2 or 3 nuclei), increased number
of nucleoli and aneuploidy (variations in the number of chromosomes, which are reported
especially in some important lymphomas or leukaemias).
Functional aspects
Since cells are dedifferentiated, they came back to some kind of immature features, they can express
again some genes that are normally suppressed in the genome (if they were specialised for a type
of function some genes have been repressed). These very important genes are called tumour
markers: they induce the synthesis of specific proteins that normally are in low amount in our tissues
or completely absent; therefore, if we have an increase in this type of proteins in the tissues or in the
blood, it means that in that specific tissue it is true that there is a tumour. The re-expression of
foetal proteins, like α-fetoprotein in the liver (AFP), prostate specific antigen in the prostate (PSA)
and carcinoembryonic antigen in the colon (CEA), is important to see if there is a primary or
secondary tumour in our body. However, this type of proteins is normally expressed during
morphogenesis (i.e. during foetal and embryonic age), so if a lady comes and has high levels of α -
fetoprotein it is possible that she doesn’t have a tumour but is pregnant, because the foetus is
synthesising this type of antigen (same case for the carcinoembryonic antigen). PSA is re-expressed
in prostate cancer but, for example in the case of chronic inflammation in aged men, there can be
an increase in this marker that is not due to a tumour but to the chronic inflammation, like for instance
a prostatitis; however, if we see it in young or middle-aged people we have to think it is possible that
there is a tumour.
Tumour markers are very important not to diagnose a tumour but to follow up the development of
the cancer in a cancer patient after a surgical resection: if the tumour has been completely removed
and we assess the value of PSA in the blood, for instance, PSA must be 0 or very low; if, for instance,
during the follow up in chemotherapeutic interventions we see an increase, we have to think that the
therapy is not working well.
Q: Is anaplasia different from dedifferentiation or are they the same thing?
Anaplasia can be used as a synonym of dedifferentiation, although they are not exactly the same:
anaplasia refers to the morphological aspects, while dedifferentiation refers to the functional ones.
However, they are parallel because morphological aspects induce or go in parallel with the functional
aspects.
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In the functional aspects, it is also important to mention the synthesis of substances that are not
normally produced in the tissue where there is the tumour. In particular, this unusual synthesis is
related to malignant tumours that are for example in a tissue which is normally not able to synthesise
certain hormones. When they become malignant, they change their function and become able to
synthesise said hormones (ectopic secretion of hormones, i.e. the secretion of hormones in a
tissue that typically does not occur). For instance, in small cell lung carcinoma there is an ectopic
synthesis of ADH or ACTH, in kidney and hepatocellular cancer there is the production of parathyroid
hormone, in breast cancer of prostaglandins, in the pituitary gland tumour of gonadotropins. So, the
ectopic secretion of hormones is a feature of malignant cancers.
Atypia
Anaplasia involves a lot of types of morphological changes; a number of these changes that induce
differences from normal cells are called atypias.
Atypia can be defined as all the types of changes of tumour cells compared to normal cells, so
everything that differs from what is normal.
Atypia, like anaplasia, can correspond to morphological, chemical, metabolic, functional and
behavioural changes. The variations in atypia can depend on the degree of malignancy and the
degree of tumour progression (slow or speedy).
Malignant vs. Benign tumours
The criteria that distinguish benign tumours from malignant tumours can be related to morphological,
structural, functional and behavioural features.
In the benign tumours the growth is slow and is expansive (figure n.11), there
is an increase of the volume, and the benign tumour has no capacity to invade
the other tissue, it does not spread out of the tissue where it is formed (very
important). In the malignant tumour the growth is rapid (although it depends
on the type of tumour) and absolutely random, infiltrating (for instance in a
solid tumour, like a carcinoma, the next step after the in situ phase is the
spreading of the carcinoma, which goes out of the basement to reach other
districts of the body).
In benign tumours, there is a reaction named fibrous reaction which delimits Figure 11 the
tumour; it is an inflammatory reaction, which in this case is a defensive
reaction, that induces the formation of a kind of fibrous capsule that can delimit and confine the
tumour. In malignant tumours this type of reaction is very rare, scarce and not delimiting, so tumours
can spread.
Atypias are moderated in benign tumours while in malignant are present and different depending
on the stage of the tumour and on the malignancy.
Mitosis is normal in benign tumours, while frequent and atypical in malignant tumours.
The function of the tissue is preserved in benign tumours; for example, in secretory tumours like
mammary adenoma, a benign tumour of the breast, the cells continue to secrete because the
synthesis of the hormones is up regulated. On the contrary, the function is often not preserved in
malignant tumours, like for instance in the case of ectopic synthesis of hormones.
Relapse is rare in benign tumours while it is frequent in malignant tumours.
Metastases20 are completely absent in benign tumour while are typical of malignant tumours.
20
Secondary tumours that develop in other sites of the body than the primary malignant tumour
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Cachexia is very rare in benign tumours but is present and rapid in malignant tumour; it is a
symptomatology typical of tumour patients during the late phases of tumours which brings many
dysfunctions related to the tumour but also to the body. A high percentage of cancer patients die for
cachexia, not for the tumour itself.
Figure 12
NORMAL AND NEOPLASTIC CELL PROLIFERATION
Today we will discuss the importance of proliferation and changes in genes involved in proliferation.
These genes are the first effectors involved in tumorigenesis and carcinogenesis.
Differentiated cells are divided in different types of populations; each population of cells has a
specialized cell morphology and function, which changes according to the different specific tissue in
which the cells are located.
This type of specialization is induced by the expression or repression of specific genes involved in
the proliferative control. In normal conditions somatic cells express somatic genes, which allow
renewal if the tissue needs to be repaired. Somatic cells have the ability to replicate and this ability
can be due both to stem cells and non-stem cells. In particular non-stem cells are present in all
tissues and are only partially differentiated (precursor cells). They stay in the tissue and if ever the
tissue needs to be repaired, they can enter the cell cycle and induce proliferation.
Proliferation depends on two types of factors:

- Competence factors (especially growth factors): they are molecules that make cells
“competent” (able) to divide meaning that they prepare cells to the division. Of course, there
are different types of competence factors; we focus on the one related to tumor development:
EGF (Epithelial growth factor), NGF (Nerve growth factor), FGF (Fibroblast growth factor),
PDGF (Platelet-Derived growth factor) are particularly involved in the first step of activation.
These molecules enable cells to be competent to enter the division;
- Progression factors: in the second step, they are other factors that allow cells to progress
through the cell cycle and eventually divide (undergo mitosis). They stimulate proliferation of
competent cells so that cells can progress along the cell cycle. Some examples: insulin,
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insulin-like growth factor (IGF-1 also called somatomedin), hormones and estrogen in
particular (estrogen has a strong function in inducing proliferation), PGF2 and calcium.
What is the function of these two types of factors in cell proliferation? They induce the cell-signaling
pathway. This means that they induce the signal activation, the transduction of the signal and the
transcription of those genes responsible for inducing the synthesis of molecules involved in the
progression phase (through the cell cycle); in particular they allow cells to go from the G1 phase to
the S phase (G1-S transition).
The cell proliferation process is constituted by four steps:
1. Signal activation;
2. Transduction signal;
3. Gene transcription;
4. G1-phase to S-phase transition.
1. Surface receptors (external signaling)

There are three types of induction of cell signaling due to external signaling:
- GF: growth factors bind to a receptor that can be phosphorylated21. Upon phosphorylation
the receptor changes its structure in order to activate transduction molecules (in this case for
example they induce phosphorylation - and therefore activation - of transduction molecules
such as AMP, GMP or PI-4,5 phosphate that once phosphorylated act as transduction signal
coming into the nucleus thus activating transcription factors. GF receptors have an intrinsic
tyrosine kinase activity. Examples: EGF, FGF, PDGF.
- Cytokines: induce the activation of this particular receptor called JAK, which triggers the
signaling of the cytokine-dependent JAK-STAT pathway in order to activate TFs.
Cytokine do NOT have an intrinsic tyrosine kinase activity.
- Hormones: act trough G-protein receptor proteins. The G-protein activates PLC which
results in the production Inositol 3 phosphate and diacylglycerol. Diacylglycerol is responsible
for the function of protein kinase C (PKC) which is controlled by the levels of calcium in the
cytoplasm. This kinase is a transduction molecule that can activate transcription factors.
Remember that GPCR are characterized by 7 transmembrane domains. Examples:
adrenaline and glucagon
21
For example, in the JAK STAT pathway, the ligand brings closer the two domains on the kinases so that they can cross-
phosphorylate each other. Consequently, the receptor changes conformation and is able to link STATs and phosphorylate them.
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To sum up surface receptor are of three kinds: those with intrinsic tyrosine-kinase activity (GF
receptors), without intrinsic kinase activity (cytokines), G-protein related receptors (hormones).
2. Signal transduction
Signal transduction also depends on different molecules:
- MAP-kinases (mitogen activated kinases) type I;
- Inositol-1,4,5-triphosphate (IP3);
- cAMP and cGMP: linkage with receptors having tyrosine kinase activity or with G-proteins;
- JAK/STAT: linkage with receptors for cytokines with no intrinsic tyrosine kinase activity.
Some of these molecules act both as transduction and transcription molecules while others are only
transduction22 molecules.
3. Gene transcription
Gene transcription has two phases:
- Immediate (early) response: induction of production of TFs;
- Delayed (late) response: regulation of activity of TFs.
Thanks to these factors cells are able to progress towards the cell cycle. Cell cycle progression is
regulated at very important points of control (checkpoints):
- G0-G1 transition phase: mainly due to environmental factors (for example damages to a
tissue induce cells to re-enter the cell cycle in order to proliferate and regenerate the tissue);
- G1-S transition phase (also called start point): in this phase cells are competent because
they are actively involved in the production proteins fundamental for DNA duplication;
- G2-M transition phase: induces quantitative and qualitative changes in DNA and control of
DNA, in particular induces DNA division e.g. mitotic spindle assembly.
So, cell proliferation has different points of control which can be GFs, receptors, signal transduction
molecules and nuclear (transcriptional) factors.
22
She actually said “transcription” but since we are talking about transduction molecules it does not make sense.
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All these molecules are synthesized through the transcription

of their genes, which are either proto-oncogenes or anti-
oncogenes.
Oncogenes are genes related to the production of all the
molecules involved in all the different steps of the signaling
which induce proliferation. In normal cells these oncogenes
are defined as “proto-oncogenes” (= genes involved in
proliferation of normal cells).
If we think about proliferation and all the other actions that our
cells can perform, we can think about two
compartmentalizations: cells can either go into the cell cycle
(the progression into the cell cycle results in proliferation) or
go out of the cell cycle – in this case the final result will be
either differentiation or cell death. Consequently, we can define as proto-oncogenes all those
genes that induce the progression of the cells into the cell cycle, resulting in cell proliferation.
Conversely onco-suppressor genes (anti-oncogenes) induce cells to go towards the other “box”
(see picture below), out of the cell cycle towards differentiation (G0 phase) or, if the cell has some
DNA damage, towards cell death.
Therefore, proto-oncogenes and onco-suppressor genes are involved in the balance between cell
cycle progression and differentiation and cell death.
Alterations of the mechanisms of control of normal proliferation induce neoplastic proliferation.

Therefore, neoplastic proliferation can be due to:
- Activation of proto-oncogenes;
- Inactivation of onco-suppressor genes.
Oncogenes
Proto-oncogenes are normal genes related to induction of proliferation.
They are cell DNA sequences that can stimulate cell proliferation and are similar to viral genomic
sequences. Instead the term “oncogenes” is related to the alteration of proto-oncogenes in a
neoplastic way: the carcinogenicity of these genes is due to some mutational changes. Proto-
oncogenes and oncogenes were discovered in viral genomic sequences and they were called v-onc
(viral oncogenes). The researchers discovered that also in our cells there are similar genomic
sequences and they called them c-onc (cellular oncogenes).
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We can have different types of activation of proto-oncogenes responsible for the strong proliferation
typical of neoplastic phenomena.
- Class I oncogenes: related to the receptor.

o Non-receptor tyrosine kinase molecules: the most important three are src, abl,
fps/fes;
o Receptor tyrosine kinase molecules: (GFs are their ligand) they are genes which code
for EGF receptor (erb-B)23, PDGF receptor (kit), CSF receptor (fms), Insulin receptor
(ros), HGF receptor (met);
- Class II oncogenes: related to the ligands.
For example, sis in the case of PDGB β chain and ks3 for FGF;
- Class III oncogenes: related to G proteins.
In particular H-p21ras, K-p21ras and N-p21ras induced respectively by the genes H-ras, K-
ras and N-ras;
- Class IV oncogenes: related to nuclear proteins (TFs).
The two nuclear proteins related to G0-G1 transition are Fos and Jun. Together these two
molecules form the AP1 transcription factor. The other two factors involved in oncogenic
activity are myc and myb, which are responsible for the G1-S transition in the cell cycle.
Moreover, proto-oncogenes can become oncogenes through different type of mutations, mostly
insertion of a promoter, translocation and amplification. These types of mutation can be point
or large mutation. Another type of mutation is the deletion of tumor-suppressor genes; however,
this mutation is related just to onco-suppressor genes.
Proto-oncogene activation or anti-oncogene deletion are important especially in the early phases of
carcinogenesis because this strong induction of proliferation due to a mutation can induce some
cellular instability (DNA-wise) that in turn can induce further DNA mutations. The first mutations of
oncogenes (proto- or anti-) can explain only one step but in order to have the development of a
malignant tumor the cells need further additional mutations (this is a very important concept).
The process through which further DNA mutations induce development of a tumor mass is called
“tumor progression”. Tumors can develop after the first mutation (and this mutation always
involves the proliferation-related genes) but cells need other mutations in order to become very
insensitive to trauma, antioxidants, body defense mechanisms and to avoid apoptosis.
Here there are some examples related to the different classes of oncogenes.
23
In parenthesis you can find the corresponding genes.
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Translocation Bcr-Abl genes (class I)

Translocation 9-22 chromosome.
In the sequences of these chromosomes there
are some genes called abl (Abelson tyrosine
kinase) and bcr (breakpoint cluster region).
When there is a pathological translocation
between these two chromosomes there is the
formation of a chimeric gene. The protein that is
therefore encoded by this gene is a chimeric
protein which has a strong tyrosine kinase activity
that acts as an oncogene inducing a strong
proliferation of the cells. This type of mutation and
translocation is typical of leukemias and
lymphomas: in particular it was discovered in chronic myelocytic leukemia (CML) and is also
present in acute lymphoblastic leukemia. The chromosome congaing the fusion of these gene
sequences is called Philadelphia chromosome. This is very important for diagnostic purposes: in
diagnoses for CML we can divide the disease as Philadelphia + or – according to the presence of
this very short chromosome (during the translocation chromosome 9 becomes longer while 22 even
shorter containing bcr-abl genes named Philadelphia chromosome, look at the picture).
Ras oncogene
Another important oncogene is Ras, a transduction molecule activated by both JAK/STAT pathway
and receptors of growth factors. Different types of external signals from the environmental can induce
the activation and regulation of Ras. Ras normally can be in a quiescent form as Ras-GDP which
can be phosphorylated and transform the GDP into GTP (normal cycle). When GTP is
dephosphorylated to GDP, there is the availability in the cytoplasm of a phosphate which can be
used for the activation of other molecules. On the other hand, if GDP is transformed in GTP there is
the inactivation of these proteins because the phosphate is no longer available. [Ras molecules
normally can be found in two forms: a GTP bound form and a GDP bound form. Ras–GTP can
hydrolyze the GTP into GDP. The phosphate group is then freed in the cytoplasm where it can be
used for the phosphorylation (and activation) of other molecules. Then GDP has to be exchanged
for a GTP through a Ras-GEF].
The Ras pathway includes different effectors, some of which are considered very important
oncogenes, like PI3K and MEKK, which are another type of transduction molecule involved in cell
proliferation.
If the Ras gene undergoes a mutation, cells
are unable to control the phosphorylation of
effector proteins (unable to control the
enzymes). Uncontrolled Ras GTP activity
(gene mutation) is found in a number of
different tumors: colon cancer, pancreas
cancer, thyroid cancer (K-Ras mutation), or
in general hematological malignancies like
lymphomas and leukemias (N-Ras mutation,
the “N” stands for “nerve Ras mutation”).
This is a point-mutation.
Myc oncogene
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Myc is an important transcription factor (class IV oncogene) fundamental for the progression of the
cell to mitosis. Mutation of Myc molecules causes strong proliferation and cancer development. Myc
is regulated by other molecules, normally present in the cytoplasm: Max and Mad. The network that
regulates proliferation is dependent on the bond between Myc and max and Max and mad.
In normal conditions there is a controlled level of the dimer Max-Max in the cytoplasm. If instead Max
binds Mad, the cell activates pathways responsible for differentiation or apoptosis; this dimer also
acts as a transcriptional repressor for genes involved in proliferation. Conversely if Max is linked
to Myc there is a transcriptional activation of those genes. In conclusion Myc needs Max to be
activated and to induce proliferation; moreover, growth factors stimuli can induce activation of myc
and consequent proliferation through its link with Max.
Therefore c-Myc over activation can be due to any of these two:
- Overexpression of the Myc protein caused by gene amplification;
- Inactivation of the heterodimer Max-Mad which is responsible for the negative control of c-myc
(anti-oncogenic activity) caused by a deletion.
Another important mutation to take into account is the c-myc translocation, which is in many
aspects similar to the translocation responsible for the Philadelphia chromosome. This translocation
is related to Burkitt lymphoma t (8,14) and involves chromosome 8 and 14 (from chromosome 8 to
14). On chromosome 14 there are sequences related to enhancer of antibody heavy chain genes;
due to the translocation these sequences (fundamental for the building of antibodies) find themselves
close together with c-myc genes located on chromosome 8. c-myc can therefore induce proliferation
of lymphocytes, in particular B cells, causing the lymphoma.
Cell cycle control → cyclin-CDK complexes

Positive control of cell progression depends on the level of cyclins, which are substrates of the
Cyclin-dependent kinases (CDKs). High-level of cyclins in the cytoplasm result in the induction of
cell progression. Moreover, specific cyclins are able to act as substrate for specific kinase isoforms,
which control different stages of the progression through the cell cycle.
Cyclin A → CDK1 and 2 → S-G2 phase

Cyclin B → CDK1 →G2-M phase
Cyclin E → CDK2 →G1-S phase
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Cyclin D → CDK 4 and 224 →G1 phase
In this picture you can also notice that there are proteins (p21, p16, p27, p53, RB) that have a
regulatory activity in regard to cyclin-CDK complexes. Due to their inhibitory activity they are
considered onco-suppressors: they regulate the levels of cyclin-CDK complexes and can arrest the
progression of cell cycle at different phases: early G1, late G1, control point G1-S and control point
G2-M.
Positive regulation of cell-cycle progression is induced by cyclin-CDK complexes; those
proteins are therefore considered as oncogenes because their overexpression can induce an
over proliferation. In human cyclin D amplification is linked with breast cancer, esophagus cancer,
hepatocellular carcinoma and lymphomas; CDK4 gene amplification can instead be linked to
melanoma, sarcoma and glioblastoma.
Another important onco-suppressor is RB

(retinoblastoma protein). Normally is linked to another
factor E2F, a transcription factor involved in proliferation.
When they are linked, this factor is unable to the induce
transcription, therefore proliferation is inhibited (that’s
why it is an onco-suppressor). The regulation of RB-E2F
depends on the levels of cyclin-CDK complexes; high
levels of cyclins and CDK induce phosphorylation of
different molecules in the cell included RB (which normally is located in the nucleus).
Hyperphosphorylated RB is unable to bind E2F, that is finally free and able to reach the nucleus
24
At first she said CDK 4 and 2 like it is written on slide 25, but then she said CDK 4 and 6 like it is on slide 26.
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where it can act as a transcription factor and induce gene transcription: in particular it is responsible
for the G1-S transition phase.
Being an anti-oncogene RB mutations (deletions) result in uncontrolled proliferation. If the mutation
is induced during morphogenesis (germ-line mutation), it causes retinoblastoma, while if it is induced
in somatic cells, it results in different cancers: glioblastoma, breast, bladder and prostate tumors.
RB protein inactivation is also related to DNA virus transformation-related proteins (important for
proliferation), especially EA1 of Adenovirus, SV40 of large T antigen and E7 HPV.
It is very important to underline that to alter the function of onco-suppressor genes the deletion
of BOTH alleles is necessary. If only one allele is deleted the other allele is still enough to endure
the anti-oncogenic effect. This is a defense mechanism of the cells of our body against uncontrolled
proliferation. On the contrary mutation of only one allele is sufficient to alter the function of
proto-oncogenes. In fact, mutations of anti-oncogenes are called dominant25 mutations.
In the case of neoplastic proliferation, the alteration of RB protein activity can be due to:
- Mutation of RB (deletion);
- Amplification of cyclin-CDK complexes (oncogenes);
- Mutation of a tumor suppressor gene called p53 which in turn controls cyclins levels.
Onco-suppressor gene mutations
P53, p21, p16 and APC

P53 is a pro-apoptotic protein, which has the function to regulate the cell cycle (for example, when
a cell is becoming old) and regulate cellular division. P53 induces cell cycle arrest in G1 late phase
in order to allow DNA repair and it can induce apoptosis if the damage is not reversible. It activates
directly another onco-suppressor gene, which is p21, an inhibitor of CDKs.
P53 inactivation is important because it is present in many types of tumors. Besides, the mutation
of p53 induces the shift of cell behavior from benign to malignant; if p53 is deleted it is
IMPOSSIBLE to control the cell cycle progress. Furthermore, gene inactivation of p53 can result
in the inability to control targets of p53 like p21, GADD45 (a protein involved in mismatch DNA repair)
or Bax (a molecule with a pro-apoptotic activity).
Of course, p53 is not the only onco-suppressor gene involved in cell-cycle progression that can
undergo mutation. Onco-suppressor gene mutations involve:
- p21 family members: p21, p 27, p57 → bind and inhibit CDK2 and CDK4;
- p16 family members: p15, p16, p18, p19 → bind and inhibit CDK4 and CDK6;
- APC (Adenomatous Poliposis Coli).
25Do not confuse this concept of “dominant” with dominant Mendelian conditions. In this case, dominant means that if
the gene does not function there must be a double mutation (on both alleles).
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APC is a negative regulator that controls β-catenin, a protein involved in anchorage between cell-
adhesion molecules to the cytoskeleton; additionally, β-catenin also functions as transcription factor,
therefore its quantity has to be tightly controlled. APC controls the degradation of the excess of β-
catenin that does not work as anchorage26. Deletion of APC results in an excess of catenin, which
in turn causes the development of a familial polyposis in which the patients show hundreds of polyps
in the intestinal mucosa. This type of hereditary familial polyposis is considered a pre-cancerous
condition.
MicroRNA
Another type of molecules involved in the
development of tumors.
MiRNAs are a family of very small sequences of
noncoding RNA (up to 25 nucleotides) that control
post-transcriptional activities. They perform both
positive and negative control, so they act both as
oncogenes and anti-oncogenes; their activity
depends on the type of miRNA involved.
For example, if I have an oncogene, whose mRNA
activity is controlled by the (negative) action of a
certain miRNA, a decrease in miRNA activity will
result in more mRNA and therefore in an increase in the oncoprotein expression. On the other hand,
an increase of the viability of the same miRNA will result in the suppression of mRNA activity and a
decrease in the oncoprotein expression.
Nowadays numerous studies investigate the different levels of miRNA expression in cancer; in the
future we will be able to use these levels for tumor classification and diagnosis.
Survival pathway
Cell signaling molecules and consequently
cellular pathways are related to three main
processes: differentiation, survival and
proliferation.
Up to now we talked about the proliferation
pathway, while now we will deal with another
pathway, which is closely related to proliferation,
the survival pathway. This pathway is related to
the activity of P-AKT kinase.
Survival is not only important for the first steps of

cancer development but is fundamental for
cancer progression; thanks to the activation of this pathway cancer cells are able to overcome the
defenses of the organism.
P-AKT kinase is activated by a phosphorylation controlled by Ras (indirectly).
26 If cell adhesion is not present β-catenin acts as a transcription factor and induces proliferation. If cell adhesion is
present β-catenin is retained at the plasma membrane level (because if cell-cell adhesion is present, there is no space
for the cell to proliferate). APC makes sure that β-catenin is never present both at the plasma membrane and at the
nuclear level (if it is not clear, give a look at the histology sbobina from Giordano’s lesson on cell adhesion – paragraph
on Cadherins).
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Ras activation results in the activation of PI3K, which in turn is responsible for the phosphorylation
of P-AKT. This signal is negatively controlled by PTEN (an onco-suppressor gene) that controls the
dephosphorylation (inactivation) of P-AKT.
P-AKT activation results in the intensification of the survival signaling:

- Increase of anti-apoptotic molecules of the Bcl family (Bcl-2 Bcl-Xl);
- Block of apoptotic molecules of the Bcl family (Bad);
- Decrease of apoptosis due to blocking of caspase-9 (involved in intrinsic apoptosis, the one
that involves mitochondria);
- Blocking FRK, which is related to the decrease of Fas ligand dependent apoptosis;
- Increase IKK α protein, which activates NF-kB and E2F. NF-kB is a pro-inflammatory
transcription factor. E2F is a transcription factor involved in the progression through the cell
cycle27.
If PTEN is deleted, we have a continuous activation of survival pathway meaning that P-AKT is
always active.
Growth factors: TGF-β1

We have investigated the control related to transcription factor receptors, transduction molecules
and transcription molecules, while now we focus on the negative control performed by ligands,
especially growth factors.
Among the different growth factors (EGF, FGF, PDGF) we have to mention TGF-β1 (transforming
growth factor beta 1) because it is fundamental in many steps of tumor progression, not only in the
development but also in the latest phases.
TGF-β1 is involved in repair (it is a pro-fibrotic cytokine) induced after tissue damage; an excess of
TGF-β causes an excess of collagen deposition and results in fibrosis (pro-fibrogenic effect). It has
also a pro-apoptotic effect in specific cells and a chemotactic effect (for example, for
myofibroblasts). It mitotically activates fibroblasts, which synthesize collagen and other components
of ECM. The main functions of TGF-β1 are cell proliferation controller and fibrogenesis mediator.
It is produced by mesenchymal cells (platelets, macrophages, endothelial cells, T lymphocytes) and
it can act on both mesenchymal stem cells and epithelial cells. On parenchyma epithelial cells it
induces apoptosis (it is pro-differentiating and pro-apoptotic factor), while when the targets are
mesenchymal cells it induces proliferation and differentiation. This is important for epithelial
mesenchymal transition of tumor cells, a concept we will discuss in the next lectures.
The signal induced by this ligand is mediated by specific receptors (M1, M2, M3) and specific
transduction molecules, called SMADS.
These types of transduction proteins are
also able to act as transcription factors,
moving from the cytoplasm to the nucleus.
TGF-β is also pro-apoptotic and pro-
differentiating in parenchymal cells;
therefore, for these types of tissues, it can
be considered an onco-suppressor
gene. In fact, through SMADS activity it is
able to induce the transcription of p21,
p27 and p15 in order to increase the
induction of these other anti-oncogenes,
involved in the control of cell cycles. It is
27 it is the same one we have seen talking about RB

134
considered an anti-oncogene because it induces indirectly G1-S transition inhibition. TGF-β

signaling alteration is very frequent in different types of human cancers: mutations of the receptors
are found in breast cancer, colorectal cancer and gastric cancer, while mutations of transduction
molecules are found in lung cancer, colorectal cancer and pancreas cancer. It is present in 30% of
colorectal cancers (which is a very high percentage).
NEOPLASTIC PROLIFERATION
Monoclonal origin of a neoplasia
Yesterday we talked about proliferation and oncogenes or onco-suppressors genes and proto-
oncogenes that become oncogenes. Therefore, the neoplastic proliferation is centred, for its first
step, around a kind of mutation of the oncogenes. Neoplastic proliferation and tumor mass have two
different definitions because they are not synonyms. In relation to the origins of neoplasia there is
this unanimously considered hypothesis: the monoclonal origin of neoplasia. According to this
hypothesis, we consider as the first step of the formation of the neoplasia, the mutation of a cell
(which is therefore mutated) and presents one mutation regarding an oncogene with proliferative
activity. These mutated cells can divide into two, four, eight and so on.
After this first division we will have cells that present other mutations for
example another additional mutation apart from the first one. This
additional mutation is due to the DNA instability induced by the first
mutation (this is the second step). During these further mitosis, the other
cells which make up this small mass can accumulate different additional
mutations (step three). When proliferation of these mutated cells
continues, a group of cells that has similar mutations that can present
defence mechanisms against surrounding normal cells can grow and
can proliferate more compared to the other cells which can have only 2
or 3 mutations that are not so important. This group of cells is called
clone of mutated cells. The first cell which presents the first mutation of
a gene implicated in cell proliferation is called initiating cell, then there
are further divisions that induce the formation of a mass in which the cells
present important mutations able to induce proliferation independently
from the surrounding cells of the tissue. In summary we can have the first
mutation of the cell in which the cell gene is malfunctioning, this cell will
have an irregular phenotype. This malfunctioning can induce a selective
advantage for the mutated cell, related to proliferation. These cells can
proliferate in an uncontrolled way compared to normal cells. This uncontrolled way of proliferation
can cause the formation of clones of cells with the same features and that can live without the
different mechanisms of control proper of the surrounding cells.
Tumor stem cells
Metaplasia and dysplasia are two different concepts. Metaplasia is a change from one type of
specialized tissue to another one: two types of differentiated tissue. Severe dysplasia is a
synonym of neoplasia on the other hand, and it presents cells that go back to their origin
(undifferentiation) in terms of function and structure. In this case stem cells have an important role,
because the tumor mass is composed of heterogeneous types of cells with different types of
mutations. Most of these types of mutations are related to proliferative control, but there is a
population of cells that can support tumor growth, called tumor stem cells or tumor initiating
cells. Therefore, there are two types of stem cells: tumor stem cells and normal stem cells. In the
tumor mass we can find a small population of tumor stem cells and another of normal stem cells.
There is no technical definition to understand the difference between these two. However, there is a
135
functional definition: tumor stem cells can self-propagate (proliferate) particularly in vitro. We
tried to cultivate the stem cell and we noticed that they are able to self-propagate. This type of self-
propagation can induce, on one hand, the formation of more cells that present more mutations and
on the other, more differentiated cells.
Tumor and normal stem cells exists in two hierarchical subpopulations: a quiescent population and
a proliferating population. Normal stem cells are mainly quiescent, on the other hand cancer stem
cells are mainly proliferating. Tumour cells have been shown in different types of cancer (leukaemia,
melanoma, brain, pancreas and breast cancer and so on) and can have two origins:
1. Normal stem cells of mature tissues that have undergone alteration of the oncogenes;
2. Normal differentiated cells of mature tissues that accumulate different types of genetic
lesions and mutations and therefore they de-differentiate28.
The peculiar role in this phenomenon is given by p53 oncogenic effect. In a population of normal
stem cells, where most cells are quiescent, the cells undergo an asymmetric division. Asymmetric
because in these population they divide asymmetrically: one of the daughter becomes the mature,
specialized cell whereas the other continues to be immature/stem. Therefore, we end up with a
population of mature cells and a small population of cells that have remained immature,
unspecialized stem cells. These different points are controlled by three important genes: p53, c-Myc
and p21.
1. p53 is the gene that blocks the exaggerated induction/activation of c-Myc. It regulates the
path. When there is an asymmetric division it also regulates p21;
2. c-Myc induces the progression of the cell cycle;
3. p21 is an anti-oncogene or onco-suppressor gene in the cell cycle, is responsible for limiting
replication of these cells. After six or seven replications for instance, p21 blocks also this type
of division. p21 is dependent and controlled by p53.
In cancer stem cells, as any other stem cells we have an asymmetric division. Cancer stem cells
will divide, and this division will induce a larger population of cancer stem cells because p53 is
deleted, c-Myc induces and accelerates the progression into the cell cycle and p21 is now
independent from p53. So, p21 normal function would be to control the number of replications, and
induce replication when needed, but since there is a mutation of p53, p21 is uncontrolled and
therefore it continues to induce unlimited replication thus becoming oncogenic. In tumor cells we
have an unlimited and asymmetrical replication.
Figure n.1 shows the regulation of self-renewal in
normal and cancer stem cells. Normal SCs divide
mainly asymmetrically giving rise to SCs and
progenitor (P) cells. Their self-renewal potential is
intrinsically restricted, therefore, they functionally
exhaust once they reach the limit of six or seven
divisions. In normal SCs, p53-dependent
regulation of c-Myc imposes an asymmetric mode
of division and p21 maintains self-renewal. In
cancer stem cells (Csc), self-renewal capability is
profoundly de-regulated. Critical to tumor
expansion, loss of p53 results in a switch to the
symmetric mode of cell division, and upregulation
of p21 extends the self-renewal ability of CSCs.
Figure 3
28
Saying “de-differentiated” or “undifferentiated” is the same thing.
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The CSCs undergo an indefinite number of rounds of cell division, which ultimately, results in the
expansion of the stem cell pool.
Therapy
Considering that in the tumor mass there is this small population of cancer stem cells if we use a
normal standard therapy, which is chemotherapy, this will target something that is present both in
normal and tumoral stem cells. In this way cancer stem cells can escape form the chemical attack.
Since the treatment is not tailored to tumor stem cells, those cells can escape/survive and tumor can
relapse and continue to proliferate. If we target therapy to tumor stem cells in the first place, we can
kill this type of cells, which have this ability to be hyper-proliferating, and in this way the tumor can
be destroyed. Therefore, it is important to try to find a therapy which must possibly be targeted to
tumor stem cells and not generally stem
cells.
Cancer progression
The most important concept between those
mentioned before is cancer progression.
Cancer progression takes place from the
first mutated cell to the clinical evidence
of tumor mass.
What are the features of these cells? (This is a summary of the previous lessons). Essential cell
changes that make cells cancerous are:
1. Increase of specific mutations related to proliferation which lead to uncontrolled proliferation;
2. Escape from normal regulation of division and growth compared from the surrounding normal
cells and they can remain undetected from those. We have different types of control
mechanisms that defend the surrounding tissues from abnormally proliferating cells. Those
cells are able to escape these defence mechanisms;
3. Gaining proliferative autonomy;
4. Reduction or loss of differentiation. The cells become undifferentiated: they go back to their
origin. The hypothesis is that these tumor cells can function with different specializations of a
different tissue. They are able of have different functions very similar to those of the
original/precursor cells in which all the genes, but especially those regarding proliferation are
de-repressed (so genes regarding proliferation are activated), therefore, they can induce
activity of proteins, enzymes, hormones and so on;
5. Reduction or even loss of chance of death by apoptosis: escape from control for instance
of p53 which induces apoptosis.
The features of tumor malignant cells are many, different. They are:
1. Invasiveness of tumor cells;
2. Ability of invading surrounding tissue and spread into the body and metastasize.
Metastases are also considered secondary tumors which are similar to the first primary one,
but they grow in another site of the body;
3. Morphology differences. Differences in ratio between nuclei and cytoplasm;
4. High mitotic index;
5. Presence inside the tumor mass of necrosis: part of the tissue presents necrosis. This
regards the invasiveness of the tumor as well and we will talk about it in the next lesson.
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6. These cells can have different degrees of differentiation. Normally they are “very young”
cells, meaning that the degree of differentiation is very low, since they de-differentiate;
7. Chromosomal abnormalities and mutations, which are random depending on the kind of
mutations. These mutations are also to be considered for the evaluation of the degree of
malignancy of the cancer;
8. Gene expression abnormalities. The tumor markers come into play: the abnormal gene
expression can induce the re-expression of some genes that normally are not expressed in
the adult body, such as α-fetoprotein (AFP), carcinoembryonic antigen (CEA) and prostate
specific antigen (PSA).
We have evaluated these peculiarities of the cells microscopically in the laboratory, in order to
determine the stage of tumor development and progression. These features distinguish the first steps
of a tumor cell. The cell cultures were subjected to further analysis: the culture of neoplastic cells
was tested to evaluate their behavioural alterations compared to normal cells:
1. Immortality: ability of replicate indefinitely e.g. if we introduce nutrients and growth factors
in a tumor cell culture, the cells continue to grow/divide;
2. Loss of anchorage dependence;
3. Loss of contact inhibition;
4. Loss of substrate-dependant orientation;
5. Decreased need for growth factors to proliferate compared to normal cells.
N.B. There are types of differences in cell cultures that we have studied in the cellular biology course.
Contact inhibition video transcript:
When normal cells are introduced into a petri dish at low numbers they begin to divide and proliferate.
As the cells begin to touch one another they slow their rate of division: this behaviour is a
consequence of the process called contact inhibition. Once the cells fill up the bottom of the dish,
the rate of cell division slows further and is balanced by the rate of cell death such that the total cell
number remains constant: this state is called confluence. Contact inhibition ensures that the cells
create a layer only one cell thick, a mono layer. The behaviour of cancer cells is quite different: if a
cancer cell is seated among normal cells, all of the cells will proliferate as before. However, once
confluence is reached, the normal cells will regulate their growth, while the cancer cells continue to
divide in an unregulated manner yielding a clump of cells which is often called a focus. Contact
inhibition can be demonstrated in vitro by removing cells from a confluent monolayer. In this
experiment cells are removed by scratching the monolayer with a needle; the surviving cells at the
edge of the wound now do two things:
- they begin to proliferate more rapidly since they are no longer fully contact inhibited,
- they migrated into the empty area of the wound attempting to fill it up.
Therefore, there are different features related to the first phase of tumor development, mainly
microscopically, and then other steps are needed to have a clinical evidence of tumor mass.
Multistep process of tumor growth

Thanks to the studies and researches first on cell cultures and then on animals and human cancer
cells, we can consider the progression of the tumor from its first moment to the last divided in three
steps:
1. Initiation;
2. Promotion;
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3. Progression: it is a chemical progression. “Cancer progression” refers to the whole process

of evolution of the tumor, whereas the other definition is related to the third phase of the
cancer progression.
We can define the whole process composed of these three phases as tumor growth.
Initiation: often in this phase environmental factors, such as chemical compounds that are able to
induce cancer and are therefore called carcinogenic, can induce a permanent DNA damage, so a
mutation related to proliferation (monoclonal origin).
Promotion: is the second part in which these mutated cells can undergo different divisions and can
produce a clone of cells. There are other factors, environmental and chemical, that can induce this
selective clonal expansion of the initiated cells. These factors can be considered non-specific,
irritating factors.
The last step is the progression: step in which clonal mutated cells acquire additional mutations that
allow cells to become more aggressive and to invade and be invasive into other surrounding
tissue. The progression is defined as:
1. acquisition of other mutations;
2. increased aggressiveness;
3. metastatic phenotypes (invasion).
The cells in this stage present:

1. unstable karyotype;
2. further multiple mutations, which occur independently;
3. appearance of heterogeneous cellular sub-clones, which differ from each other due to the
different phenotypic characteristics and the presence of heterogeneous cellular mutations.
The sub-clones can differ from one another, which is an important aspect because one clone
can be more aggressive than another clone or can present higher invasiveness compared to
another sub-clone;
4. subsequent qualitative changes with progressive acquisition of new permanent
features that induce selective advantage for cancer cell survival, you remember that there
are also signalling pathways related to akt-dependant survival which is important in cells.
These characteristics induce selective advantage for the cell survival, independency, the
undifferentiation and increase the aggressiveness of the tumors.
What are the different features that during progression can induce this aggressiveness?
1. variation of number of chromosomes with increasing DNA content;
2. additional mutations with deletions, duplications, translocations of chromosomal fragments:
o activation of proto-oncogenes;
o loss of tumor suppressor genes;
3. increased grow rate: normally when cancer cells become more undifferentiated they are
able to increase the rate of division, so they accelerate the progress toward malignant
invasiveness;
4. independency from cell control: they manage to escape the control mechanisms;
moreover, they re-express other genes with other function and become independent from
hormones and effector of the normal control of proliferation, because for example there is a
loss of receptors or transduction molecules. TGF-β for example partakes into a signalling
pathway which in cancer cells is modified due to receptors deletions or SMAD deletion (small
mother against decapentaplegic are the transduction molecules of the signalling induced by
TGF-β1.
Antigenic function modifications:
o Loss of histocompatibility antigens;
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o Acquisition of new antigens;

o Acquisition of foetal antigens for example PSA, CDA, α-fetoprotein and so on;
5. Changes in metabolic features: cancer cells need a lot of energy, so they must change
their metabolism in order to retain as much energy as possible in order to proliferate;
6. Loss of drug sensitivity: different sub-clone populations present different features i.e. one
can be more aggressive than another one. This aggressiveness is important particularly in
tumor therapy: by administering chemotherapy we believe to kill all types of clones, but
among different clones with different features there can be one clone that doesn’t have
sensitivity to the chemotherapeutic drugs. That clone therefore can re-express and can
relapse even after chemotherapy.
7. Infiltrative capacity of adjacent normal tissue (capacity to infiltrate the normal surrounding
tissue);
8. Ability to develop distant tumor growth, so capacity to metastasize (induce metastases);
Step 7,8 and 9 are the three last steps of tumor progression, in which the tumor acquires the
maximum of the malignancy.
Kinetics of tumor growth

The growth rate and the progressive expansion of tumor mass are not associated: they depend on
the type of tumor. These two events depend on the number of cells present in the proliferative
compartment and also on the ratio between the tumor cells that are produced and the tumor cells
that die i.e. presence of necrosis in tumors. In fact, the tumor mass could present areas where there
is a lack of vascularization, where therefore the oxygenation is insufficient so that some tumor cells
in those areas undergo hypoxia and die. In the tumor mass there are some sites in which there is
necrosis. The period between the onset of neoplasia and the clinical evidence of tumor mass can
vary: it depends on the tumor, but generally is long. Although the period is generally considered long,
we are not really able to understand and individuate the time at which the first mutation happened;
we are only able to evaluate the clinical aspects of the tumor mass when we have evidence of said
tumor mass.
When clinical diagnosis of tumor occurs:
1. The tumor presents with an extremely heterogeneous cellular population because it is in
the last part of progression (it is old);
2. The tumor has already competed most if its life cycle. It is already well advanced and therefore
implies high malignancy.
The three phases of tumor development

Initiation:
o is not reversible
because it is due to the
first mutation;
o is immediate because
there is a mutation
which is immediate;
o is due to direct
modifications induced
on DNA by the
carcinogens, like
environmental factors
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or genetic factor for certain tumors;

o there is no close relationship between the dose and the effect of a carcinogen.
Promotion:
o can be reversible: for example, metaplasia can become dysplasia, particularly
moderate dysplasia and this happens in the promotion phase, but if we remove the
cause of the induction of promotion, the dysplasia can be reversible;
o the rate of this phase is slow and repetitive. These cells, in order to develop
malignant clones, need different/several periods of exposure to the carcinogenic
compound;
o Can be influenced or modulated by environmental factors;
o Sufficiently continuous administration and exposure to the agent. In this way the cells
are induced to continue to divide. These carcinogenic chemical compounds are called
promoting factors, because they promote proliferation and increase the number of
divisions.
Progression:
o Is irreversible, because the clones are at this point malignant;
o Is very slow, although this again depends on the type of tumor;
o It is autonomous because it is independent from any kind of control.
Epithelial cancer development

In this case normal cells (germinative layer) mutate,
becoming initiating cells that then form the clone. This
clone, as a morphological tissue feature, shows
hyperplastic lesions and anaplastic as well, where the
initiated cells begin to proliferate. These lesions grow
in hyperplastic nodules, some of which can be
clinically evaluated, can be seen. Hyperplastic
nodules can become either benign or malignant
tumors. In order to progress toward malignancy, they
require different other steps such as metaplasia with
severe dysplasia, then carcinoma in situ, so a
malignant tumor of epithelial cells (i.e. carcinoma) that
is not invasive (i.e. in situ) and then invasive
carcinoma. It is very difficult that benign tumors progress toward malignant tumors, but it can happen
for certain epithelial tumors e.g. polyps are benign epithelial tumors.
Development and progression of melanoma
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Melanoma is a very aggressive cancer of melanocytes.
Progression:
1. It starts with
mutated
cells from
junctional melanocytic hyperplasia;

2. Then becomes junctional nevus, in which these dysplastic cells are still in the epithelial
layer;
3. They acquire different atypias and this type of tissue alteration is called dysplastic nevus
because it presents cells that begin to divide without orientation;
4. They start to proliferate in a radial and tri-dimensional way. This is the first phase of a
primary melanoma;
5. In the last stage of advanced melanoma, cells present not only a radial but also an advanced
vertical growth and invade the dermal layer. Advanced melanoma is malignant and
aggressive because the metastases are mainly in the brain, because the embryonic origin of
melanocytes is at the level of the neural crest (which gives rise to brain cells as well). They
originate from that layer and follow a process named organotropism: some tumors
metastasize in particular tissue. In this case tumoral melanocytes go mainly in the NS and
particularly in the brain because of their origin (in the brain, neural crest).
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Experimental hepatocarcinogenesis in rats

Another example is the experimental carcinogenesis which can be currently performed in animals
such as mice or rats. Hepatocarcinogenesis, for instance, requires initiating factors which can be
chemicals like diethylnitrosamine. This chemical compound induces the rise of initiating cells.
These rats eat this promoting chemical factor which is 2-acetylaminofluorene that induces the
alteration of severe dysplasias in liver tissue/cells and induces formation of foci of altered
hepatocytes, dysplastic (or dysplasic) nodules and clonal expansion, which in turn induces
neoplastic nodules. The rate of this part of the progression process can initially be moderate but then
can accelerate leading toward the invasive phenotype.
In the bottom of the picture on the right, we can see specimen of the liver whose parts can be stained
in brown with an enzyme which is re-expressed
in the liver when there is a tumor. We stain the
liver with this enzyme and after four weeks of
treatment we can see that only foci are present.
After more weeks (8-12) the staining is present
in a larger area of the tissue and spreads all
over the tissue in about 54 weeks, so little over
a year. At this point the rat must be suppressed
because it presents an extremely aggressive
hepatocarcinoma. Still, in this way, we have the
chance to see what happens during the
different phases of tumor progression.
Tumor progression of colorectal
carcinoma in humans
Figure n.2 is related to human
tumor (not experimental ones)
and depicts the progressive
cancer process of colorectal
carcinoma which is important for
our studies because of its very
large life span: some colorectal
carcinomas can have and display
aggressiveness even after 10
years from the first diagnosis.
Figure 4 The morphological changes and

steps that induce malignant and
invasive colorectal carcinoma, starting from normal epithelium, are:
1. Moderate hyperplasia;
2. Low degree of dysplasia, also called early adenoma, which is a benign tumor;
3. Moderated degree of dysplasia, called intermediate adenoma;
4. high degree of dysplasia, called advanced adenoma. This is the starting point of the
carcinoma because it is a very severe dysplasia. More than 60% of tumors in this stage have
a mutated p53: deletions and loss of p53 accelerates the changes that lead to the
development of carcinoma in situ;
5. Carcinoma in situ;
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6. Metastases. The carcinoma in situ becomes then even more invasive and metastasizes.
Same identical steps are related to important mutations:

1. Mutation of APC. This mutation is found in hereditary colorectal cancer29 and if it is present,
like in familial adenomatous polyposis, the normal epithelium becomes hyperplastic;
2. This hyperplastic epithelium induces a low grade of hyperplasia and early adenoma. Early
adenoma corresponds to polyps. The patient at this stage has hundreds of polyps inside his
colon;
3. The passage from early adenoma to intermediate adenoma involves a Ras mutation (proto-
oncogene becomes oncogene). This alteration induces intermediate adenoma;
4. Deletion of TGF-β signaling pathway, such as smad4 deletion or receptor RII deletion, can
then induce advanced adenoma. In this phase, between the intermediate and the advanced
adenoma, there is another important oncogene suppressor, DCC (deleted in colon cancer)
that has anti-oncogene function. This anti-oncogene DCC is deleted in the sporadic colon
cancer for example. Remember that APC is deleted in hereditary colon cancer whereas DCC
can be deleted in the sporadic form. At this step the rate of progression is very fast and the
acceleration is due to the deficiency in the defective mismatch repair that induces additional
mutation, a DNA instability;
5. The deletion of p53 is the main turning point that changes the advanced adenoma into
carcinoma in situ;
6. Mutations of adhesion molecules leads to metastatization such as cadherins: if they
present mutations, tumor cells are independent from mechanisms of control and blockage
given by the other cells of the tissue and therefore they can move into the mesenchymal
tissue, they can move across the basal membrane and give rise to metastases.
Figure n.3 is a reminder which tells us that different tumors have different progression stages and
timelines. The normal progression of epithelial tissue is: normal, initiated cell, mild dysplasia,
Figure 5
29
Colorectal cancer could be either hereditary or sporadic.
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moderate dysplasia, severe dysplasia, carcinoma in situ and invasive cancer.
THERMOREGULATION
Today’s lesson is about hyperthermia, going through situations in which the body temperature is
increased. However, in general, it’s about thermoregulation.
All animals are divided into two groups:

1. Hemeoterme animals: this category includes mammals and birds. This type of animals is
able to maintain the body temperature at a constant temperature, around 36-38°C. In fact,
humans, for example, are able to maintain their body temperature independently from the
temperature of the environment;
2. Poichiloterme animals: this category includes lower animals, reptiles, fishes, amphibians.
This type of animals is not able to maintain a constant body temperature; indeed, their
temperature is similar to the one of the environment e.g. if the environmental temperature is
cold, the body will be cold as well.
Thermoregulation is the body’s ability to maintain a constant body temperature despite of the
variations in the temperature of the environment, this ability of our body is due to an equilibrium
between the amount of heat produced (thermogenesis) and the amount of heat dispersed
(thermodispersion).
Thermoregulation (thermogenesis + thermodispersion) is under the control of thermoregulatory
centres located in the brain, in the CNS. The hypothalamus, and in particular the preoptic region,
plays a central role in the thermoregulation. Together with the hypothalamus that controls the body
temperature, other structures are involved: the inferior tract of encephalic trunk, the reticular
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formation, the spinal cord, the sympathetic ganglia and the peripheral nerve ends. In particular,
neurons present in the preoptic anterior and posterior hypothalamus receive two types of signals:
1. The first one derives from the peripheral nerve ends which transmit the information to the
hypothalamus. Thanks to thermoregulatory receptors and/or peripheral nerve ends, the
hypothalamus can receive information on whether it’s hot or cold from warmth or cold
receptors present in the skin;
2. The second type of signal derives from the blood temperature.
All these signals are then integrated by the hypothalamus (by the thermoregulatory centre) and
elaborated in order to start the control of the temperature. For example, if the presence of cold is
sensed by the hypothalamus, it will try to activate the mechanisms to produce heat i.e. to increase
the body temperature. Whereas if heat is sensed, the hypothalamus will activate mechanisms in
order to lose heat and decrease the body temperature.
Concerning the thermoregulatory mechanisms of mammals, we can distinguish:
1. A peripheral and a central temperature sensor: sensors able to detect information deriving
from the periphery (thanks to nerve ends) and from the circulating blood, this information will
later reach the hypothalamus. In particular, warm neurons can be activated for
thermodispersion;
2. Preoptic thermoregulatory nuclei: they are present in the preoptic anterior hypothalamus.
In this region, humans receive all the information, in fact, it’s where the set point of our
temperature is located. The body temperature is maintained constant thanks to the presence
of this set point in the hypothalamus. Neurons present in the hypothalamus know that our
body temperature must be around 36-37°C, thus are adjusted to maintain the T in this range.
Thus, in this area, there are:
- Warm neurons for thermodispersion;
- Cold neurons for thermogenesis. They can forecast that the body temperature is going
to decrease because they can sense that it’s cold outside and thus, they will activate the
mechanisms to produce heat: thermogenesis, in order to increase the body temperature;
- Neurons I or insensitive to temperature: they have only integrative functions, they
don’t sense if it’s cold or warm outside.
3. The third mechanism is formed by a series of effectors and peripheral and central
reactions, which also act to increase (neo thermogenesis) or decrease the body temperature
(thermodispersion).
Body temperature regulation

Different ways to regulate and maintain our body temperature depending on that of the environment,
either by increasing or decreasing it:
1. Thermogenesis is used to produce heat: chemical event which is based on redox reactions
that occur at the level of the mitochondrial respiratory chain. Substances mainly involved in
this process are lipids, glucides and ethyl alcohol, therefore their metabolism is increased.
From 1 gram of lipids we obtain 9 calories, from 1 gram of glucides we obtain 4 calories, from
1 gram of ethyl alcohol we obtain 7 calories. It is important to understand that what we eat
(carbohydrates, proteins, lipids) is necessary in order to produce heat in our body. Moreover,
the heat is also produced by muscles contraction which can be voluntary or involuntary e.g.
shivering is a form of involuntary muscle contraction to produce heat when we are cold. All
these events are under the control of the brain. In particular, in this case, the increase of
muscle tone/contraction and shivering are under the control of the posterior hypothalamus.
Regarding thermogenesis, some glands are important too:
- Thyroid has an important role thanks to the release of hormones such as thyroxine and
thyroiodin;
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- Adrenal glands release glucocorticoids such as cortisol, cortisone, adrenalin;

- Hypophysis is important for the release of adrenocorticropin, somatotropin and
thyrotropin hormones.
2. Thermodispersion is used to lose heat, thus reduce our body temperature. It occurs through
some physical events such as:
- Irradiation: heat transfer to a colder solid object by infrared rays;
- Convection: heat is transferred from the body surface into the surrounding air, especially
it can occur when there is an air current;
- Conduction: heat from one solid object to another without a direct contact;
- Evaporation: elimination of water as water vapour by breathing, perspiratio insensibilis
of the skin and the mucous membranes;
- Sweating: it’s the most effective system to lose heat from our body and it occurs when
environmental temperature is quite high, above 30-35°C. This mechanism works very
well if there’s not a lot of humidity in the air e.g. in the summer it is hot but most of the
time also humid, it is not ideal. In fact, humidity stops the elimination of water through
sweat, thus, the body is not able to eliminate the heat accumulated inside the body.
Evaporation and sweating are the best ways through which lose heat.
Normal values of body temperature in human

Humans produce more heat than it is necessary to maintain the core body temperature in the range
of 36.5-37.5°C.
- Body temperature can change if we measure it from the surface of the body or from
internal areas of the body, in fact, every temperature is around 36.8°C but can vary during
the day. Usually, in the morning and in the night T it’s lower than during the day, it can
increase during the day and has its maximum in the afternoon.
o Tympanic T is lower than the oral one;
o Rectum T is higher than the oral T;
- In deep tissues, T is higher than the oral one, it’s around 37.5°C;
- On the surface, the temperature can change: there can be a variation depending on the
possibility of heat loss. For example, we can measure the T in the groin or the axilla and
it will be different; the temperature at the extremities is lower than that of the other parts;
- The internal temperature differs in response to activity, to environmental temperature, to
fluctuation (of the circadian rhythm) etc;
- Also, physiological variations of the body temperature need to be kept in mind: for
example, after muscle exercise or after sport, the body temperature can rise by 2-3°C; it
can increase during digestion, or in women depending on menstrual cycle phase etc. too.
Thermoregulation was defined as the capacity of our body to keep our its T at the range 36.5-37.5°C.
When the temperature rises above 37°C we can talk about hyperthermia.
Hyperthermia
Hyperthermia can be of two different types:
1. Without fever: body temperature can increase for excessive heat production (increased
thermogenesis) or can increase because thermodispersion mechanisms are altered, thus,
the body is not able to lose heat. It can have different causes:
- Exogenous causes: sun stroke or insulation and heat stroke;
- Endogenous causes: increase of body temperature following muscular effort, activity,
or for endocrine diseases. In fact, it was mentioned before that some glands are important
in thermogenesis, they play a fundamental role in producing heat. For example, in
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patients with hypercorticosteroidism or hyperthyroidism, the thyroid and the adrenal gland
produce too many hormones, thus there’s an increment of basal metabolism leading to a
heat increase. The increase of the body temperature can occur also for a rare autosomal
disease called malignant hyperthermia: patients are characterised by increased
muscular contraction that, as mentioned before, causes a heat increase;
2. With fever: it occurs for the presence of fever, it is the “normal” situation in any flu and of
many types of diseases or infection and so on. In this case, the body temperature increases
due to the action of substances called pyrogens. They can be of endogenous or exogenous
origins and they can act directly or indirectly on the thermoregulatory centre.
Hyperthermia without fever

In our preoptic region, at the level of the hypothalamus, there is the set point of our body temperature
where neurons are set to maintain the T around 36-37°C. In the hyperthermia without fever, the set
point of the body temperature remains unchanged. The temperature increase above 37°C is due to
excessive production of heat or to a reduced thermal dispersion. Among the causes (exogenous
ones) there is sun stroke and heat stroke. In this case, the body temperature increases because the
organism is exposed for a long time to a certain high temperature. Sometimes, the pathological
manifestations can be serious and/or fatal.
Sun stroke or insulation

Causes: people stayed for a long time under the sunlight. There are some categories of humans
which are more sensitive to the sunshine, for example, children and old people compared to others.
They need to cover their head properly in order to prevent sun stroke: they have a bareheaded.
Insulation can occur especially in tropical region.
Symptoms: increase in body temperature, headache, physical alterations e.g. these people can
have also pathological manifestations, for example, delirium, insulation and coma.
Effects: one important part of the body that can be interested by insulation is the head. It can suffer
for meningeal inflammation, cerebral coma, cerebral oedema. Insulation can also lead to death,
since, as we have said, it can be quite severe
Therapy: it’s necessary to cull down the body temperature, to refresh the subject and to prevent the
insulation it is important, especially for more exposed people, to cover the head with insulating
materials.
Heat stroke
Causes: excessive exposure to high environmental temperature associated with high degree of
humidity. In fact, when humidity is high in the air, it can prevent heat loss (=dispersion of heat from
our body). Body temperature can also increase following muscles efforts, producing increase in basal
metabolism: this is called heat stroke at rest. In this case, there’s no movement nor use our muscles
nor perform any activity but the basal metabolism is increased thanks to hormones released by
different glands.
There are different types of heat stroke:
1. Tropical heat stroke: affected subjects are most likely people who live in tropical regions
where temperatures are high (sometimes above 40°C) and where often there is high
humidity. For this people the risk can increase with muscular effort which cause increase in
thermogenesis.
Symptoms: increase in body T (up to 44°C), feeling of nausea, vomiting, delirium,
convulsion, loss of consciousness, dizziness, coma and death. Patients lose a lot of water by
sweating so they can die for a hypovolemic shock, which is a reduction of blood volume, or
because of renal failure.
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Effects: alteration of hydro-saline balance due to the loss of liquids and electrolytes because
of excessive sweating; for the same reason, blood volume decreases while blood viscosity
increases leading to hypovolemia which is one of the major cause of drop in blood pressure,
so these people have both hypovolemia and hypotension, in fact, patients can die because
of hypovolemic shock due to the great drop in blood pressure. If in the environment there is
a high humidity, this prevents the release of heat from the body and the situation can get
worst. There’s also an alteration in equilibrium of acid-base balance, but especially there’s
severe loss of potassium.
Therapy: it’s important to decrease body temperature immediately, but not in a rapid way,
indeed, this may cause a peripheral vasoconstriction making it not be possible to reduce the
heat in internal organs. Then, it’s also important to do a rehydration or administration of
potassium. In order to avoid heat stroke, it’s important to get used to the temperature of the
area in which someone is going. For the first days, it’s important not to make much muscular
effort, that may cause body temperature increase. During this period, which is called
acclimation, also our kidney may get used to the temperature: adaptation by retaining more
sodium chloride, consequently patients have to drink more and the water in our body should
increase;
2. Common heat stroke: symptoms, effects and therapy are similar to the previous one, but
the situation is less severe. It interests especially people who spend a lot of time in a closed
poorly ventilated room and especially where humidity is high e.g. people who are working in
a laundry, boiler rooms and so on.
Subjects who are more exposed are:
o Old people because they have less capacity to lose heat from the body, less capacity to
sweat. They have structural and functions changes of the skin and, since they are old,
they are more likely to have different diseases and alterations at the thermoregulatory
centres;
o Children are also very exposed because they have lesser capacity for sweating, a high
body/body mass ratio and a greater production of metabolic heat during physical activity.
Mothers have the habit to cover children a lot, without knowing that children produce more
heat when they move compared to us, thus, it’s not useful to cover them too much;
o Subjects in which mechanisms of thermodispersion are altered (increase body T even
only for basal metabolic increase, heat stroke at rest);
o Subjects with severe heart failure;
o Subjects with ectodermic dysplasia because they cannot sweat;
o Subjects under pharmacological treatment that can interfere with sweating;
o Subjects who use drugs e.g. amphetamines, cocaine because that can increase the body
T. In this last case, we talk about heat stroke caused by drugs;
3. Heat exhaustion: subjects more exposed are elderly people during summer, when there’s
humidity. In these people we can find an increase in body temperature even only for basal
metabolic increase, thus, we talk about heat stroke at rest. However, in this case also young
people who make a lot of muscles efforts during summer may be affected: in this case we
talk about heat stroke during exercise.
Symptoms: hypotension, cardio circulatory failure, headache, violent sweating etc.
Therapy: interruption of physical activity. Example: sometimes during summer, people use
their bike when there’s 40°C at one o’ clock, they are being stupid because it would be better
for them to do sports in the morning in order to prevent heat stroke not in the hottest period
of the day!
Rehydration is important too: patients may drink a lot in order to recover all the water that
they have lost sweating.
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Hyperthermia with fever

Hyperthermia with fever is so-called because it is characterized by fever. It’s important to remember
that fever is NOT a disease: it’s a reactive healing reaction of the body against either an infection or
another real disease. In this case, the body temperature set point is changed while in the case of
heat and sun stroke, the body T set point is unchanged, and the body T maintains the normal set
point: T increases because people stay for a long period in a very hot place. On the other hand, in
case of hyperthermia with fever, we have fever even when the environment is cold: it is our set point
which is changed, our regulatory centre is set at a higher point, not at 36-37°C but at 38-39°C. This
change is however temporary, indeed, when there’s the recovery from the fever the set point returns
in a normal condition.
Fever is a systemic sign of inflammation. In fact, it occurs in every disease characterized by
inflammation, starting from a simple flu, a tumour or an autoimmune disease. It’s characterized by
decreased thermodispersion, because of which people are not able to lose heat from their body.
There is an adaptation of the circulation and an increase in neothermogenesis, thus, there’s an
increase in body heat. In this condition, the affected subject is therefore not able to lose heat, but
rather keeps producing it. For this reason, there is a resetting of the hypothalamic thermoregulatory
centres, but it is a reversible functional alteration of neurons in the hypothalamus that is temporary.
The fever is caused by release or presence of pyrogens which can be either:
- Exogenous: formed outside the body e.g. endotoxins and other bacterial constituents,
heterologous protein especially of milk etc.;
- Endogenous: produced by inflammatory cells, especially by monocytes-macrophagic
system and leukocytes. These monocytes can release endogenous pyrogens which are
mainly cytokines, and other inflammatory mediators such as prostaglandins E2. The
release of these endogenous pyrogens occurs thanks to different stimuli e.g. exogenous
pyrogens can stimulate inflammatory cells to release cytokines but also complement
components, bile acids, lymphocyte products etc. The release of these endogenous
pyrogens can also occur following endogenous damage such as necrosis, haemorrhage,
any kind of inflammatory process, tumours and autoimmune damage. Following these
stimuli, cells (monocytes, macrophages, neutrophils, lymphocytes, endothelial cells, etc.)
can release cytokines which can act through two types of mechanisms:
o Apocrine mechanisms: they amplify the release of themselves i.e. stimulate
inflammatory cells to release more cytokines;
o Paracrine mechanisms: they act on target cells e.g. on hepatocytes, stimulating
them to release acute phase proteins (sign of inflammatory process).
The main endogenous pyrogens are:
- Cytokines: especially many interleukins 1,2,6,8;
- Interferon;
- Tumour necrosis factors;
- Prostaglandin E2: it has a key role in this type of hyperthermia.
General mechanisms of fever

We can talk about fever when the body temperature increases of 2-3°C. It can be due to two principle
mechanisms:
1. In the early phase, in the body, there is a thermoconservation. Thus, the heat is preserved,
and it cannot be released by the body, which is able to prevent this release thanks to
superficial vasoconstrictions which as a consequence, causes a vasodilation of internal
organs;
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2. In the second phase, there is a neothermogenesis: production of heat thanks to the

increased basal metabolism. Body temperature is regulated by thermoregulatory centres
that, in this case, are activated by endogenous pyrogens that are released during any kind
of inflammatory process, including the flu.
Some of the cytokines are able to cross the blood brain barrier at the preoptic hypothalamus at the
level of the organum vasculosum of the lamina terminalis because in this area the endothelium
is fenestrated, thus there is a higher permeability. Thus, cytokines can cross the barrier, reach
directly the hypothalamus and to act directly on the thermoregulatory centre.
However, most of the cytokines are not able to cross the blood brain barrier and to act directly on
the thermoregulatory centre, but they need another molecule as intermediate: prostaglandin E2
(PGE2). Inflammatory cells release these endogenous cytokines. Pyrogenic cytokines can be
released by epithelia, monocyte-macrophages, endothelial, neuronal and glia cells. Some of them
are able to cross the blood brain barrier and act directly on the thermoregulatory centres in the
hypothalamus which is the principal thermoregulatory centre able to regulate the body T. other
cytokines cannot cross the barrier, so they stimulate the nervous cells (in the CNS, endothelial cells,
neurons, glial cells) to release prostaglandin E2. At the same time, endothelial cells are able to
release other cytokines such as interleukins 1, after stimulation by the already released cytokines,
in order to stimulate PGE2 production.
Even if there are four receptors for PGE2, the one involved in this mechanism is the receptor EP3.
After the interaction of PGE2
with this receptor, there’s an
increase of cyclic adenosine
monophosphate (cAMP), which
acts as a neurotransmitter. In
this way, indirectly (using
PGE2), these cytokines can act
on the hypothalamus. The
result of the action of cytokines,
both direct and indirect, is the
raising of thermal sensitivity
threshold, thus, the temporary
increase of the set point.
To sum up: during the early phase there’s thermoconservation, the preservation of the heat. In the
second part there’s neothermogenesis, the production of heat. So, at the beginning we keep the
heat inside our body and we produce new one. After the release of cytokines, there’s the production
of PGE2 which activates thermoregulatory neurons that leads to the increase of the set point. Other
signals can reach the vasolocal centre and the glands: they start from the activated pituitary gland
or from the neurohypophysis. These are sent for the redistribution of the blood circulation in the body:
when there’s thermoconservation, peripheral vasoconstriction occurs meaning that the blood is shift
from the superficial circulation to the deep circulation. Later, other signals can activate the
adenohypophysis for neothermogenesis: new heat is produced thanks to the release of the
hormones such as T3 and T4 (thyroid), but also other secerned by adrenal glands and hypophysis
whose aim is the increase of basal metabolism (important to produce heat).
Figure 1
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To better understand, look at figure n.1. At the beginning, we can

say that we have some thermosensory afferents, such as
peripheral and central temperature sensors, peripheral nerve
ends and thermoreceptors present in the skin, which are able to
recognize if the environmental temperature is cold or hot. This
information is sent from these sensors to the brain as signals that
activate the thermoregulatory centres. At the same time, the
thyroid gland is also activated to produce and release hormones
important for basal metabolism; the heat can be produced by
contraction of muscles etc. too. Then, we have some effector
organs: in fact, the heat can be released also through sweating
by sweat gland which is accompanied by superficial vasodilation.
If you want to summarize what has been said until now, look at
figure n.2 on the next page, there is another scheme. Signals
from the skin and blood temperature reach the brain thus
activating the hypothalamus. As a consequence, the activation of some glands will follow, thus
producing and releasing hormones which are involved in the increase of the basal metabolism. On
the other hand, the heat can also increase thanks to muscle contraction and shivering; but we can
lose heat thanks to superficial vasodilation and activation of sweat glands i.e. through sweating.
Figure 2
The course
of fever
The course of
fever has three
main phases:
1. Thermal increase phase or prodromal phase: there is the increase of thermogenesis and
drastic reduction of thermodispersion, thus, heat is kept, stored (thermoconservation) and
not released. During this first phase, before having the “real” fever, a cutaneous (superficial)
vasoconstriction occurs due to thermoconservation in order to preserve the heat which can
be released only when there is vasodilation. Vasoconstriction starts at the level of the skin,
patients appear pale, they have cold, they shiver, blood pressure and heart rate increase. It
is known that there are 8 pulsations/min for each additional degree of fever. Here the body
temperature starts to increase;
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2. Fastigium or stationary phase: it’s the phase during which the body temperature is stable
at high levels, above 37°C, for a few days because there is a balance between
neothermogenesis and thermodispersion. The subject doesn’t feel cold anymore (in the first
phase), he feels hot.
There are different variations of fever:
- Low fever: if body temperature increases by only one degree;
- Medium;
- High;
- Very high fever or hyperpyrexia: if body temperature reaches 41°C.
Values of fever can change during the day depending on the type of infection or disease
that stimulates the appearance of the fever. For example, after an infection due to
Escherichia coli e.g. cystatis, fever may appear only for few hours. In case of bacterial
pneumonia or viral influence, fever can be stable for three or five days. In case of tumours,
fever can stay stable for weeks or years;
3. Defervescence phase: temperature starts decreasing, there’s a reduction of thermogenesis
and a marked increase in thermodispersion. The loss of heat occurs thanks to superficial
vasodilation that favours the sweating.
It’s important to know the development/course of the fever because it depends on the disease that
first induced it. Thus, it’s important to know if, for example, the first phase appears immediately, or if
the second phase is stable or not and if the fever changes during the day, in a 24-hours range. For
that reason, sometimes, it’s not good to start immediately the therapy, it can only be useful for
specific categories of individuals e.g. children or old people and for the ones who have other
diseases, while for others (young adults) is better to wait and see the evolvement of the fever to
detect what caused it in the first place so do not take aspirins, antibiotics and so on right away
because it will make it impossible for you to detect the disease behind the fever which, we must
remember, is only a physiological protective reaction of our body against the pathology.
Principle types of fever

1. Continuous fever: it’s characterised by a rise in
temperature. In few days, fever reaches high
temperature, around 40-41°C, in the thermal
increase phase. During fastigium fever remains
constant at high level and during defervescence
phase it starts to decrease. It’s typical of bacterial
pneumonia and salmonella typhi.
2. Remitting fever: it’s totally different from the first

one. During fastigium there are daily oscillations of more
than 1°C: in the morning there could be no fever then
high fever, then nothing again and so on. It’s typical of
septicaemia, bacterial endocarditis, all the bacterial
infections.
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3. Continuous-remitting fever: between first and second type.

4. Intermittent fever: it’s characterised by period of
fever and period without it.
- Daily intermittent fever: there’s a thermal
increase everyday, with apyrexia in the morning
and fever in the afternoon. It’s typical of
tuberculosis.
- Tertian intermittent fever: one day of fever, the

second without it (apyrexia) and the third with it.
Typical of malaria from Plasmodium vivax and
oval.
- Quartan intermittent fever: one day is

characterized by the presence of fever, two by its absence and the fourth by its presence
again. it’s typical of malaria from Plasmodium malariae and falciparum.
Again, it’s important to know the course of each fever in order to guess which the cause of the fever
is.
5. Odd fever: fever periods with daily variations, few days
with it and few without. It’s typical of tumours,
lymphomas and so on.
6. Recurrent or periodic fever: there are days

characterized by the presence of fever and days by its
absence. The difference with odd fever is the transition
between the two periods: in the first case transition is
gradual soft, in the second it’s drastic. It’s typical of
infections transmitted by mites and lice, some
treponemes.
Fever treatment
We mentioned before that fever is not a disease but a reaction
of the body, defence response against any kind of infection. The therapeutic strategies are not useful
except for specific people e.g. children, old people, because in case of healthy people it is better to
wait for the treatment in order to detect the underlying cause of the fever. There are different
antipyretic drugs:
- Cyclooxygenase inhibitors: most of anti-inflammatory drugs, called non-steroids anti-
inflammatory drugs, can induce the blockage of cyclooxygenase thus preventing the
production of prostaglandins (PGE2, necessary for T increase). Aspirin and acetyl-
salicylic acid are cyclooxygenase inhibitors too;
- Glucocorticoids: synthetic steroid hormones.
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Metabolic alterations
- Basal metabolism: of course, in any type of fever that appears following any kind of
disease, I can have alterations concerning the basal metabolism which will increase,
condition necessary for the production of heat, it is important for the neothermogenesis;
- Glucidic metabolism: increased hepatic and muscle glycogenolysis, as a consequence,
there will be glucose increase in the blood (hyperglycaemia);
- Lipids metabolism: mobilization of lipids from their deposits (adipose tissue) which can
trigger metabolic acidosis because as a consequence there is a high production of ketone
bodies;
- Increase in protein metabolism;
- Hydro-saline metabolism can be altered because of the sweating: sweat is used to
eliminate heat during defervescence phase.
Systemic effects of fever

Fever is not a disease/pathology, but high fever can cause effects on other organs and other parts
of the body:
- Cardio-circulatory system;
- Respiratory system: increase of respiration rate, difficulty in the respiration i.e. dyspnea
when there’s high fever;
- Digestive system impairment: as a consequence, loss of appetite, nausea, vomiting;
- Nervous system: especially during very high fever e.g. agitation, muscle problems,
asthenia, hallucinations, delirium;
- Immune system: both humoral and cell-mediated responses can be stimulated because
the inflammatory response is parallel to the immune response.
Hypothermia is the opposite situation (see lecture n.11 for further details). It can occur when there
is increased thermodispersion and loss of heat from the body.
- External causes:
o Frostbite, freezing: involves only parts of our body which are most exposed to
cold: usually hands, ears, etc.;
o Systemic hypothermia: it occurs when I stay for a long time in a cold
environment, all the body gets cold and until 25°C I can die;
- Intrinsic causes:
o Drugs;
o Alcohol;
o Circulatory shock.
- It can also occur for decreased thermogenesis, when the body is not able to produce
heat:
o Hyperthyroidism e.g. thyroid is not able to release T3 and T4;
o Hypocortisolism e.g. adrenal glands do not release the hormones;
o Anaemia;
o Insufficient nutrition: in fact, the body is able to produce heat using nutrients
(metabolism). I eat because it is a pleasure to (SANTE PAROLE) but also
because I need to produce calories that are needed for heat production.
GLUCOSE METABOLISM IN CANCER CELLS
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There are differences in the metabolisms of tumor cells compared to normal ones. Tumor cells need
a lot of energy to induce cell invasion, spreading, to have metastasis, in other words, to become
“more malignant”. Related to this energy, from the biochemical point of view, what is important is the
Warburg effect30. It is related to glucose metabolism in cancer cells and it is very similar to the
normal glycolysis that we have in our normal cells: in fact, during this reaction, the glucose is
transformed into pyruvate and then into lactate.
In normal cells which are exposed to high concentrations of O2, glycolysis induces mitochondrial
oxidation, in this way pyruvate in converted into CO2 and H2O. In this case, we have a kind of
inhibition of glycolysis because our cells use the O2 for mitochondrial oxidation, putting it to better
use. This oxygen-dependent glycolysis inhibition is in normal cells called Pasteur effect.
On the other hand, in tumor cells, the Pasteur effect decreases and the Warburg effect (aerobic
glycolysis) increases; that is: even in presence of high (different) concentration of oxygen molecules,
the conversion from glucose to lactate increases through glycolysis. So, the glycolysis is the main
metabolic pathway that is used by cancer cells to have the maximum energy possible.
As regards the energy connected to the molecules of ATP that are produced, in the normal cells we
have glycolysis in which 1 molecule of glucose yields 2 molecules of pyruvate which in turn yield 2
ATP and 2 NADH.
Another metabolic pathway that occurs in normal cells is the Krebs cycle (citrate cycle). It consists
of eight enzymatic reactions in the inner part of the mitochondrial matrix and it converts acetyl-CoA
to CO2 and H2O with generation of energy. The main source of oxygen however, is the oxidative
phosphorylation in mitochondria which is the final phase of respiration after glycolysis and Krebs
cycle. In this case, we have formation of ATP (through linkage with phosphate), which is a high
energy molecule, through the redox changes and oxidation of NADH and FADH2, with electrons
release.
In cancer cells, glucose is the main substrate meaning that it is captured by cancer cells though two
specific membrane transporters: GLUT1 and GLUT3. These transporters are receptors which
capture glucose that therefore enters the tumor cells where
it is phosphorylated by hexokinase yielding glucose-6-
phosphate which in turn undergoes glycolysis, so it is
transformed in pyruvate and lactate.
Therefore, the transformation of glucose in pyruvate and
lactate through glycolysis generates only 2 ATP molecules.
The transformation of glucose through mitochondrial chain
respiration instead produces 36 ATP molecules. In a tumor
cell, the main metabolic pathway used is glycolysis which
means that this tumor cell induces a “loss” of 34 ATP
molecules compared to normal cells. Therefore, just one
cancer cell induces a waste in our body of 34 ATP
molecules.
Mitochondrial defects in tumor cells
The important differences between aerobic and anaerobic glycolysis are those related to
mitochondrial defects.
Structural changes:
- Elongation;
- Swelling;
- Pyknosis;
- Abnormal arrangement of ridges;
30
It was named “Warburg effect” after the professor Otto Warburg who discovered it in 1920.
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- Cristae (ridges) fragmentation and subsequent defect of one or more enzymes that are
structurally present in those membranes;
- Increased fragility of membranes for altered lipid composition.
Biochemical alterations:
- Lower oxidation of fatty acids;
- Prevalence of stage 4: during mitochondria respiration there is one phase which is the
consumption of oxygen, then another that consists in the phosphorylation or linkage for
energy, these steps are called stages; for example, stage 3 is when mitochondria have the
oxygen from the external environment and changes in respiratory chain of electron ions begin
i.e. electrons continue to cycle from one flavoprotein to the other. This stage 3 is dependent
on the oxygen which is consumed/arrives by mitochondria; this oxygen induces
phosphorylation from ADP to ATP triggering stage 4 which is a kind of feedback control of
oxygen and energy production. Therefore, when there are enough molecules of ATP formed,
respiration is lower but when we need more energy respiration is higher. Stage 4 increases
in this case because there is a disconnection between oxidation and phosphorylation, it
continues independently from oxygen consumption. In this case, tumor cells are always in
stage 3 phase of mitochondrial activity;
- Defect of electron transporters;
- Citrate extrusion which is involved in the Krebs cycle which is always linked to mitochondrial
respiration.
Remember: Warburg effect is an aerobic glycolysis, while our normal glycolysis is anaerobic; this
means that even if there is the possibility for the cell to assume oxygen, tumor cells prefer to perform
glycolysis. It is indeed called aerobic because it occurs also in presence of oxygen.
Positron electron tomography imaging with 18-fluorodeoxyglucose of a patient with
lymphoma.
Based on the need of cancer cells to use glucose, we can analyze different types of tumors in order
to evaluate the possible different tumor sites to which the tumor can spread all around the body
(metastases).
We use PET (positron-emission tomography) in which GLUT1 and
GLUT3 in cancer cells induce a cell-intake of large quantities of
glucose in tumor cells.
If we introduce 18-fluorodeoxyglucose (FdG, radionuclide similar to
glucose) in a patient with a tumor, this will be captured by the tumor
cells. The concentration of radioactive glucose can be observed in
the sites where the tumor metastases are. For example, in the side,
there’s the image of a lymphoma with different metastases at lymph
nodes that are full of tumor cells which can be detected through PET
imagining.
This type of analysis is used especially for brain, lung and colorectal
tumors. It is important to evaluate and detect the stages of tumor
meaning the degree of malignancy of tumors. Tumors can in fact be
classified in stages and grading:
- For stages, depending on localization and volume of the
tumor;
- For grading, depending on the degree of the dysplasia.
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Staging and grading normally go together to evaluate the malignancy progression of this tumor. It is
useful to check on patients who underwent surgical tumor resections, in order to evaluate if there
are metastases which have spread out through the body after the surgery.
Another PET purpose is to monitor patients post-ischemia and/or radiotherapy which is an attempt
to counteract the tumor: it is used instead of or together chemotherapy depending on the type of
tissue in which tumor in present e.g. in the brain, radiotherapy is very important (for the localization
of the tumor) while in other sites such as lungs chemotherapy and radiotherapy can be performed
together.
During radiotherapy, some sites of the tissues which are surrounding the tumor, will undergo
necrosis. So, it is common that these tissues begin to develop phenomena of ischemia. What is
ischemia? Lack of blood flow, or better, the lack of both oxygen (anoxia) and nutrients and it can
be due to a necrosis e.g. during the cardiac necrosis, there is an ischemia.
Other than glucose metabolism, also the other two types of metabolisms of our tissues are impaired
in tumor cells: tumor protein metabolism and tumor lipid metabolism.
Tumor protein metabolism
In the tumor there is an increase of protein synthesis because cancer cells synthesize different
proteins, especially those important for the proliferative function of the cells; they increase very fast
the protein synthesis but there is a slow protein and amino acid catabolism.
There is also an increase of nucleoproteins metabolism (those proteins located into the nucleus
which play an important role for division and proliferation) meaning:
- Increased enzyme synthesis and decreased enzymes catabolism;
- Cancer cells with glycolytic ATP;
- Tumors consume many more substrates to obtain the same quantity of metabolic energy
(ATP molecules) obtained by normal cells (18-fold, they need a higher energy to exert their
metabolic functions; they work harder, in a less efficient way);
- Active and selective amino acid transport is lacking or reduced.
Tumor lipid metabolism

As regards tumor lipid metabolism, the changes are due to changes in the composition of
membranes. In these cancer cells, the composition is quite different compared to that of normal cells.
there is a decrease in synthesis of fatty acids, decrease in oxidation of fatty acids (in both
structure, and substrate for the oxidation important for the function), then there’s deprivation of fatty
acids from the host because they need also lipids from the host to have gluconeogenesis and have
much more glucose.
The three metabolisms in the body are connected i.e. if cancer cells need glucose, the sources of
glucose can be carbohydrates (diet) or the lipid tissues because from lipids we have lipogenesis that
enables the cells to perform gluconeogenesis yielding new glucose formation. Cancer cells therefore
get their substrates also from the body of the host, more specifically also from its lipid reserves.
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Another important change is related

to the cholesterol synthesis which
follows this pathway (in the picture)
from acetyl CoA to cholesterol. The
important enzymes involved in this
pathway are synthetases and
reductases:
hydroxymethylglutarylcoA
synthase and
hydroxymethylglutarylCoA
reductase. If there is no availability
of this molecule (acetyl CoA) or if
there are changes/mutations, like it
happens in some tumors, of these
enzymes (of synthetase in
particular), there is no feedback
control on the formation of endogenous cholesterol. Cholesterol is very important in our membranes
as well as omega-3 and omega-6 fatty acids, but cholesterol is particularly important because it is
not just a compound of our membranes: it is also involved in the formation of steroid hormones, bile
acids, activation and production and maintenance of many molecules of cholesterol into the brain
which is very rich in cholesterol.
In this case, there is a process which is reverse cholesterol transport: a negative feedback control
comes into play if we have a strong quantity (high levels) of cholesterol into our tissues (and into the
blood subsequently). This feedback mechanism is important to maintain the exact concentration of
cholesterol in the body, in different tissues and it is determined by HMGCoA synthetase. If there is
a high concentration of cholesterol, endogenous synthesis of cholesterol is blocked (remember: we
can also introduce cholesterol from food, not just by metabolic processes; the balance between the
two cholesterol intake mechanisms is important).
In tumor cells, if this feedback control pathway is blocked, the formation of cholesterol increases. So
high quantities of cholesterol are inserted into the bilayer of membranes. Cholesterol normally is
important in the formation of the so-called raft membranes which are platforms of cholesterol that
bind to important proteins such as tight junctions and adhesion molecules, to maintain the exact
plasticity and elasticity of the membranes. But if there is an increase of cholesterol, also these raft
membranes are changed in their composition which means that also the adhesion molecules and
tight junctions and their functions are impaired.
Alterations of membranes (e.g. lipids and cholesterol concentration alterations) depend on the level
of atypia of the tumor. If the atypia (tumor malignancy) increases, we have:
- Reduced fluidity of membranes;
- Reduced synthesis of complex lipids;
- Rafts alterations;
- Impaired function of proteins, also of the membrane layer;
- Altered cell-cell and cell-matrix interactions: these interactions are very very important in
the spreading of tumor cells in our body.
All these impaired metabolisms that involve not only tumor cells but also normal cells (those from
the host) are due to the high energy which tumor requires from us. There is removal of glucose
and fatty acids from host, reduced active and selective amino acid transport, reduced protein
concentration and then there is another external factor which is the inadequate food intake like in
the case of a tumor patient affected by colorectal cancer (e.g. obstructions) or that of a patient who
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underwent chemotherapy, whose stomach has chronic and acute gastritis due to the killing action of
chemotherapy. In these instances, in addition to the impairing action of the tumors endogenously,
we have an important factor that is the difficulty of energy intake through dietary foods. In Italian, this
deprivation is called “azione spoliatrice”: deprivation action, stripping action, in which the body of
the tumor patient in the last phases of the tumor is undergoing this big azione spoliatrice.
Induction of invasiveness and different types of molecules which are involved in it. Tumor growth, as
we have already seen, can be of two types:
- Benign tumor: expansile growth (expansive), there is an expansion;
- Malignant tumor: infiltrating, there is an infiltration, invasion.
Expansile growth
The expansion of benign tumor induces the
increase of local pressure on the surrounding
normal cells and you can observe only an
increase of tumor mass in diameter (due to
new cell production and less destruction of old
cells) with central portions of the tumor being
necrotic because they are not getting enough
nutrients and oxygen (due to the fact that there
are in the innermost part). This type of growth is
due to the formation of a fibrous capsule which
surrounds the tumor so that it cannot invade
other parts of the tissue. There is tumor pushing: there are some necrotic zones; it is due to vessel
compression by tumor mass.
Infiltrating growth
It starts with the “in situ cancer” (1st figure). The
tumor invades the membrane basement
destroying it with proteases. In this case, we can
talk about epithelial malignant tumors and solid
tumors, not hematological ones, coming from the
epithelia that must pass this membrane. In the
connective tissue, the malignant cells can form
cellular spreads (in Italian, zaffi cellulari) and
perle or cordoni epiteliali (2nd figure).
Then there is the maximum of invasion which is
reached by the tumor mass when the tumor cells
invade the vessels (3rd figure). In this case, this
type of process needs different factors i.e.
chemical changes and also cell signaling
activation. When there is cancer in situ there is an
increase of local pressure due to the increase
of cell number. In order to cross the cell
membrane (necessary for spreading), they need
to have motility: they must get away from other
cells, there must be low adhesion between
tumoral and normal cells. Also, to walk around the
connective tissue, they must produce
substances which facilitate the tissue
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invasion: enzymes able to destroy connective tissue (e.g. lysosomal enzymes, cathepsins,
collagenases, hyaluronidases) which can be produced by both tumor cells and host cells whose first
mechanism of defense is inflammation (e.g. phagocytes); indeed, normally, in the surrounding
environment of the tumor, there is inflammation. A tumor patient can have a light fever 31 that is due
to the inflammation that occurs in the microenvironment of the tumor. Indeed, these enzymes are
activated during inflammatory processes also. Tumor cells can produce them from the necrotic
(central) portion or by tumor cells of peripheral portions which are more aggressive than the others.
Invasiveness
Invasiveness is a typical feature of malignant cells. Steps of the process are:
- Invasion: passage through basal membrane;
- Intravasation: tumor cells enter vessels (general circulation) leading to formation of
circulating tumor cells;
- Extravasation: to reach other sites of the body, other tissues; formation of a secondary
tumor32 similar to the primary one i.e. metastasis.
This process and its phases are similar to white blood cells extravasation in inflammation (when
there is an increase in permeability of the vessels): same mechanisms are used by tumor cells to
invade connective tissue in case of solid tumors and
inflammation process. Tumor cells need to attach and
destroy the basal membrane in order to migrate. Therefore,
we need four phases of invasion that:
- Tumor cells detachment;
- Cell attachment to matrix components;
- Extracellular matrix degradation;
- Tumor cell migration.
The first two need adhesion molecules to occur. However,

these are needed also in the second part of this process. In
fact, when the secondary tumors need to stop in a site, they
need adhesion molecules but in the opposite way: when
they have to spread, they increase some types of adhesion
molecules but when they have to invade and grow stability
in one site, they have to decrease these same molecules
modulating the increase and decrease of these molecules.
31
Fever itself is a general type of inflammation.
32
A secondary tumor is the same of the primary one but located in another region of the body. Secondary tumor =
metastasis.
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Adhesion molecules overview

Cell adhesion molecules are called cell-cell adhesion molecules, in general CAMs. They are
located on the cell surface, and most of them have structures similar to receptors e.g.
immunoglobulins. The four main families of CAMs are:
- Integrins;
- Cadherins;
- Immunoglobulins superfamily;
- Selectins.
All of these CAMs have an extracellular portion (NH2

function, amino groove), an intracellular domain (COOH
function) and an intermediate transmembrane domain.
Their function is to link one cell to the other. CAMs in
general are oriented in a correct way and distributed in
focal loci of the cells: along specific parts of the
membrane. Other adhesion molecules named tight
junctions33 are important in cell physical permeability for
passage of water, ions and so on).
CAMs function by binding other molecules that can be
either equal, similar to them or different. This type of
binding can be either:
- Homophilic: they interact with the same type of
molecule located on an adjacent cell;
- Heterophilic: they interact with different types of molecules of adjacent cells.
They can be activated by calcium or not, so they can function in a calcium-dependent way or calcium-
independent way.
Some CAMs are also tissue-specific: those of a particular tissue normally prefer to bind with
proteins of their same kind e.g. I-CAMs, N-CAMs, E-CAMs; cells reaggregate into different groups
according to their embryonic origin. From the slide: cells of the same tissue preferentially attach
together, thus maintaining tissue architecture.
Integrins
Integrins have a very big extracellular domain that consists of a
sequence of two chains:
- α: subunit whose NH2 extracellular portion interacts with
extracellular matrix molecules in particular those containing the
sequence “arginine, glycine and aspartic acid” aka SAM (substrate
adhesion molecules). These molecules are involved in invasiveness
of the cancer e.g. fibronectin, laminin and thrombospondin-1;
- β: subunits whose function is related to linkage of adhesion
molecules to cytoskeleton, this binding causes rigidity of the cells;
contains four cysteine-rich repeated sequences.
The mechanism of action is calcium-dependent and mediate tumor

cell adhesion to endothelium during tumor progression.
33
They are named “tight junctions” because they strictly link one cell to the other.
162
Cadherins (CAD)
Cadherins are transmembrane glycoproteins (700-750 aa) composed of
a single chain. They have an extracellular portion with high quantity of
calcium ions (at least 3 domains for calcium binding out of 5). They are
present in different tissues in different isoforms (up to 12) which are named
differently according to the name of the tissue in which they were first
discovered e.g. E-cadherin (epithelial tissue), N-cadherin (neurons, but
later discovered in muscle cells and crystalline cells as well) and P-cadherin
(placenta). They are calcium-dependent and link through homophilic
binding. Cadherins, like integrins, link to cytoskeleton through the
interaction of another protein i.e. catenin that acts as a chain between
receptors and cytoskeleton (E-cadherin-catenin complex).
Different types of cadherins are involved in different types of tumors and the
increase of invasiveness is induced by deletion or malfunctioning of
these cadherins: E-cadherin loss for epithelial tumors, or it can function well, but catenin gene might
be mutated thus causing a decrease activity of cadherins. Changes in cadherin levels are very
important for the metastatic process.
Immunoglobulin superfamily
They are CAMs but are very structurally similar to Igs (conservation in folding and in sequence of
specific features found within the Ig molecules). They are calcium-independent and can link
through both homo- or heterophilic binding. They have a very long, five-folded extracellular portion
and different amino acids in the intracellular portion (functioning as cytoskeleton binding and/or
signal transduction). They are glycoproteins like the others whose family comprises several different
types. Among these, the most known are:
- N-CAM (at least 27): homophilic binding, is important during morphogenesis but also for
tumor spreading, mutations of this are important;
- I-CAM: heterophilic binding, is important especially for the
inflammatory process.
The regulation of molecules of this superfamily is induced by sialic acid

which has negative charge and induces a negative regulation of the
anchorage of these immunoglobulin-like molecules. Polysialytated N-
CAMs (with many sialic acids) induce less adhesion counteracting the
effect of glycosylation of the middle part of the molecule, that very close
to the membrane.
Mutations of N-CAMs are found in neuroblastoma (malignant tumor of the
brain), small cell lung cancer (microcitoma, like in Italian), Wilms’ tumor
(nephroblastoma, malignant tumor of the kidney). N.B. blastoma means
malignant for a particular type of mesenchymal tissue (there is another
kind which is sarcoma) while carcinoma is used when referring to
epithelial malignant tumors.
Selectins
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Selectins are single transmembrane proteins. They have a middle part of

the external chain which is a domain similar to epidermal growth factor.
They are of different types depending on the kind of tissue where they are
located e.g. L-Selectins (on lymphocytes), E-Selectins (on endothelial
cells, ELAM-1) and P-Selectins (on platelets and endothelial cells). In
general, they are very important in rolling and calling for leukocytes in the
inflammatory process thanks to their capability of interacting with sialyl
Lewis X (SLex) protein. In fact, epithelial cancer activates selectins when
they have to spread in other sites which they want to “colonize” (sit there).
Cancer cells detachment from their parent tumor can be found in the first
state as:
- lack of E-cadherin synthesis;
- catenin gene mutation in some neuroblastoma cell cancer and
whilst tumors;
- N-CAM with highest content of sialic acid counteracting attachment and function of N-
CAM;
Tumor cells attachment to extracellular matrix:

- In different types of tumors due to the attachment with the extracellular matrix e.g. melanoma,
colorectal and breast cancer (integrin receptors and laminin interaction) and colorectal
carcinoma (cell adhesion related to selectins).
Subsequently to the involvement of adhesion molecules, extracellular matrix degradation can go

on through:
- Passive pressure by the tumor mass;
- Tumor cell production of specific proteinases (produced by tumor);
- Induction of proteinases’ production by host cells e.g. fibroblasts and macrophages.
Proteinases can be therefore produced by both tumors and host.
Among different types of proteinases, very important are:

- Collagenase type IV (also named matrix metalloproteinase MMP-2) which disrupts
collagen;
- Cathepsin D: it is a lysosomal proteinase found to be increased in breast cancer (high levels
in both cells and serum);
- Plasmin is a serine proteinase;
- Plasminogen activator: metalloproteinase, can be of different types (urokinase-type, uPA
and tissue type tPA, plasminogen activator), it is a serine proteinase that converts
plasminogen into plasmin and is able to degrade extracellular matrix glycoproteins.
These four proteinases are therefore important in the tumor metastatization process and extracellular
matrix degradation.
NORMAL AND TUMOUR ANGIOGENESIS
Introduction
During the lesson, normal and tumour angiogenesis, which have already been mentioned talking
about wound healing and also by professor Biasi, will be explained. Moreover, the mechanisms
responsible for the formation of new vessels during tumour progression will also be covered.
The lesson starts with a video about professor Folkman, a very important scientist who discovered
the process of angiogenesis.
Video link: https://www.dailymotion.com/video/xjh8co
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Angiogenesis
Figure 1
Angiogenesis is also called neovascularisation. In figure n.1 you can see
an example of angiogenesis during tumour development. Tumours need new
blood vessels to grow: when tumours grow and reach a dimension in which
cells are too far from the blood vessels existing in the tissues, they release
pro-angiogenic molecules that stimulate the formation of new vessels starting
from pre-existing ones.
Angiogenesis is not only a pathologic process that occurs during tumour
development, it also occurs in physiologic conditions, in particular during
embryo development, when there is the formation of new vessels starting from the staminal cells
coming from the bone marrow. Moreover, there are some tissues that require angiogenesis during
their physiological processes, for example the female organs that are growing or when a tissue is
damaged and needs wound healing34.
Usually, however, our tissues in physiologic conditions don’t require angiogenesis, because the
vessels are quite stable. In normal healthy tissues, angiogenesis mainly happens when tissues need
oxygen (when there is a state of hypoxia). In pathologic conditions, there are several situations in
which angiogenesis occurs, but we focus on cancer.
Physiologic angiogenesis
Sprouting angiogenesis is initiated in poorly perfused tissues with hypoxia: it’s really hypoxia that
stimulates the activation of a specific transcription factor able to activate the expression of genes
coding for the molecules responsible for the formation of new vessels. Therefore, hypoxia is what
stimulates angiogenesis, both in physiologic and in pathologic conditions.
The difference between the physiologic and pathologic
process of angiogenesis is that in physiologic conditions the
balance in the tissue between the levels of pro-
angiogenic and anti-angiogenic molecules is tightly
regulated: in some physiologic conditions, the amount of
activators may rise and so there is activation of angiogenesis,
but in a short period of time this process is stabilized and it
stops; in pathologic conditions there is a great imbalance
between activators and inhibitors of angiogenesis and the
process is not regulated, it doesn’t stop or it is very difficult to
stop.
Different phases of angiogenesis are shown in figure n.335:
Figure 2
34 What happens in the tissue during wound healing has been seen in lesson 7
35 The same image has already been seen in the lesson about wound healing
165
1. The first phase is vasodilation of the pre-

existing vessels in response to
vasodilatative molecules, for example NO. In
figure n.3, the cells in violet are
periendothelial cells: their function is to
stabilize the vessels and they are called
pericytes when we are in the
microcirculation and smooth muscle cells
in the bigger vessels;
2. Then there is the separation of the
pericytes and the degradation of the
basement membrane thanks to the action
of enzymes like matrix metalloproteinases
(MMPs);
3. A guide cell, i.e. a cell that initiates the
sprouting of the vessel, is able to migrate
following the signals coming from the
damaged tissue that is suffering from
hypoxia;
4. Then endothelial cells are able to Figure 3
proliferate; these cells have particular
receptors on their membranes that are able
to interact with the ECM, allowing them to
proliferate in a specific direction;
5. Endothelial cells adhere to the ECM;
6. Then a process called tubulogenesis takes place, which is essentially the endothelial cells
tube formation;
7. New periendothelial cells are recruited, in particular pericytes, since the main processes
regarding angiogenesis happen in the microcirculation, and so the new vessel is formed;
8. The final step requires the deposition of a new basement membrane.
In figure n.4 there are several receptors

present on the membrane (yellow line) that
have a kinase domain intracellularly and that
are responsible for endothelial cells
proliferation, for migration, for survival, for
the recruitment of pericytes, for the
formation of new basement membrane i.e.
for all the processes that are necessary for
angiogenesis.
For example, the most famous and Figure 4

important molecule, VEGF (vascular
endothelial growth factor) A, B or C (there
are several isoforms), binds to its specific
receptors present on the membrane of
endothelial cells, VEGFR1, 2 and 3, which
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has a tyrosine kinase domain on its intracytoplasmic portion. The binding of VEGF activates the
kinase domain in the intracytoplasmic portion of the receptor, leading to downstream pathways which
imply the activation of other enzymes in a cascade through the phosphorylation of the substrates
like Ras, MAP kinase, PI3K, Akt, etc.36. This leads to the translocation in the nucleus of NF-kB, a
transcription factor that has been demonstrated to be most involved in the processes needed for
angiogenesis, like the proliferation of endothelial cells (that normally don’t proliferate), migration and
also survival (we have already studied cell death; to induce a cell to die for apoptosis, a lot of
molecular pathways have to be activated, but there are also pathways that are necessary to induce
cell survival). Therefore, by specific drugs or by other specific molecules we can stimulate a cell to
survive and these pathways, in particular PI3K and Akt, if activated, can induce a cell to survive.
Endothelial cells have to survive to hypoxic conditions, migrate and create new vessels.
Then angiopoietins 1 and 2 are also important molecules involved in angiogenesis that have
opposite effects: angiopoietin 1 doesn’t induce angiogenesis while angiopoietin 2 does; they
compete for the same receptor, a very famous one called Tie-2, which also has a kinase domain, so
the binding of angiopoietins with Tie-2 leads to the activation of a downstream pathway.
Then ephrins, the same as their antibodies.
On the surface of endothelial cells there are integrins, which have the role of adhering to the ECM
thus favouring the migration of endothelial cells.
There are also two growth factors, the basic fibroblast growth factor (FGF) with its specific
receptor on the membrane of endothelial cells, and the platelet derived growth factor (PDGF),
which also in this case binds to its specific receptor. These two pro-angiogenic factors are
responsible for the recruitment of smooth muscle cells and pericytes, the perivascular cells
necessary to stabilize the new vessel that has formed. Although VEGF is important and has been
studied a lot and therefore a lot of drugs against it or its receptor exist to stop tumour angiogenesis,
now also drugs against FGF and PDGF are available.
To sum up, several angiogenic factors are now recognized:
- VEGF (vascular endothelial growth factor), important in vasculogenesis37/angiogenesis; its
receptor, present on the cell membranes of endothelial cells, has a tyrosine kinase activity
and so it transduces the signal that leads to vasculogenesis. The release of VEGF is tightly
controlled by angiopoietins 1 and 2, that interact with their receptor Tie-2: when angiopoietin
1 binds to Tie-2 there is no angiogenesis while when angiopoietin 2 binds the contacts
between endothelial cells are reduced and the process of angiogenesis is stimulated.
There is another video: https://www.youtube.com/watch?v=8BMvcK7HRGc38
The following explanation is based on Figure 11 and is only to show why the video is wrong:
Angiopoietin 1 and 2 bind to the same receptors, called Tie-2. When angiopoietin 1 binds Tie-2
there is no angiogenesis because the vessels are stabilized by this interaction. When
angiopoietin 2 is present in higher amounts than angiopoietin 1, it competes for the same
receptor, so angiopoietin 2 binds to Tie-2 and this binding favours the formation of new vessels.
All these pro-angiogenic factors are released by tumours or by tissues that need oxygen (in
physiologic conditions).
Other angiogenic factors released by hypoxic tissues (besides VEGF) are:

- Ephrins, which have receptors on the endothelial cell membrane with a tyrosine kinase
domain in the cytoplasm. Their function is to stabilize tight junction vessel orientation to
36 The professor said it doesn’t matter if we don’t remember all these pathways
37 https://en.wikipedia.org/wiki/Vasculogenesis
38 The professor said she is confused, she has to see the video again because she heard that angiopoietin 1 promotes
angiogenesis when it is actually the exact opposite (the video is not so clear, but it had beautiful images)
167
modulate the orientation of the migration of endothelial cells because endothelial cells must
proliferate and migrate but in a specific way, not in a disordered fashion;
- FGF also in this case its receptor has tyrosine kinase activity. It is able to induce all the
phases of angiogenesis;
- PDGF; receptors have tyrosine kinase activity so binding of PDGF activates the receptor,
which phosphorylates the substrates until several phases of angiogenesis are activated:
migration, proliferation of fibroblasts, smooth muscle cells, monocytes, etc.;
- TGF-β (transforming growth factor beta) is also important39. During inflammation, it stimulates
the secretion of ECM proteins by fibroblasts, therefore playing an important role in
fibrogenesis and scar formation, which is also important during repair and angiogenesis. In
the case of TGFβ, the receptors40 have serine/threonine kinase activity, meaning that they
phosphorylate the substrate on serine threonine. It has a dual effect: low levels of TGFβ are
able to induce the synthesis of PDGF in fibroblast (so to stimulate angiogenesis), high levels
of TGFβ lead to a stronger stimulation of the TGFβ receptor and therefore to the inhibition of
the synthesis of PDGF by fibroblasts (so it inhibits angiogenesis). Therefore, it is considered
to be a sort of a regulator of angiogenesis since it is able to switch it on and off, in particular
during physiologic processes.
- Extracellular matrix proteins that are able to control the migration of endothelial cells. For
example:
o Integrins, which are present on the membrane of endothelial cells, but they interact
with other proteins present in the ECM and give the possibility to endothelial cells to
migrate;
o Other proteins, called SPARC, the most famous one being thrombospondin 1 (but
there are also tenascin, osteonectin and osteopontin), have anti-angiogenic
activity: therefore, the interaction of SPARCs with endothelial cells gives stability and
inhibits the process of angiogenesis.
Tumour angiogenesis
The same molecules involved in physiologic
processes are also involved in pathologic
conditions, for example in tumour progression. The
difference is that in pathologic conditions the
balance between anti-angiogenic proteins and pro-
angiogenic molecules is not controlled, so
Figure 5 angiogenesis starts but doesn’t stop, it is never
stabilized and is continuously stimulated since there
are lots of activators of angiogenesis released by
the tumour.
New tumour blood vessels, formed to favour tumour growth, are growing inside the tumour but they
are not the same as in physiologic conditions, they are completely different from normal vessels.
They have a chaotic architecture, so they are not regular; they are dilated and the normal tight
junctions between the endothelial cells (which leave no spaces between endothelial cells in normal
conditions) are absent, the cells are separated and so there is an increased permeability of the
39 Professor Biasi loves it

40 Not shown in the image, but still important and to be remembered
168
vessel, which also leads to a higher interstitial pressure because it causes the release of liquids
from the vessel. The basal membrane is discontinuous, sometimes even absent, and the same
is for pericytes, which are less numerous, absent or have loose interactions with each other.
Blood in tumour new vessels flows irregularly, more slowly than in normal vessels and sometimes
with a discontinuous flow. In this way, the new clones of tumour cells are more resistant to hypoxia
because the blood arrives and then goes away, and then arrives flowing slowly, so the new vessels
give oxygen and nutrients to the tumour cells but not in a “completely great” manner so the cells are
resistant to hypoxia, they survive but they suffer and resist.
Another video: https://www.youtube.com/watch?v=mvsF_pIClaI
In figure n.6 it is possible to see the
Figure 6
characteristics of a normal blood vessel and a
tumour blood vessel. Normal blood vessels are
characterized by a regular shape and, at a
microscopic level, it can be seen see that
endothelial cells are tightly bound to each other
thanks to the presence of tight junctions, which
can also be found between endothelial cells and
the basement membrane and between the
basement membrane and pericytes, providing a
structure that is highly stable.
In tumours (figure n.6, right side), blood vessels
are chaotic, tortuous, vasodilated and, as also
seen in the video, pericytes are loosely in
contact with each other, tight junction are not present anymore or are less than in normal conditions
and the same happens between pericytes and the basement membrane; sometimes pericytes are
not even present and are replaced by tumour cells, which locate around blood vessels to stabilize
them. The basement membrane is discontinuous, it can be present or not and, in any case, the
junctions between endothelial cells and the basement membrane are less present; therefore,
endothelial cells are separated from each other and fluids from the blood go in the interstitial space,
creating a higher interstitial pressure inside the tumour, so a higher blood permeability in these
vessels with respect to normal ones (IP=interstitial pressure). VEGF is high because we are inside
a tumour and the tumour cells release all the pro-angiogenic factors that we have already seen
including VEGF, although VEGF is not only produced by tumour cells but also by inflammatory cells
that can be found inside the tumour. As already explained in previous lessons, chronic inflammation
is important in tumour development, since tumours can be also considered chronic inflammatory
processes because inside there are lots of molecules of inflammation like monocytes, macrophages,
lymphocytes, etc.
Figure 7
169
Figure n.7 is a nice picture that gives

the idea of what is happening when a
drug created against VEGF is
administered to a patient who has a
tumour. Nowadays there are a lot of
therapeutic strategies that are
directed to stop, prevent or delay the
process of angiogenesis because it
has been discovered that, if you block
angiogenesis, the tumour stops
growing, as can be seen in professor
Folkman’s experiment in the cornea:
if you take the tumour away from the
cornea, the vessels disappear; the
same happens if you inhibit angiogenesis, because the tumour stops growing and its cells die
because they don’t receive oxygen.
Bevacizumab is a monoclonal antibody against VEGF; these antibodies bind to VEGF and, in this
way, VEGF is not able to bind to its receptor anymore because it is covered by the antibodies.
Therefore, the early treatment with Bevacizumab destroys the new vessels that have formed in the
tumour (comparison with figure n.6) and new tight junctions between endothelial cells and the
basement membrane and between pericytes and basement membrane appear again. Later when
treatment is continued, we can see that endothelial cells are destroyed, tumour cells die, and the
basement membrane disappears, causing the tumour’s vascular collapse. In fact, we don’t want the
tumour vessels to become simply physiologic, we want them to disappear completely.
The guide cell, or the tip cell, is the first cell of the vasculature that starts to proliferate and migrate,
followed by the destruction of the basement membrane, separation of pericytes and tubulogenesis.
That is what happens in physiologic angiogenesis. In tumours, the process is almost the same, but
the new vessels are strange, not normal, and some processes are peculiar.
For example, in figure n.8 there is a pre-existing
vessel; in violet there are the new vessels that are
forming inside the tumour. When there is a tumour,
at first cells inside the mass are suffering from
hypoxia because the tumour is growing and the
vessels are far away, in particular from the cells in
the middle of the mass; hypoxia leads to the
translocation and activation of a transcription factor
that leads to the expression and synthesis of pro-
angiogenic factors (VEGF, PDGF, FGF, etc.). In
parallel to the release of pro-angiogenic factors
there is the migration from the bone marrow to the
tumour of precursors of endothelial cells (CEP,
circulating endothelial precursors); they arrive
inside the tumour and they differentiate into
endothelial cells thus creating new endothelial cells
(in green) starting from the precursors. Moreover,
new sprouts are formed from pre-existing vessels
because of VEGF or angiopoietin 2 released from
the tumour cells and integrins are upregulated on
the cell membrane. Besides, HSC (hematopoietic Figure 8
170
stem cells, in grey in figure n.8), stem cells coming from the bone marrow, go inside the tumour and
differentiate into macrophages (but also other cells), which have the role to produce matrix
metalloproteinases (MMPs). These enzymes are responsible for the degradation of the ECM by
creating a road inside the matrix, allowing endothelial cells to migrate in a specific direction, and also
for the degradation of the basement membrane to allow the separation of pericytes, thus favouring
proliferation and migration of endothelial cells. MMPs are negatively important because when the
tumour cells start producing them, they can detach from the other cells migrate from the principal
mass and give a metastasis in other tissues or organs.
When the tumour mass is little, Figure 9
angiogenesis is not stimulated; however,
when the tumour grows, the cells inside
the tumour are suffering from hypoxia and
so they start releasing pro-angiogenic
factors that initiate angiogenesis. In figure
n.9 we can see a tumour cell that is able
to migrate and thanks to matrix
degradation (mainly by MMPs) is able to
give metastases in other tissues and
organs, also very far from the original
mass.
In 1974 Folkman, the professor that we

have seen at the beginning of this lesson,
hypothesized the presence of a soluble
factor he called TAF, tumour angiogenic
factor. This factor, which was still
unidentified, was able in his opinion to
stimulate angiogenesis. Now we know
that TAF are all those pro-angiogenic factors that we’ve already seen, like VEGF, FGF, etc. In figure
n.10 there is a little tumour, veins and arteries, formation of new blood vessels and, consequently,
tumour growth.
Figure 10
Figure n.11 summarizes what we have seen so far. It is possible to see some of the pro-angiogenic
factors like angiopoietin 1, VEGF, PDGF, FGF, etc., all factors that are released by tumour cells.
Therefore, as already mentioned, tumour cells produce all these pro-angiogenic factors, but also the
inflammatory cells present inside the tumours do. These factors bind to receptors on the surface of
endothelial cells. When angiopoietin 1 binds to Tie-2 there is no angiogenesis, when angiogenesis
2 binds to the same receptor you have the sprouting of new vessels, the same when PDGF binds to
171
its receptor, when VEGF binds to VEGFR (VEGFR 2 is the main receptor for VEGF), the same for
integrins which bind to ECM and so on.
Figure 11
It has been demonstrated that inside platelets there are granules called α-granules that contain all
those pro- and anti-angiogenic factors. Figure n.11 comes from a review that was written by Folkman
in 2007, so it can be considered a quite recent review41.
Pro-angiogenic factors can be directly released from tumour cells but also indirectly produced by the
inflammatory cells that arrive in the site of a tumour trying to destroy aberrant cells.
Anti-angiogenic factors can be produced by tumours and by the inflammatory molecules but, when
the balance is in favour of the pro-angiogenic factors, they don’t work. Anyway, in normal conditions
anti-angiogenic factors work and at a certain moment angiogenesis is stabilized. The most famous
anti-angiogenic factor is thrombospondin 2, but there are also three classes of statins that have
been demonstrated to inhibit angiogenesis. Statins are also important (although this characteristic is
not relevant in the case of angiogenesis) in cholesterol metabolism, since they are the drugs used
to inhibit the synthesis of cholesterol, acting as inhibitors of the enzyme responsible for cholesterol
synthesis, hydroxy-methyl-glutaryl-CoA reductase.
When tumours are very little (less than 2 mm3) their cells don’t suffer from hypoxia and so
angiogenesis is not activated. When they exceed the size of 2 mm3, they acquire an angiogenic
phenotype because they start releasing pro-angiogenic factors, becoming responsible for the
activation of angiogenesis.
The level of angiogenesis is correlated with the degree of malignancy. The density of the
microcirculation is a prognostic indicator: the more angiogenesis is present, the worse the tumour
progression is.
The first phase of tumour development, when the tumour is littler than 2 mm3, is called avascular
phase; the vascular phase is when the tumour is rich in new vessels, i.e. when there is the
acquisition of the angiogenic phenotype by the cells and the formation of new vessels.
When the tumour mass is bigger than 2 mm3 new vessels start forming because hypoxia induces
the release of angiogenic factors that bind the same tumour cells.
41 Folkman then died in 2008, so this is his last review

172
In figure n.12 there is a pre-existing

normal vessel near to a tumour. The
cells of the tumour that are nearer to
the vessel can live and proliferate
normally because they are near
oxygen and nutrients, while the cells
that are far away from the vessels are
suffering and so immediately acquire
the pro-angiogenic phenotype; hypoxia
leads to the activation of transcription
factors, called HIF-1α and HIF-1β42,
which translocate inside the nucleus,
bind to specific parts of the DNA and in
this case lead to the overexpression of
genes coding for several pro- Figure 12
angiogenic factors like VEGF, angiopoietin, NO synthase (nitric oxide is the molecule
that gives the initial vasodilation of the vessel, promoting angiogenesis ), PDGF, FGF and so on.
Other genes involved in cell survival are activated so that endothelial cells survive better, don’t die
and start proliferating.
Angiogenesis is now a target for antitumor therapy; therapies against tumour growth and progression
are mainly directed against these processes, e.g. tumour cell proliferation. Tumour cells are not able
to go in apoptosis, they are very resistant, and they continue to live and proliferate, so that they are
also called “never ending cells”. Therefore, therapies are directed to control the cell cycle, to try
and stop tumour cells proliferation and invasion. In figure n.13 it is possible to see tumour cells that
are invading the blood vessel and are giving metastases and in green are written some of the
possible targets for antitumor therapy:
- signal transduction pathways
important in several processes
activated during tumour
progression;
- several molecules like growth
factors (that make cells grow a
lot);
- several drugs are directed Figure 13
against angiogenesis to try to
counteract the growth of a
tumour.
A lot of drugs have to be administered, but it is very important to stop angiogenesis.
42HIF=hypoxia inducible transcription factor, a transcription factor that is activated and translocated in the nucleus only
when there is a state of hypoxia
173
Folkman had already proposed some strategies: inhibit the synthesis of angiogenic factors in the
tumour, inhibit the translocation of these factors from the tumour to the vessels or inhibit blood vessel
formation.
Figure n.14 shows a vascularized tumour mass when anti-angiogenic drugs are Figure 14
administered to the patient:
in little time you can see
that the vessels disappear
and also the tumour
disappears or becomes
smaller because some of
the cells die, since they
don’t have oxygen
anymore. Therefore, it can
be considered to be a good
therapy.
However, also
Figure 15
resistance to these
drugs can appear.
For example, in
figure n.15 there is a
tumour with vessels
inside; tumoral cells
are releasing a lot of
VEGF, stimulating
angiogenesis. One
possible therapy is
the use of anti-
VEGF monoclonal
antibodies or also
antibodies
directed against the receptor for VEGF: if the antibody binds to VEGF, VEGF is not able to bind
to the receptor anymore because it is covered by the antibody; if the antibody is directed against the
receptor, the receptor is covered and VEGF, even if it is present, is not able to bind, and so it doesn’t
work anymore. In both cases it is an anti-VEGF therapy that leads to the disappearance of the
vessels; in these cases, you can perhaps observe and increase of VEGF, since the cells are suffering
and, therefore, are trying to produce new VEGF even though it doesn’t function since antibodies
against it are being given.
In the early phases the tumour responds well, it is reducing in mass and the vessels are
disappearing, but, at a certain moment, the cells of the tumour start producing lots of the other pro-
angiogenic factors, like PDGF, FGF, angiopoietin 2 and integrins are overexpressed, so even if
anti-VEGF therapies are continued, new vessels are forming thanks to the action of these new
angiogenic factors. In this case, what can be done is to give new drugs that have been developed
against FGF or against PDGF together with the anti-VEGF therapies; these new therapies are not
particular drugs, they are simply antibodies against FGF or against PDGF.
Other therapies besides monoclonal antibodies anti-VEGF or anti-VEGFR are used; some of the
agents are interferon α, which is a molecule that blocks FGF, inhibitors of MMPs or plasminogen
activator, that is also important in the degradation of the matrix, and antibodies against integrins,
which are covered by these antibodies thus preventing endothelial cells binding to the ECM.
174
In figure n.16, anti-VEGF monoclonal antibodies bind to VEGF or its receptor on endothelial cells;
the one represented is an endothelial cell where there are also inhibitors of angiopoietins, of ephrins,
of MMPs and of integrins.
Figure 16
Video: https://www.youtube.com/watch?v=95n4uIS06Qc
METASTASIS
Metastasis is the development of a secondary malignant growth at a distance from a primary

cancer (malignant tumor). Metastasis is the reproduction of the primary malignant tumor in another
place at a distance from the primary area/focus.
This type of tumor is called metastatic tumor or simply secondary tumor.
A primary tumor, the first tumor will develop in a tissue or in an organ but some of the tumor cells
are able to migrate in another organ or tissue far away from the primary focus and they can stop and
develop another tumor, called secondary or metastatic tumor.
Some cancer cells from the primary tumor are to break away, migrate through blood or lymph system
and form a secondary (metastatic) tumor in tissues of other parts of the body.
When this happens, prognosis for the patient worsens. This is because several secondary tumors
form and these are difficult to treat. It is difficult or impossible to recover from this situation:
metastases are responsible for 90% tumor-related mortality.
Formation of metastases indicates that cells from the primary tumor, from the site of first tumor
development, are able to reach another part of the body. Sometimes this event is not predictable. It
also indicates that our immune system was not able to stop the migration of tumor cells, our immune
defenses were not strong enough to block this event.
Keep in mind that the secondary tumor is by morphology, behavior, biochemical aspect, completely
identical to the primary tumor. For example, if breast cancer cells metastasize in the lung, cancer
cells in the lung are breast cancer cells and not lung cancer cells and will be treated with breast
cancer drugs. This allows to identify a primary tumor from a secondary metastatic tumor: breast cells
found in lung, brain or bones indicate a metastasis and not a primary tumor.
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Of course, this is possible as long as the malignant tumor has a grade of differentiation. Malignant
tumors have different grades of differentiation. We can have highly differentiated cancers that still
retain the phenotype (almost the same morphology, behavior, metabolism) of the cells they
originated from and in this case recognition is easier, or more undifferentiated cancers (anaplastic
tumors), whose cells underwent dedifferentiation, in this case it might be difficult to determine the
cells of origin.
For recognition, it is necessary that some characteristic of the cells of origin is maintained.
Ability to metastasize varies among the types of tumor. All malignant tumors can potentially spread
and develop metastases, some cancers do it more frequently and rapidly, some do it slowly, others
(although only a few) statistically do not metastasize, for example the basocellular epithelioma (or
basal cell epithelioma, malignant tumor of the skin) is malignant, but it is known (statistically) that it
doesn’t give origin to metastasis. However, most of malignant tumors do eventually metastasize with
various speeds, some slower some faster. Approximately 30% of newly diagnosed solid tumors
(excluding skin cancers other than melanomas) present with metastases.
But why do only some tumors metastasize and why the metastatic process itself can be inefficient?
Thanks to studies in animals or humans, it has been demonstrated that millions of cells are released
into the circulation or lymphatic system each day from the primary tumor. But only few of them are
able to settle and give origin to a metastasis (tumor heterogeneity), which is due to several reasons.
Most of the circulating cancer cells die immediately as the leave the primary tumor, others will be
killed by the immune system, very few will be able to reach another area, organ, tissue of our body.
Only some specific tumor cells are able to acquire a metastatic phenotype (or metastatic
potential) and are therefore able to produce a metastasis.
Different competing theories have been proposed to explain the origin of the metastatic phenotype:
A. Metastasis is caused by rare variant clones

that develop in the primary tumor. The most
diffused idea is that during the progression of a
tumor, several mutations appear and
accumulate. Initially, we have a tumor
homogeneity: all the cancer cells present the
same mutations, those that allowed the cancer to
develop (light blue cells in figure n.1a); as the
tumor develops, several different mutations
accumulate in genetically unstable cancer cells
and give rise to what is called tumor
heterogeneity (Clonal evolution model).
Therefore, tumor metastatic variants (in pink in
figure n.1a) appear inside the primary tumor: they
are the cells which have acquired a metastatic
phenotype and represent the only ones able to
give origin to a metastasis;
B. Metastasis is caused by the gene expression
pattern of most cells of the primary tumor,
referred to as a metastatic signature. A second Figure 1
theory states that metastasis is caused by the gene expression pattern of most cells of the
primary tumor: all tumor cells have a metastatic signature and are thus potentially able to produce
a metastasis. It has been observed that tumor cells of a subset of primary breast cancer all have
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the same gene expression pattern, similar to that of tumor cells of breast cancer metastasis. It
has been hypothesized that in primary tumors with a “metastatic signature” most, if not all, cells
have the potential to spread and form a new metastasis during early stages of carcinogenesis;
C. A combination of A and B, in which metastatic variants appear in a tumor with a metastatic
gene signature. A third theory combines the first two. The primary tumor has already a
metastatic signature, so potentially all the cells can form a metastasis, but this is not sufficient:
inside the primary tumor, some metastatic variants must appear and, in the end, only the cells
with these variants are able to form a new metastasis. The metastatic signature is thus
necessary but not sufficient for metastasis, additional mutations are needed for metastasis
to occur;
D. Metastasis development is greatly influenced by the tumor stroma, which may regulate
angiogenesis, local invasiveness, and resistance to immune elimination, allowing cells of
the primary tumor, as in C, to become metastatic. Another theory is that metastasis
development is greatly influenced by the tumor stroma, which may regulate angiogenesis, local
invasiveness, resistance to immune elimination. Tumor cells need nutrients and support from the
stroma, that might allow cells of the primary tumor to become metastatic. The ability to
metastasize requires not only properties intrinsic to the cancer cells but also the
characteristics of their microenvironment, such as the components of the stroma, the
presence of infiltrating immune cells, and angiogenesis (necessary to provide the needed
nutrients).
The metastatic cascade

The structural organization and function of normal tissues is determined by interactions between
cells and the ECM. Normal tissues are organized into compartments separated from each other by
two types of ECM: basement membrane and interstitial connective tissue. Although organized
differently, each of these components of ECM is made up of collagens, glycoproteins, and
proteoglycans.
Concerning tumor tissue, it is very important the interaction of tumor cells with extracellular matrix,
at several stages of the metastatic process. Tumor cells, once acquired the metastatic phenotype,
must be able to detach from primary tumor and extracellular matrix, to breach and cross the
underlying basement membrane, to pass through the interstitial connective tissue and finally gain
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access to the vascular circulation or lymphatic system (principal routes of dissemination) by

penetrating the vascular basement membrane. Of course,
tumor cells must then be able to arrest in the tissue where they
will proliferate in a secondary tumor. Therefore, this process is
repeated in reverse when tumor cell emboli extravasate at a
distant site.
Metastases are the results of complex interactions between

cancer cells, extracellular microenvironment and other cells.
Metastatization is a very complex process, the following events

constitute what is called the metastatic cascade:
1. Cell detachment (separation);
2. Disruption of the basement membrane;
3. Cell mobility;
4. Invasion;
5. Penetration of the vascular/lymphatic system, depending on
dissemination route;
6. Circulating tumor cells;
7. Arrest (stasis);
Figure 2
8. Extravasation and proliferation.
Changes in cellular properties occur to allow the development

of an invasive phenotype and progression through the
metastatic cascade.
Different key events of the metastatic cascade are:
1. Carcinoma development (primary cancer) at the
primary focus and acquisition of invasive potential (metastatic phenotype) in some of the
cells, specific gene expression (necessary to allow the detachment from the primary tumor):
1. Tumor metastatic genes upregulated;
2. Metastatic suppressor genes downregulated;
2. Expansive growth, detachment from primary tumor, invasion of the basement membrane
and surrounding tissues (so they must cross the interstitial connective tissue):
1. Enhanched protease activity (e.g. MMPs);
2. Enhanched cell motility interaction with surrounding tissue ECM/stromal cells;
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3. Decrease integrity/strength of cell-cell

contacts (E-cadherin, TJ’s, etc);
3. Angiogenesis, intravasation (blood or
lymphatic vessels), and thus the
crossing of the vascular basement
membrane, and transport in the
circulation around the body:
1. Migration/interaction through ECM;
2. Interaction with vascular cells;
3. Interaction with cavity mesothelial cells
(during transcoelomic metastasis);
4. Invasion into blood vessel;
5. Survival in circulation/immune evasion;
4. Arrest and extravasation (exiting from the
blood vessel) at secondary site, cross of
vascular basement membrane,
movement in the interstitial connective
tissue:
1. Interaction with vascular cells;
2. Interaction with mesothelial cells during Figure 3
transcoelomic metastasis;
3. Invasion and settling into secondary
tissue;
5. Invasion of secondary tissue,
proliferation, angiogenesis and
formation of micro- or macrometastasis with its stroma and circulation:
1. Invasion/migration into secondary tissue;
2. Interaction and adaption to tissue microenvironment;
3. Establishment of new vasculature;
4. Secondary tumor establishment or dormancy.
What is important during the metastatic cascade is tumor cells interaction with the tumor
microenvironment and other types of cells, for example fibroblasts, epithelial cells and adipocytes.
During the process, many molecules are produced and released by each type of cell, in particular
for the mobility: proteolytic enzymes are necessary for the degradation of the extracellular matrix.
Communication between main components of the surrounding microenvironment play vital roles in
enhancing:
- metastatic potential;
- epithelial to mesenchymal transition (EMT);
• immune-evation;
• mesenchymal to epithelial transition (MET);
• angio- and lymphangio-genesis.
We can divide the metastatic cascade into two phases:

1. invasion of the extracellular matrix;
2. vascular dissemination, homing of tumor cells and colonization.
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Invasion of extracellular matrix

Event that initiates the metastatic cascade. It is an active process.
Neoplastic cells with metastatic phenotype must be able to:
- detach from the primary tumor: cells start a reduced or altered synthesis of adhesion
molecules (e.g. thanks to e-cadherins, one of the most important adhesion molecules, cells
are tightly bound to each other) therefore they become less adherent to each other and
mobility is increased. Loosening of tumor cell-tumor cell interactions;
- synthesize and release an excess of proteolytic enzymes (e.g. MMPs, matrix
metalloproteinases, able to degrade ECM) to attack and degrade the components of the
ECM, favoring tumour cell migration. This event is favoured by an increased release of lactic
acid, which decreases the pH allowing the proteolytic enzymes to work better;
- cross the basement membrane, move into interstitial connective tissue and invade the
blood or lymphatic capillaries;
- produce a large amount of lactic acid, which promotes activity of proteases by lowering
the pH;
- release other adhesion molecules (e.g. integrins): to move in the interstitial connective
tissue, cells need the interaction with molecules of the
fundamental substance (e.g. collagen) and with the cells of Figure 4
the connective tissue. These adhesion molecules are
necessary for tumor cells to interact with ECM components
and migrate. It is like when a person walks on the stairs and
needs to hold in order not to fall.
Vascular dissemination, homing of tumor cells and

colonization
In the second phase of the metastatic event, it is fundamental that
tumoral cells are able to reach one of the routes for dissemination. In
the hematogenous spread of a tumor, the cells cross the basement
membrane and reach the blood stream where most of them die; in
fact, in the blood they are destroyed by the immune system because
they enter into contact with the lymphocytes. However, tumor cells
are clever and have found a solution to escape the immune defenses:
they aggregate with each other, forming a sort of an embolus, and
they cover themselves with platelets (see figure n.2), which are
normally present in the blood and therefore are not recognized by the
immune system. In this way, some of the tumor cells43 are able to
reach the end of the metastatic cascade.
Therefore, neoplastic cells with metastatic phenotype must be able
to:
- Survive the immune defense systems (natural and
acquired) once they arrive in the blood or in the lymph, where
most cells are usually killed by the immune system.
Metastatic cells are able aggregate with platelets and form a sort of embolus so that the
immune system can’t recognize them;
43
Around 1 million tumor cells reach the blood stream every day
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- Stop at a certain site of the blood stream (or lymphatic), attach to the endothelium thank
to expression of certain adhesion molecules, to cross again the vascular basement
membrane and enter a specific tissue or organ;
- Proliferate and invade the tissues;
- Produce pro-angiogenesis factors (e.g. VEGF), stimulate fibroblasts and endothelial cells
of neighboring capillaries to grow, which leads to the development of a stroma and
vasculature of the cancer cells that can proliferate and form a secondary tumor.
The metastatic process can be inefficient, at any point of the metastatic cascade (that involves many
steps, each of which may be inefficient and is subject to a multitude of controls), cancer cells can die
if something goes wrong (let’s say if something goes right).
Most of patients with cancer soon or later develop metastases, it is a quite frequent event.
There are different factors that favor the engraftment of metastasis:
- Immunosuppressive factors: they lead to inhibition of immune response, alteration of
natural killer cells (NK cells are important in the recognition and destruction of tumor cells);
- Adhesion factors: homotypic binding between neoplastic cell-neoplastic cell; adhesion
molecules (integrins, laminin receptors, CD44 expressed on normal T lymphocytes and used
by neoplastic cells to promote metastasis);
- Procoagulant and platelet aggregating factors: help the migration of the tumor cells
because of the heterotypic binding between neoplastic cell and platelets leading to aggregate
formation, which protect circulating cancer cells from the immune system;
- Proteolytic enzymes.
Dissemination routes
Tumor cells of the primary tumor can use different modalities of metastatic dissemination:
- Canalicular routes (most common ways of spreading):
o Vascular route, through blood circulation;
o Lymphatic route, through the lymphatic system;
- Transcoelomic route, penetrating the surface of the peritoneal, pleural, pericardial,
subarachnoid spaces (for example ovarian tumors spread transperitoneally to the surface of
the liver);
- By contact or contiguity;
- By grafting.
Vascular Route
Cells enter into blood vessels and exploit bloodstream to disseminate.
Cancers that have a high probability and frequency of metastatic spread by vascular route:
- Sarcomas: all of them; they are the malignant tumors of mesenchymal tissues (i.e. basically
all the tissues of the body except epithelia and brain tissue), sarcomas use only the vascular
route;
- Epithelial tumors of the digestive tract (stomach, intestine);
- Tumors of internal secretion glands (carcinomas, breast cancer mostly use lymphatic
route, more rarely the vascular route, but it is not impossible).
Figure 5
Cancer cells usually intravasate into capillaries or venules, rarely arteries and
arterioles because their wall is thicker (higher muscular component) and more difficult
to cross.
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The direction of the vascular flow is important as

it causes corresponding movement of cancer
cells arising in different organs (figure n.5); for
instance, different types of metastases can be
distinguished according to their origin:
- Portal Vein Type Metastases: the portal
vein collects blood coming from stomach,
intestine, spleen, colon, pancreas, etc.
Primary tumors that develop in these
organs (stomach, small intestine, colon,
large intestine and sigma tumors) can
give rise to liver metastases (because the
first organ that they reach is the liver);
- Vena Cava Type Metastasis: they come from primary tumors localized in organs whose
blood is collected by the vena cava, including the liver. The vena cava, then, through the right
side of the heart can reach the lung: pulmonary metastases can come from neoplastic cells
from a primary tumor in the liver (e.g. liver carcinoma) through the vena cava; this doesn’t
mean that other organs will not be metastasized, but only that there is a higher probability to
find a liver metastasis in the lung;
- Lung Vein Type Metastasis: neoplastic cells coming from a lung tumor reach the left side
of the heart through the pulmonary veins, from where they can theoretically reach all of the
body through the arteries and cause metastases in all organs.
Figure n.6 shows the preferential sites of metastasis by blood route:

in the brain, lung, liver, adrenal glands and bones.
Breast cancer cells that leave the primary tumor by blood vessels
will be carried by the blood flow first through the heart and then to
the capillary beds of the lungs. Some cancer cells might pass
through the lung to enter the systemic arterial system, where they
are transported to remote organs, such as bone. Others might form
metastases in the lung, which might then shed cells to the arterial
flow. By contrast, colon cancer cells will be taken by the hepatic-
portal circulatory system first to the liver. There is no direct flow from
the lymphatic system to other organs, so cancer cells within it — for
example breast cancer cells — must enter the venous system to be Figure 6
transported to remote organs.
Lymphatic route
The tumors with a greater probability and frequency of metastatic diffusion by lymphatic route are
the carcinomas (epithelial tumors), of which breast tumor is the typical example (some others will
instead use the vascular route).
Neoplastic cells enter mainly through lymphatic capillaries (small, thin wall). After the first phase of
diffusion, in a second moment from the lymphatic system neoplastic cells can enter blood circulation
at the level of the thoracic duct and right lymphatic duct which drain into the systemic venous system.
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Regional lymph nodes act as a filter and

first protective barrier; when tumor cells
Figure 7
reach one of them, it is possible to see
hyperplasia in the lymph node due to
lymphocyte proliferation, histiocytic and
plasma cell reaction and the lymphocyte
is enlarged. However, most of the times,
tumor cells are more aggressive and can
carry on to reach other lymph nodes in
other parts of the body. For example, if a
breast carcinoma is diagnosed, lymph
nodes will be immediately checked for the
presence of neoplastic cells. If neoplastic
cells are present, it means that the
metastatic cascade has already started.
Distant breast cancer metastases occur in the bone, liver, brain, lung and distant lymph nodes.
Transcoelomic route
Primary tumors of organs lined with serous membrane (for example the thoracic and abdominal
cavities, lung, heart, intestine) can spread through the celomatic spaces (pleura, pericardium,
peritoneum) giving metastases in these serous membranes.
- Lung tumors metastasize in the parietal pleura;
- Heart tumors are very rare, metastases at the level of pericardium are thus rare;
- Tumors of the organs contained in the peritoneal cavity (e.g. stomach and intestine tumors)
metastasize in the intraperitoneal serum (parietal and/or visceral peritoneum). Krukenberg
tumor (rare): it is a particular metastasis present at the level of both ovaries resulting from
primary tumors of the stomach and large intestine exfoliating into the peritoneum; possible
presence of ascites and intrapleural fluid.
Primary tumors of abdominal organs: fluid accumulation
(ascites or transudate) containing fallen neoplastic cells for
dripping into the cavity, called ascites-tumors.
Lung neoplastic cells can diffuse in three ways: vascular

route, lymphatic route and transcoelomic route, by which a
metastasis can develop in the space between the two layers
of the pleura. Lung tumor and metastasis from primary lung
tumor are quite frequent.
By contact (Or contiguity) Figure 8

Tumor cells spread, even without contiguity of
tissue, from one organ to another adjacent.
For example:
- metastasis in the palate due to contact with a primary tumor of the tongue;
- metastasis in the lower lip by contact with a primary tumor of the upper lip (or vice versa);
- metastasis in the gum due to contact with a cheek primary tumor;
- metastasis in the liver by contact with a primary tumor of the head of the pancreas.
By grafting
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Sometimes, although very rarely, neoplastic cells are accidentally disseminated during surgery by
surgical instruments or gloves, and so sometimes contamination occurs and occasionally a
metastasis develops.
The secondary tumor occurs at the scar or in other regions of the operative field away from the
primary tumor.
Organ tropism of metastatic localization

Most of metastasis don’t occur at random sites, there’s a preferential localization of metastasis
due to many factors that favor invasion of specific organs or tissues. This phenomenon in called
organ tropism.
Organ tropism is the preferential localization of metastases in certain organs rather than in others.
For instance, if it is known that there is a primary tumor in the intestine, one of the organs that can
be affected by metastases is the liver, although it is not the only site for metastases: cells can stop
there, or some can go on and reach the heart through the vena cava.
Many factors are responsible of organ tropism:
- General Hydrodynamic Factors;
- Local Hydrodynamic Factors;
- Local Humoral Factors;
- Tissue Cellular Factors;
- Embryogenetic Factors.
General Hydrodynamic Factors

Metastases depend on the direction of the vascular flow (as already explained before).
It is important to know which is the first organ that metastatic cells will encounter after detachment
from the primary tumor, because there is a higher probability that they will stop there and give rise
to a secondary tumor.
The probability that the neoplastic cells arrive and metastasize in an organ rather than another is
strictly dependent on the amount of blood that flows to that organ (abundant circulation is
frequently linked to metastases).
Local Hydrodynamic factors

Contractility is an impediment to the localization of metastasis. Metastases at the level of muscles,
including the heart, are very rare for hemodynamic conditions of the local circulation due to frequent
local contraction and decontraction. In fact, the contraction of the muscles can disturb the attachment
of the tumor cells.
On the contrary, stiffness is a contributing factor. Bones, especially spongy bone tissues (because
the trabeculae can block the tumor cells), are stiff and highly irrorated and, therefore, metastases
are frequent (e.g. vertebrae).
Tissue Cellular Factors
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Metastases can develop in tissues

which have less defenses, for instance
the brain is one of the principal sites for
metastases because it is very poor in
defenses, while it is very difficult for
metastases to develop in the spleen or
in lymphatic. Reticular-histiocytic
tissues and immunological defenses
prevent the diffusion and engraftment
of metastases.
Local Humoral Factors

Metastases develop in tissues that
favor neoplastic cells proliferation, in
which there is an adequate
environment for growth.
- Factors with chemotactic
activity;
- Factors that increase affinity of neoplastic cell for the tissue, interaction between cell receptor
and ECM components (e.g. fibronectin);
- Factors that allow neoplastic cell adhesion to vessel walls and to nearby structures (e.g.
integrins, VCAM, ICAM, cadherins, selectins);
- Growth factors and hormones.
Embryogenetic Factors
Some cells during embryogenesis derive from the same embryonic tissue, for example brain cells
and melanocytes. There is some affinity between these very different cell types. Melanoma, a very
aggressive tumor of the skin, often firstly metastasizes in the brain (that is also a tissue with poor
defenses), which is why brain metastasis must be immediately checked after a melanoma is
diagnosed.
Table: organ specificity of metastasis

These are the most common metastatization sites for each cancer type, however no organ must
ever be excluded, in rare cases metastases develop in other sites.
For example:
- Breast cancer can metastasize very easily the bone, the brain, the liver and the lung;
- Melanoma can metastasize in the brain, the bone the liver, the lung, the skin and the muscle;
- Lung cancer can metastasize in the adrenal gland, the bone, the liver, the other lung, the skin
and the muscle;
- Colon cancer mainly gives origin to metastasis in liver, lung and peritoneum, but metastasis
in the bone can also occur more rarely.
Particular examples:
- Tumors of the upper rectum: hepatic metastases (by upper tributary hemorrhoidal plexus of
the portal system);
- Tumors of the inferior rectum: pulmonary metastases (by lower hemorrhoidal plexus tributary
of the surface vessels of the body and therefore of the vena cava);
- Middle rectal tumors: both hepatic and pulmonary metastases;
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- Prostate and thyroid tumors: bone metastases (by antero-venous anastomoses or by the
dissemination of neoplastic cells through the finer pulmonary capillaries)
TUMOR CLASSIFICATION
Today’s lesson will be about the different types of tumors, their general classification and which
criteria are used. It depends mainly on the histogenetic aspect, so on the characteristics of the
cell/tissue in which the tumor cell grows. The other criteria which is close to the histological and
histogenetic characteristics is the biological behaviors that defines whether a tumor is benign or
malignant. The most used criterium is the histogenetic aspect, which indicates from which tissue the
tumour is originating. If we can’t recognize the type of tissue from which the tumor arises, so its
histogenetic origin, we define the tumour as anaplastic.
In this case the characteristics of the cells are important, so whether they are big, giant or small, their
volume and their shape. To recognize this type of tumors the presence of tumor markers is important,
not the serum tumor markers, but the immunohistochemical tumors markers, the antigens that
can be inside the membrane of the tumor cell. Histogenetic classification follows the directives of the
World Health Organization, according to whom we can divide tumors in:
1. Epithelial tumors
- of skin and mucosa (in Italian, tumori epiteliali di rivestimento e delle mucose), they can
be divided in:
o Benign: polyp, papilloma, wart, condyloma and acanthoma;
o Malignant: there are two of importance which belong to the class of epitheliomas
(also called carcinomas): basal epithelioma and squamo-cellular epithelioma;
- glandular epithelium:
o Benign: adenoma;
o Malignant: carcinoma;
2. Mesenchymal origin tumors: they are all types of tumors of the connective tissue, so fibrous
from chondrocytes, cartilaginous or bone-related and so on, as well as the ones of the blood
since blood is of mesenchymal derivation. Blood tumors belong although to a separate
subgroup with respect to the other connective tissues.
- Non-hemolymphopoietic origin:
o Benign: name is related to which type of connective they come from i.e. fibroma,
chondroma, osteoma, lipoma and so on;
o Malignant: can be of two types depending on the type of dysplasia and its severity.
Blastic sarcoma (fibrosarcoma, osteosarcoma) presents a minor degree of
differentiation compared to the second type which is anaplastic sarcoma, in this
undifferentiated sarcoma;
- Hemolymphopoietic tumors: blood and lymphatic vessels (in Italian, tumori dei tessuti
emolinfopoietici);
3. Nervous tissue tumors;
4. Mixed tumors.
Natural history of a tumor

The history of a tumor can be divided into steps: the first step, which is considered a preclinical
phase, is observed only microscopically whereas the second step is the symptomatic phase with
the presence of a localized mass and is related to the nodular tissue (foci of tumors); finally, there is
the advanced phase where there is metastatization.
Epidemiology
Different types of studies related to the condition of pre-cancerosis are very important because they
can help us realize if the tumor is particularly aggressive or not and whether or not a population is
more subjected to a particular tumor. These types of quests belong to the field of epidemiology and
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are of great importance when it comes to tumor development and its type. The considered factors
are: age, sex, diet, habits and so on and their correlation with the onset of certain tumors. We have
to consider the incidence, prevalence and survival as well as number of deaths.
Incidence is related to the risk of the development of tumors. It is the number of new cases of tumor
that occur in a particular population in a certain period of time. Prevalence is the number of
individuals affected by a certain disease in a specific time in a given population. In the same way
survival is related to the number of individuals that are affected by a particular tumor but can survive
in a precise population and time. Number of dead is the opposite of survival.
The incidence is generally calculated as a percentage of number of new cases in a unit of time, such
as year. It is important because it gives us the knowledge of the probability
of getting sick in a particular range of time or age.
The epidemiological data in figure n. 1 about the
incidence of cancer come from Globocan,
which is a source of statistic data of the
international agency for cancer research. It is
important to consider these data because for
instance it allows us to see if a specific
population is more subject to cancer or not.
Here it is shown the incidence of new cases in
2018. The main types of cancer that we can find
in the pie chart are: breasts, lung, colorectal,
prostate and stomach. These cancers are the
ones presenting the highest risk factors.
Looking at the pie chart of the prevalence in
Figure 6 figure n.2, always related to different types of
cancers in 2018, the main cancers that
developed in 2018 are still breast, colorectal, and lung. Finally, looking at the pie charts of the
mortality in figure n.3, we can see that lung and colorectal cancer have the highest mortality, whereas
breast cancer has a lower mortality compared to its incidence and prevalence due to different types
of prevention, secondary prevention and early diagnosis.
Figure 8 Figure 7
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Epithelial tumors
Figure n.4 reminds us that every layer of our skin can develop tumors. We can have melanocytic,
keratinocytic or epithelial tumors which in general can all invade the dermis and hypodermis. It is
important to understand the anatomy of the skin and consider the different layers. In particular the
presence of a basal and a squamous layer: squamous, basal and horny layers are involved in
development of tumours. Moreover, melanocytes are also involved in the development of a very
aggressive tumor, probably the most aggressive epithelial tumor, i.e. melanoma.
Carcinogenesis
There are many factors that damage the skin layers leading to development of cancer. UV radiation
coming from the sun is considered to be the main factor that can induce the development of epithelial
tumors. The incidence of different types of skin cancer is increasing. The epithelial tumor is very
important also incidence and Figure 9
prevalence-wise, especially because
strong UV radiations can induce
different types of mutations. This factor
(UV radiation) can or cannot be
associated with epithelial cancer, but
there is also another risk factor which
is represented by particular viruses
(viral infection). Those viruses are, in
particular, papilloma virus.
Associated with viral infections and UV
radiation coming from the sun, there is
also immunosuppression: for
instance, in transplant patients or
individuals who have certain
occupations, immunosuppression can easily lead to this type of cancer. Therefore, carcinogenesis
related to epithelial tumors can be related to genetic factors, X ray radiation and chemicals.
In this lesson we will consider benign and malignant cancers, but also the pre-cancerosis condition.
There are a number of different pre-cancerosis lesions that are considered malignant because they
easily develop into one of the most malignant tumors, the squamo-cellular carcinoma (in Italian,
carcinoma spino- o squamo-cellulare). Those pre-cancerous conditions are: xeroderma
pigmentosum, actinic keratosis, viral keratosis (Bowenoid papulosis, similar to Bowen disease,
another type of pre-carcerosis and epidermolysis…), leucoplakia and erythroplakia.
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Xeroderma pigmentosum (discussed in lecture n.9) is hereditary and due to impaired DNA repair
system; we can see this type of pre-cancerosis especially in children exposed to UV, who, since they
cannot repair the damage made to DNA by the radiation, can develop freckles and with time
squamous cancer, as shown in figure n.5.
Figure 10
Actinic keratosis (in Italian, cheratosi attinica) is again

due to prolonged exposure to radiation. Ranging from
1-20% of the actinic keratosis can evolve to squamous
cells carcinoma. It is called active keratosis because
it shows an increase in the number of layer of keratin,
called hyperkeratosis, which can present necrotic
Figure 11 parts. Actinic keratosis is especially seen in this part
of the head because of its exposure to the sun; it is
very visible and easy to detect. Shown in figure n.6
and 7.
Figure 12: actinic keratosis
Bowenoid disease, shown in figure n.8: the

name comes from Bowen’s disease which is
not only considered a pre-cancerosis but
also as a carcinoma in situ, intraepidermal.
This tumor develops commonly between 60
and 80 years of life and its origin can have a
degree of familiarity: it has been noticed
that in certain populations that presents a
major risk compared to others, for example
80% of affected people in the UK are
women. They are common in areas of the
head and neck due to exposure to UV rays
and, normally, the lines between/of tumors
Figure 13
and pre-tumoral lesions are well defined and
clear. The growth of this precancerous
lesion is very slow, only 1 or 2% of Bowen’s disease cases develop into cancer. Nevertheless, it is
still considered carcinoma in situ because it can directly become malignant, it doesn’t first pass
through the benign tumour phase, as other types of benign
diseases do, it directly acquires the malignant features.
Leucoplakia, shown in figure n.9, is a condition afflicting
the oral or genital mucosa. Very few cases of this type of
pre-cancerous disease can induce the progression toward
the malignant tumor. Different factors can induce this type
of lesion to move toward malignancy, such as papilloma
virus (especially in relation to genital mucosa) and tobacco
abuse (in relation to the oral mucosa). What is important is Figure 9
189
that it is very difficult that this precancerous lesion will progress toward malignancy by itself, but if it
does (due to stimulation of
abovementioned factors) it is very
aggressive.
Benign tumors
Can be divided in:
- Warts;
- Acanthoma;
- Condyloma;
- Papilloma;
- Polyp.
This division is related to the

morphology of the protrusion of these
new formations from the skin or
mucosa. Depending on the type, they
can originate and grow either on the skin or the mucosa. Based on the morphology there are specific
factors and characteristics, such as the axis called fibro-connective axis. It is important to maintain
the structure of the formation (in this case polyp) outside the skin and to provide nutrients, therefore
in this fibroconnective axis there is also a vascular compartment that brings nutrition to the
extruding structure, which can be called either fibroconnective axis or projection. Depending on
the structure of this axis, we can distinguish polyps from papilloma: polyps have a single axis, while
in papillomas the axis can be divided in many small axes, resembling a tree. The basal part of this
projection can be either very narrow or large. Narrow is the type of base of polyps and papilloma
whereas the large base characterises acanthoma, kerato-acanthoma, condylomas and warts.
Morphology is very important to distinguish the different benign tumors. There are different types of
warts (in Italian, verruche). They are benign tumors that can present with familiarity but also can
appear due to an infection.
1. The most known and common type of wart are the
seborrheic keratosis, (in Italian, verruche seborroiche).
Figure n.10 represents a histological preparation of this
condition, whereas the three images below represent the
phenotypical manifestation. They are absolutely benign:
they cannot develop into malignant cancer. The
epidemiology is unknown, but it is related to the familiarity.
This type of keratosis is well circumscribed and defined, can
present different colorations, although most of them are
brown and can also be part of the skin aging process and
therefore appear especially in the elderly population.
Figure 10
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Histologically they are similar to the other type that we have seen: they present
hyperkeratosis, acanthosis and papillomatosis because they enlarge like a papilloma;
2. Viral warts (verruche normali) are mainly due to the infection by papilloma virus. It is
contracted through normal contact with the virus, especially in areas of the skin where there
could be microlesions and microabrasions which allow the virus to enter and penetrate the
external barrier. HPV (human papilloma virus) is especially common in the genital areas
where the epithelium can suffer microlesions;
Common warts.
3. Common warts (in Italian, verruche
volgari), which can be cutaneous or can
also grow as a papule, are confined and
localized, the wart has this type of
morphology;
4. Another benign tumor that shares the

common wart morphology is the
keratoacanthoma; it is pretty similar to the
wart, but at the top of the lesion there is a very
strong keratinization and the part is
surrounded by chronic inflammation. It is
commonly due to sun exposure and in this
type of benign tumor, although it is benign,
the central part can undergo necrosis and it
is located in areas, like the neck. This type of
tumor can continuously undergo necrosis
and induce very large scars;
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5. Condyloma is very similar to the keratoacanthoma. In this

case, the upper part of the wart is lined around; in these other lesions,
the new formations that correspond to the benign tumor can grow
inside the connective tissue, but the boundaries are organized and
well defined, so there is no possibility that a condyloma can invade
the subcutaneous layer in an infiltrating malignant way.
Figure 11
Condyloma can also be called condylomata
acuminata, (depicted in figure n.11): they consist in
dermal and epidermal papules and can normally
extend in different sites, so they can grow and
spread; this formation is often sexually transmitted
and can be found in the vagina, urethra and extend
to the perirectal epithelium.
Figure n.12 presents a histological representation of
the condyloma. Here this type of cells is very similar
to the squamous layer, but this growth is confined
and does not spread;
Figure 12
6. Polyp: not everybody considers polyp as a
benign tumor, but a pre-cancerosis. This is one of the
type of lesions that can change and progress toward
malignancy. Its features change and express malignancy. The polyp can be both in the
mucosa (internal too, i.e. gastrointestinal) or
in the skin. The types of polyps depend on
the type of growth. For example, the two
main types are:
- Hyperplastic polyps, which are the
simplest type of polyps;
- Adenomatous polyps, which resemble
in the protrusion the inside of a gland.
7. Papilloma can also be seen in the

mucosa and skin.
NB. Both polyps and papilloma can be found

in the mucosa. Polyps are present mainly in
the gastric, whereas in the colon mucosa we
can see both polyps and papillomas. The most important
ones, however, are the polyps. The adenomatous familial
polyposis is due, for instance, to cancer progression,
because polyps can undergo malignancy from a simple
dysplasia: a hyperplastic polyp can become an edema with
severe dysplasia and then can undergo adeno-carcinosis.
Intestinal tumors that can present malignant features
present this condition called FAP, aka familial
adenomatous polyposis (in Italian, poliposi familiare
adenomatosa), as shown in figure n.13. Polyposis means Figure 13
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that there are many polyps extruding from the surface of the mucosa. It is a hereditary disease,
due to a deletion of APC, a gene that normally is important in the destruction of catenins: an excess
of catenin is normally destroyed by APC, which is therefore an anti-oncogene, an onco-suppressor
gene44. Onco-suppressor genes are mutated for deletions and must be mutated for both alleles.
In this type of patient there is a polyposis, in which the colonic mucosa develops thousands of polyps.
Not only polyps themselves are benign tumors, but
the disease is a pre-cancerous condition because it
is impossible that during a colonoscopy clinicians
can resect thousands of polyps and one of these
thousands, although very easily removed, can
change into cancer.
Papilloma: can originate in the mucosa or the skin,
but mainly in the mucosa. It can originate and be
found in the pharynx, oropharynx, larynx and urethra. Different types of papilloma viruses, which are
called like this because the virus induces morphological lesions that look like papillomas, are isolated
in fact from papilloma.
Some types of papilloma could not be
caused by a virus, for instance in the
intestinal tissue it originates without the
viral infection, in this case the causes are
unknown.
Malignant lesions of the skin

There are two types of malignant lesions:
1. Basal cell carcinoma: also called basal epithelioma (in Italian, epitelioma baso-cellulare);
2. Squamous cell carcinoma also called spino-cellular carcinoma (in Italian, epitelioma
spino-cellulare o squamo-cellulare).
The different types can be dependent on the localization, on the affected tissue: in the skin it is called
spino-carcinoma, whereas in the mucosa it is called squamo-carcinoma. However, they are the exact
same type of lesion.
Baso-cellular carcinoma
Baso-cellular epithelioma is the most common type of malignant cancer. This type of malignant
lesion develops for a pre-existing lesion such as Bowen’s disease or can also originate from the
normal exposure to the sun (so UV radiation). It is usually located on the head and neck and it is
NOT a metastatic tumor. If it doesn’t metastasize why is it malignant? Because it relapses
frequently. It continues to grow even if it is resected and causes relapses in the same areas that it
was resected from. In Italian the term relapse is “recidivare”.
In this case, due to its location, the continue relapsing and growth of this tumor in the same site can
induce large necrosis and scarring. Why is it called basal cell carcinoma? Because the cells that
compose this carcinoma are cells resembling the basal cells (of the basal layer). Those cells grow
forming islands of tumoral cells, which are confined and not infiltrating, but located in the
connective. They can have cells with many atypias: small quantity of cytoplasm, large nucleus,
abnormal morphology and rectangular shape.
44
What type of mutation can onco-suppressor genes have? A deletion.
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Baso-cellular epithelioma are non-healing lesions and can be of different types depending on the
volume they occupy, so their dimensions. The nodular type is the most common. It is very confined
and easy to resect but we still have to consider that relapse is also common.
Basal cell carcinoma (Basocellular epithelioma). Cell are similar to

those deriving from basal layer and the tissue presents malignant
Squamous cell carcinoma or spino basal cells with large nuclei.
cellular carcinoma. (in Italian,
Carcinoma squamoso o spino-cellulare)
Most of the pre-malignant lesions showed before such as actinic keratosis and Bowen’s disease can
progress into squamo-cellular carcinoma. The exposure to UV is very important and this condition
presents with a deletion in the p53 gene and there is an inverse relationship with skin pigmentation
and squamous cell pigmentation, this means that the lighter skin tone we have the higher the risk of
suffering from this tumor. The main characteristic that distinguishes this carcinoma is the
morphological one, it is very infiltrating compared to the other and can infiltrate into the connective
by forming particular processes called horny pearls (in Italian, perle cornee), which are keratinized
processes, shown in figure n.14. They also tend to form
islands whose central part is necrotic (mainly squamous
necrotic) which makes the appearance of this tumor whitish
as a pearl. In the figure we can see this type of tumor, called
squamous cell carcinoma because the tumor cells are very
similar to the cells that origin from the spinous layer of
the skin. Figure n.15 is a magnification of the horn pearls,
also called keratinic pearls. Here tumor cells continue to
grow towards the central part and here they do not have the
possibility to obtain nutrients and undergo necrosis. This is a
Figure 14: horn pearls.
keratin necrosis. This particular staining of the island is why
this type of tumor is called spino or squamocellular
epithelioma with horn pearls. The shape of the islands resembles a rose and this is why they are
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also called “rosette”. It is very malignant with a significant metastatization potential and can be
limited to lymph nodes.
Figure 15 MELANOCYTIC LESIONS

With this lesson, we finish the classification of
epithelial tumors and then we will go on with the study of the mesenchymal tumors and hematopoietic
tumors. Melanocytic lesions are related to melanocytes which derive from the neural crest, which
must be kept in mind in order to understand the organotropism of the tumor/metastases related to
melanoma. They are present in the basal layer of the epidermis and their function is to secrete
melanin. These cells are not properly considered as epithelial cells but as in a nest of epithelial
cells. This unit has ramifications, like dendritic cells, prolongs its parts in order to transfer
melanosomes into keratinocytes through these protrusions.
Melanocytic proliferative lesions can be divided into two classes:
- Benign lesions, furtherly subdivided in:
o Hyperplasia;
o Benign neoplasia;
- Malignant lesions: related to only one malignant tumor of melanocytes which is called
melanoma.
Figure n.1 shows the benign lesions where melanocytes are

hyperplastic, so they induce an increase in the mass only,
but they remain into the epidermal layer.
Figure 1
Among these types of melanocytic lesions, the most known
is the formation of lentigines (lentigo, freckles) which is
mainly due to mutation of the gene called melanocortin 1
receptor (MC1R) that is associated with a particular type of skin (fair) and red hair, due to
proliferation of melanocytes (=increase of the number of cells). Lentigo are different from another
type of process (not properly a lesion) that induces the ephelides in which there is no proliferation:
the number of melanocytes is identical, but there is only an accumulation of melanin
only after the exposure45.
Another particular type of lentigo can induce an extended change in the color of the
skin and is called caffè au lait (coffee with milk, caffè macchiato).
Lentigo solaris increases after prolonged exposure of the subjects to UV light.
45
She did not say exposure to what, my guess is UV light/rays.
195
Lentigo simplex is
enhanced
during exposure but
is also associated to induction of adult damage: it is seen in adults. [From
slides: associated with chronic cumulative sun exposure and cutaneous signs
of photodamage; found in adults.]
These types of damage can be considered hyperplastic lesions also and are not tumoral.
Benign melanocytic proliferative lesions (melanocytic neoplasia)
Benign tumors are called in general nevus, pl. nevi (mole, birthmark; in Italian, neo). Usually, they
can appear during childhood and later on in life also during old age. Sun exposure is one of the
cause factors of development of this type of benign proliferation. Some of these developments are
due to different types of mutations, the main of which is that of BRAF gene which induces high
proliferation of these melanocytes.
These types of nevi are divided in three classes based on the location where melanocytes are:
- Junctional: in the junction between the external layer and the dermal
layer (figure n.2a);
- Compound: they are deeper and can be seen into the dermis (figure
n.2b);
- Dermal: deepest (figure n.2c).
Figure 2a
Figure 2b
Melanoma
Figure 2c
The malignant
lesion is the
melanoma. It is a very aggressive tumor and is characterized by color
variations and irregular borders (important characteristic of this type
of tumor). It might arise “de novo” or it can be a change of a dysplastic
nevus e.g. the clinician will tell you to wait and see how it grows, it is
better to not resect it (remove it), because often from a dysplastic
nevus, melanoma can originate.
UV exposure is the major risk factor for development of this tumor:
there is a direct association between the sunburns during childhood and risk of melanoma.
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Other than environmental factors (e.g. sunshine), we have genetic factors: mutation of BRAF (like
in nevus) or of the receptor for melanocortin 1 and the deletion of the p16 allele since this is an
anti-oncogene. Also, the family history is important.
ABCD rule
Melanoma is classified in different classes depending on
the morphology. The classification is called ABCD rule:
- A is related to the asymmetry of the morphology;
- B is related to the even edge in benign borders
and uneven edges in malignant ones;
- C is related to the change of color which is
darker in the malignant ones;
- D is related to the diameter of the nevus: if it is
>6mm and there are changes in the border, it is
sure that this is a melanoma.
Staging Clark levels – Bleskow thickness

The staging used to assess the degree of malignancy of
melanoma is the staging Clark that is related to the
depth and thickness of the lesion. Therefore, Clark level
1 is considered an in situ melanoma which is only in the
epidermis, Clark 2 in the papillary dermis, Clark 3 also in
the reticular interphase, Clark 4 in the reticular dermis
and Clark 5 in the subcutis. The dimension and type of
tissues that this type of tumor can invade are important.
Clark staging is a particular classification used to classify
melanoma.
TNM classification
Clinical classification of malignant tumors is the staging
that identifies a particular moment in the progression of the
malignancy of a tumor. This stage is related to:
- the mass of the tumor, therefore is called “T”;
- the involvement of lymph nodes, and by nodes, this is called “N”;
- if there are metastases or not, this is called “M”.
Therefore, the staging classification, which is a general classification of all types of tumor, is called
TNM. The volume of the tumor can be 2 mm or more than 2mm and based on it, we can divide the
T in subclasses: T1, T2, T3 and T4. If there is absolutely no evidence of the presence of a tumor
mass, we can define it T0. If we think that there is a possibility that in some parts of the body there
is a tumor, but we are not able to detect it, this is called TX.
Similarly, the N, which is the involvement of lymph nodes, can be further subdivided in N1, N2, N3.
If there is involvement of 1 node only, we have N1; if there is the involvement of 2 nodes, we have
N2, if there is the involvement of 3 or more nodes, we have N3. If there is no involvement of nodes,
we define it N0.
M stands for metastases and has only two numbers: M0 and M1, that is there is no metastases or
there are metastases. If we see that there is a tumor which has a stage T1, N1 and M0, it corresponds
to only stage 1 tumor: there is involvement of lymph nodes but is not metastasizing.
The worst tumor is T4, N1/2/2 and M1.
LUNG, BREAST AND PROSTATE TUMORS
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Other epithelial lesions of three important types of tumors: breast, prostate and lung, that have been
estimated to have the major incidence worldwide. In particular, lung and breast have the major
percentage of mortality rate in the world.
Lung tumors
In the lung we have precancerous lesions, benign tumors and malignant tumors.
Bianca’s question: why is breast cancer the one with the highest incidence and mortality rate? Is it
because it is very common to have breast cancer or because it is more aggressive than other
cancers? The question arises because there are many tumors which can metastasize way more
easily than breast cancer and also you hear about people healing from it more often compared to
people healing from other types of cancer.
Answer: It is not really possible to answer, it depends on the epidemiological data. There is a high
incidence of breast cancer, and possibly the high mortality can be due to the time of diagnosis
because it is very important. So, you say that there are tumors which metastasize more than breast
cancer and do not have such a high incidence. This is because there are tumors such as melanomas
which are very aggressive, more than breast cancer, but the number of subjects that develop this
type of cancer is smaller. It also depends on risk factors: in these three cancers, the environment,
other than UV light which is a risk factor for melanoma, is very important, but also estrogen and
hormones in general. These types of cancer (lung, breast and prostate) can develop more easily
because there are many environmental factors that can induce them.
Precancerous lesions
Pneumoconiosis is considered as a precancerous
condition (seen in lesson related to inflammation).
Asbestosis is a kind of chronic inflammation, not only
related to chemicals e.g. asbestos, but also to foreign
bodies, the continue and persistent local inflammation can
induce the cells of that tissue to develop a dysplasia and
from there (low grade dysplasia) to develop a severe
dysplasia and subsequently cancer. All these types of
diseases are considered precancerous lesions.
Metaplasia is another precancerous

condition e.g. metaplasia of bronchial epithelium can be
related to an important environment factor which is air
pollution and smoking.
Another type of precancerosis is the tumorlet.

Tumorlets (in Italian, formazioni neoplastiformi) are nodules with high hyperplasia, normally develop
198
from neuroendocrine cells, they are focal, localized especially in bronchial or bronchiolar cells.
Normally the location of this area is related to the periphery of the bronchi, is composed by small
nests of cells which have some granules related to their function: neurosecretory granules. When
tumorlets are larger than 5 mm, they are called carcinoids. They are present in the advanced stages
of fibrosis (e.g. asbestosis). Chemotherapy might cause necrosis which triggers inflammation and
therefore high dysplasia of these cells.
This type of tumorlets, in particular carcinoids, can be considered as possible precursors of the small
cell lung carcinoma which is a malignant tumor linked to smoking.
Side note: you will see and be in contact with clinicians and patients who are not only from university
but also from the hospital, so it is important to know Italian.
In figure n.3, tumorlets are pointed at by the arrow. They have a particular feature: they are, as
benign tumors, spread with an expansive growth but not an
infiltrative one, they are delimited by the bronchial cells. These
carcinoids, even though in the slides she wrote malignant
tumors, are considered actually carcinoma in situ, but are still
malignant. Bronchial carcinoids derive from the development of
tumorlets and affect in particular the central airway and also, as
tumorlets, have defined margins and grow like in a bronchial
structure.
Lung carcinomas
Figure 3 Different types of malignant carcinomas:
1. Small lung cancer, SLC (divided from others): it is
normally located centrally in the larger bronchus with very few
peripheral lesions and it is anaplastic because the cells are
undifferentiated, can have sheets but not like rosettes because they
are arranged in a kind of nodule, in different micronodules. It is very
aggressive because the cells develop in many different areas which
are small in volume making it difficult to find/resect it (in Italian,
microcitoma);
2. Others are called non-small lung cancers, NSLC:
a. Adenocarcinoma: is usually bronchio-alveolar and is the
most common in non-smokers and women; it is arranged as a
gland, the central lumen has a gland formation and, like a gland, it
can secrete mucin; it is malignant because these cells are not
normally secreting cells but they now have this new function, so
there is an ectopic secretion of mucin. There are different
adenocarcinomas depending on the structure of the tumor itself:
199
i. Lepidic: tumor cells are aligned

into the parenchyma, but these
lines have a volume that is
increased if we compare it with
other ones which is scirrhous
carcinoma;
ii. Acinar: pseudo-glands;
iii. Papillary;
iv. Micropapillary;
v. Solid;
vi. Mixed;
vii. Mucinous, more invasive than
other ones.
b. Squamous cell (or epidermoid)
carcinoma: is identical to the one that we
saw on lecture n.19, presence of
squamous or horn pearl formations, with central
keratinoid necrosis that is typical of epithelial
malignant tumors; in this case, we are into the
mucosal layer, so they are squamous, non
spinocellular, carcinomas;
Adenocarcinoma and squamous cell carcinoma

can be called blastic tumors;
c. Large cell carcinoma: called also, like the
small cell lung carcinoma, anaplastic46
carcinoma; the cells’ shape cannot be exactly
recognized, they have large nuclei, many
nucleoli, poor cytoplasm and do not contain
mucous nor have squamous differentiation or
neuroendocrine properties, they are multipotent
so that we can’t understand their functions;
d. Scirrhous carcinoma (in Italian, carcinoma
scirroso): it is divided from the others, here cells
are aligned throughout the parenchyma; it is considered as non-small lung cell
carcinoma but it is very aggressive because it is spread into the parenchyma so it is
impossible to resect, so like small lung cancer it is difficult to come up with a good
therapy for it.
Estimated incidence of lung cancer in both sexes has shown that the area of the development of
these tumors is the western world so they are westernized probably because of the presence of
industries which constitute a risk factor for the development of these tumors.47
46
Anaplastic: totally undifferentiated, so that we can’t understand the original tissue from which they come.
47
Here I really had to work with my imagination, if anybody wants to hear the recording it is at minute 49:20
200
Also mortality is increase in both sexes with age in western countries.
Breast tumors
The breast contains 15-20 lobes covered by fat and filled by lobules whose
sack produces milk which can be delivered through ducts to the nipple.
There are in this case both benign and malignant lesions. Benign lesions
are also considered precancerous lesions because there is a difference
between classification worldwide and the Italian classification which is
divided in:
1. Premalignant;
2. Benign;
3. Malignant.
Normally considered that some of the lesions have several similarities, it is easy to divide them into:
- benign lesions that can also include for example dysplastic lesions and benign tumors;
- malignant lesions.
N.B. There are so many types of tumors, not just related to the breast but in general, (epithelial,
lung…) that it is easy to divide them just in two groups, but it is important that you recognize a
precancerous, a dysplastic a benign and a malignant form.
Breast benign epithelial lesions

1. Non-proliferative breast changes in the sense of fibrocystic breast changes, it is
dysplastic only;
2. Proliferative breast disease: they can be either cancerous or benign;
3. Atypical hyperplasia can be both benign and malignant. Benign lesions depending on the
type of tissue affected can be:
a. Atypical ductal hyperplasia (ADH);
b. Atypical lobular hyperplasia (ALH).
Corresponding to these two forms there are the ductal and the lobular carcinomas (explained later).
Fibrocystic breast disease (mastopatia fibrocistica) - non cancerous
201
It consists of stromal fibrosis within which there are cysts with aprocrine adenosis. These cysts can
form also hyperplastic nodules at various degrees. It is considered a disease dependent on hormonal
changes, more specifically on the ratio estrogen/progesterone.
Paget’s disease of the breast (malattia di

Paget), carcinoma in situ - precancerous
Another type of precancerous hyperplasia is the Paget’s disease which is also a carcinoma in situ.
It is characterized by the involvement of nipple and areola of the breast. Both these sites are red and
irritated, the borders are irregular. In this case, it is a carcinoma which resembles an epithelial tumor
with rosettes and corneal necrosis into them: like the squamous carcinoma. These are all the islets
with horn pearl inside and corneal degeneration.
Gynecomastia – non cancerous

Another non cancerous lesion is gynecomastia which obviously develops only in boys and men,
not in women. It is a swelling of the breast tissue due to the imbalance of hormones: increase in ratio
estrogen/testosterone induces a hyperproliferation that is not cancerous.
Breast benign tumors

Benign tumors are named according to the morphology of the lesions (described also in epithelial
tumors). Same type of benign tumors found in the epithelial/mucosal/skin tumors are found in the
benign tumors of the breast:
202
1. Simple adenoma (in Italian, adenoma semplice) shown in figure n.4a;

2. Papillary cystadenoma (in Italian, adenoma cistopapillifero) shown in figure n.4b;
3. Intraductual papilloma: in the duct, stromal proliferation is predominant, but the ducts can
be also restricted but can still function; shown in figure n.4c;
4. Pericanalicular fibrous adenoma: in this case the proliferation of the fibrous tissue is
present and it occludes the ducts; shown in figure n.4d;
5. Intraductual (intracanalicular) fibrous adenoma contrary to what said above, in the
intraductal fibrous adenoma the stromal proliferation is predominant, the ducts can be also
restricted but can still function; shown in figure n.4e.
Figure 4c
Figure 4a
Figure 4d Figure 4e
Figure 4b
Breast malignant tumors

These are similar to benign tumors, in fact they can develop from them e.g. ductal carcinoma can
arise from the duct and normally can cover the duct, grow into the duct and become malignant when
they spread out through the digestion of the membrane, therefore it is possible that the ductal benign
form can progress toward the ductal malignant form (ductal carcinoma). From a simple ductal
hyperplasia, which is a reversible phenomenon, the subject can develop a high degree dysplasia
like a benign ductal carcinoma in situ which could be still reversible but can also further develop into
ductal carcinoma which is irreversible.
Histological classification is the most frequent type of classification for breast tumors: as of now,
there are 18 best-known breast tumors. Among these there are two main malignant tumors:
- Ductal carcinoma (in Italian, carcinoma duttale): malignant cells arrange like ducts which
are not connective ducts, cells form new small ducts; shown in figure n.5a;
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- Lobular carcinoma (in Italian, carcinoma lobulare): malignant cells can grow in different
directions arranged in single lines or can grow also as rosettes generating new lobes, lobular
structures; shown in figure n.5b.
Figure 5a Figure 5b
IDC types
Side note: in Italian, carcinoma can be abbreviated also with “Ca” while worldwide it is abbreviated
with “K”
Other types of intraductal carcinomas can be classified according
to the type of cells involved in the mutation and their degree of
differentiation:
- Medullary carcinoma (in Italian, Ca midollare)
- Scirrhous carcinoma (in Italian, Ca scirroso): these lines
of cells inside the dermis are named scirris or scirri;
- Mucinous or colloid carcinoma (in Italian, Ca mucinoso
o colloide): it is very rare, with large spaces with high
concentration of mucous.
Different types of intraductal carcinomas can be classified as:

1. Purely epithelial:
a. Squamous;
b. Large cells keratinizing;
c. Spindle cells;
d. Acantholytic;
e. AdenoCa with spindle cell differentiation;
f. Adenosquamous including epidermoid;
2. Mixed epithelial and mesenchymal components:
a. Ca with chondroid metaplasia i.e. cells similar to chondrocytes;
b. Ca with osseous metaplasia i.e. cells similar to osteoblasts;
c. Carcinosarcoma (+ specifying components) i.e. “sarcoma” is the definition of the
malignant tumor of mesenchymal cells of the connective tissue, so depending on the
components of the tumor which must be identified, we specify which kind of
carcinosarcoma it is.
[From slides: Metaplastic carcinoma: an aggressive invasive breast carcinoma, characterized by

typical IDC component combining with dominant metaplastic tissue, including squamous cells with
204
or without spindle cells differentiation, or mesenchymal differentiation such as chondroid, osseous

and myoepithelial differentiation.]
Breast regional lymph nodes

In recognition of malignancy degree of these types of breast carcinomas, it is important the lymph
nodes involvement and the type of lymph nodes involved in metastatization. During classification
of TNM, we must detect the involvement of lymph nodes but also the types of lymph nodes involved.
Different types of nodes are divided in regions:
1. Pectoralis major muscle;
2. Axillary lymph nodes level 1;
5. Supraclavicular lymph nodes;
6. Internal mamillary lymph nodes.
It is important because normally the localization of the lymph nodes

involved is very important, in fact the breast is divided into four parts
when the breast tumor is classified.
Risk factors of breast cancer
Familial history and hereditariness are considered very important because if there is a familial
history of breast cancer, it is possible that we have a hereditary mutation Sporadic
of proto or anti-oncogenes e.g. BRCA1, BRCA2, TP53 and CHECK2
(anti-oncogenes). However, sporadic tumors are still 85% of the total so
other factors (other than genetic) can be responsible of breast cancer Hereditary
e.g. environmental factors, including:
1. Age: risk rises throughout the lifetime of a woman;
2. Family history; Familial
3. Menstrual history: first period (menarche, in Italian menarca) and
the menopause age i.e. if the menarche comes very early and the menopause very late,
there is an exposure to estrogen for a longer period of time which can be a risk factor;
4. Pregnancy history: if you increase the age of pregnancy (>20), you have a higher possibility
to develop cancer;
5. Breastfeeding history;
6. Oral contraceptive use (birth control pill);
7. Hormone replacement therapy (HRT) during menopause;
8. Lifestyle and eating habits.
Most of these risk factors are related to time periods in a woman’s life in which she is exposed to
changes of hormones (estrogen and progesterone).
Also in this case, the estimated- higher incidence rate is related to western industrialized countries.
Prostate cancer
It is another important tumor of which we can have both benign and malignant forms.
Benign prostate cancer
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Benign prostate cancer is also called benign prostatic hyperplasia (HBP) and is a nodular
hyperplasia (not hypertrophy), so there is an increase in proliferation of stromal and epithelial cells
in the prostate. It compresses, closes and narrows the urethral canals, this proliferation can induce
the complete obstruction of urethra (urinary obstruction). This type of hyperplasia is not considered
a premalignant lesion which is important because the hyperplasia does not progress to malignant
cancer. The important molecule involved in this benign proliferation is dihydrotestosterone (DHT)
which is similar to testosterone but is produced through the activity of 5-α-reductase enzyme: DHT
derives from testosterone through activation of this enzyme. This enzyme is normally located in the
stromal cells and, if strongly active, it can induce the formation of DHT which is able to bind to nuclear
androgen receptors with high potent activity in the stimulation/proliferation of prostatic cells (both
epithelial and stromal). This molecule is considered an important risk factor to induce benign
prostatic hyperplasia.
There are other types of tumors for prostate:

1. Prostate intraepithelial neoplasia, PIN (in Italian, neoplasia intraepiteliale prostatica): it can
be considered benign and it can be discovered if you have to diagnose an adenoma (only in
3/4% of cases); normally it is present in peripheral part of the gland and is considered as a
kind of precancerous lesion because in 30% of cases, it can progress in adenocarcinoma in
a 20 year-period;
2. Adenocarcinoma of the prostate (in Italian, carcinoma della prostate) is the “real” malignant
tumor, usually called prostate cancer. It is due to mutation of a gene that is overexpressed:
ETS (E26 transformation-specific family transcription factor genes). It is a proto-oncogene
that induces proliferation (oncogene). Cells, in case of adenocarcinoma, are normally
cuboidal and aligned in a single layer or can also form an epithelium similar to the columnar
one. In case of prostatic carcinoma, the localization of the tumor is important. This type of
classification goes parallel to TNM staging, therefore:
a. Stage I: T0 or T1, N0 and M0; tumor mass is about up to 5%. This stage can be
divided in stage A1 and A2: if the dimension of the mass is less or equal to 5% the
staging is A1, if the mass is localized but has a mass >5% the stage is A2;
b. Stage II: mass is increased but can be either small or bigger, the small one is more
than 5% and is stage 2A and the evidence is macroscopic; stronger volume of the
mass and it is intraorgan is a stage 2B. The mass fits in this category (either 2A or
2B), if the tumor mass is unilateral (involves only one lobe);
c. Stage IIC: if the mass involves both lobes;
d. Stage III: we have an extraprostatic extension which can be either extracapsular (3A)
or it can reach seminal vesicles (3B);
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e. Stage IV: when the tumor is highly invasive, metastases are found in the body.
STAGE I II C – BOTH
MICROSCOPY LOBES
A A2
1
STAGE II
MACROSCOPIC STAGE III – LOCAL
EXTRAPROSTATIC
EXPANSION
A B
ONE
LOBE A EXTRACAPSULAR
Gleason system grading B SEMINAL
“Grading” is a classification related to the histological VESCICLES
type of cells. Gleason system grading
considers the grade of differentiation of the cells, while TNM considers the localization and the mass
of the tumor. Gleason system grading is divided in five degrees of differentiation which can come
from Class I (well-differentiated cells) to Class V (poorly differentiated, infiltrating cells). Therefore,
Class I cells are considered as differentiating cells e.g. hyperplastic cells, while Class V are
metastasizing cells. Normally, there are different types of pattern of cells which can have different
degrees of differentiation. The pathologists, who want to classify the malignancy/aggressiveness of
this tumor, have to identify and investigate in two different patterns of cells from the biopsies and
combine them in the so-called Gleason score. If in the first pattern I find a degree of differentiation
which is 1 and in the second pattern I find a degree of differentiation which is still 1, the Gleason
score will be equal to the sum of the two, which in this case is 2. Well-differentiated tumors have a
Gleason score of 2 (1+1) while poor differentiated tumors have a Gleason score of 10 (5+5) because
I observe Class V of undifferentiated cells in both patterns. When Gleason score reaches 8, 9 or 10,
the prognosis is poor: these are very undifferentiated cells, so they are malignant.
Risk factors of prostatic cancer
1. Age: prostate cancer is rare in men younger than 40, but
the chance of developing it rises after 50;
2. Family history: related to different types of population.
Gene changes as well are involved (BRC1 and BRC2
genes);
3. Hormones;
4. Race/ethnicity: it occurs more often in African-American
men and also in Caribbean men than in other races, the
geography is important (more common in North America,
Australia and Caribbean islands, here the risk of tumor
development is higher);
5. Dietary fat/habits (important also in breast cancer);
6. Multivitamin use;
7. Dairy products and calcium intake;
8. Cadmium exposure (-);
9. Dioxin exposure (-);
207
10. Environmental factors and behaviors: smoking and ethanol are risk factors for the
development of this tumor.
We do not have time to discuss hematopoietic tumors which will be discussed in the next lecture.
MESENCHYMAL TUMORS
Introduction
This lesson will finish the classification of the tumors, some of which have already been seen during
the practical lesson at the microscopes.
So far, the epithelial classification has already been seen and so have the precancerous lesions,
benign lesions and the malignant lesions that characterize the epithelial classification. In particular,
two types of malignant epithelial neoplasia (squamocellular carcinoma and basocellular carcinoma)
and different types of benign tumors (papilloma, polyps, acanthoma and keratoacanthoma) have
been discussed. Also other types of epithelial tumors, such as those in the lungs but especially those
in the liver, have been seen. It is important to underline that the tumors that are found in the liver are
predominantly secondary tumors which have metastasized into the liver from other organs,
especially of the GI tract, through the hepatic portal vein. The only primary tumor that generates in
the liver is the hepatocellular carcinoma, which originates either due to a cirrhosis or due to viral
infections such as hepatitis C or B.
The topic of this lesson is the mesenchymal tumors, their nomenclature and their classification.
Mesenchymal tumors nomenclature

These tumors do not originate from the blood, hence lymphopoiesis and tumor cells that derive from
the blood are not involved in this classification and are classified in another separate classification.
Mesenchymal tumors are those that include non-epithelial tissue excluding the skeleton, joints,
central nervous system, hematopoietic and lymphoid tissues.
Benign mesenchymal tumors that are non-hemolymphopoietic have the suffix “oma” at the end of
their name; their prefix depends on whether or not the origin of the cell is known: if the cells’ origin
can be identified, then, the first part of the name refers to it. For instance:
- Fibroma, if they originate from the fibrous tissue;
- Lipoma, if they originate from adipocytes;
- Xanthoma, referring to the accumulation of cholesterol (cholesterol producing cells);
- Myxoma, referring to the accumulation of mucus (tumour cells originating from mucoid
tissues);
- Chondroma, if they originate from chondrocytes;
- Osteoid Osteoma, if they originate from osteoblasts;
- Osteoclastoma or Giant Cell tumor of bone, if they originate from osteoclasts;
- Leiomyoma, if they originate from the smooth muscle tissue;
- Rhabdomyoma, if they originate from the striated muscle tissue.
The corresponding malignant tumors have the suffix “sarcoma”, while the prefix depends on the
tissue they originate from. However, their prefix can differ as it can either be a blastic sarcoma
(meaning that the origin of the cells can still be identified) or an anaplastic sarcoma (meaning that
the origin of the cells cannot be identified).
Examples of blastic sarcomas include:
- Fibrosarcoma, corresponds to the benign fibroma;
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- Liposarcoma, corresponds to the benign lipoma;

- Xanthosarcoma, corresponds to the benign xanthoma;
- Myxosarcoma, corresponds to the benign myxoma;
- Chondrosarcoma, corresponds to the benign chondroma;
- Osteosarcoma or Osteogenic sarcoma, corresponds to the benign osteoid osteoma;
- Leiomyosarcoma, corresponds to the benign leiomyoma;
- Rhabdomyosarcoma, corresponds to the benign rhabdomyosarcoma.
To repeat, these malignant tumors are called blastic sarcomas because their origin can still be
identified. Whereas, if the embryogenetic origin cannot be identified and only the shape and the
volume of the cancer cells can be understood, then the tumor is termed anaplastic sarcoma. These
tumors are highly undifferentiated malignant tumors. The classification of these tumors is done
according to the shape of the cells:
- Round cells;
- Spindle cells;
- Pleomorphic cells, when they have different types of shapes and volumes.
A particular classification is related to the organoid tumors, which are the tumors of the endothelial
cells found in the vessels; they are called angiomas and if the tumor cells are from the blood vessels,
they are called hemangiomas, while if they come from the lymphatic vessels, the tumors are called
lymphangiomas. The corresponding malignant tumors are called hemangiosarcoma or
lymphangiosarcoma.
In addition, another particular tumor is the mesothelioma which derives from the mesothelium and
is malignant.
Arnaud’s Question: what is the difference between angiogenesis and angioma?

Answer: they are two different phenomenons. Angiogenesis is the increased vascularization of a
tumor due to the ability of a tumor to secrete angiogenic growth factors, while angiomas are a type
of tumor caused by the proliferation of endothelial cells lining the vessels. The name is similar
because in Latin “angio” means “vessel”.
Benign tumors
Fibroma
It originates from the fibrous tissue and it can be seen in the skin, in the uterus, in the ovaries and
in the bones.
There are two types of fibromas, depending on the type of tissue components:
- Soft fibromas (“fibroma molle” in Italian), which have cells as the main component;
- Hard fibromas, (“fibroma duro” in Italian), which have fibers as the main component.
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This type of fibroma can undergo changes, for instance it is possible to find
hyaline degeneration and calcification, in which case this type of tumor is called
calcified fibroma (“fibroma calcificato” in Italian); it is well defined, since it is
surrounded by a fibrous cap, and it can be present in keloid scars (a pathological
condition of tissue repair, seen in figure n.1) or in desmoid tumors, which is a hard
fibroma found in abdominal muscles.
Figure 1
In general, fibromas have different orientations of the fibers as compared to normal fibrous tissues,
for instance they can have a circular fiber orientation like in figure n.2.
Lipoma Figure 2
It forms from adipocytes.
It is a capsulated benign tumor which appears like a cone of adipose tissue

that arises from the surrounding epithelium.
As it originates from adipose tissue, it can be found in all the organs which contain adipose tissue,
therefore it can be found everywhere.
Figure 3 Figure 4
Xanthoma
Xanthoma is also called fibrous histiocytoma since it derives from
histiocytic cells.
Generally, it forms in the subcutaneous tissue, it has a slow growing rate

and it is capsulated. Figure 5
It presents with a yellow coloration since it is the site of accumulation of free
cholesterol or cholesterol esters (figure n.6).
It presents vacuolated cells due to the intracellular presence of high
concentration of cholesterol and cholesterol esters.
Myxoma Figure 6
It originates from the mucous tissues in different sites, which
depends of course on where the mucous tissue is.
It is a soft encapsulated tumor.
The cells have a stellate shape with a soft stroma rich in mucin.
Chondroma
It originates from the chondrocytes and is divided into two types of tumors:
210
- Ecchondroma (“eccondromi”), if the tumor grows outside the bone;

- Enchondroma (“encondromi”), if the tumor grows within the bone.
Normally, the cells grow disorderly in islets surrounded by fibrous fibers.
It is very difficult to have a differential diagnosis that distinguishes this tumor from another disease
called chondrosis, which instead consists in the reactive proliferation of the same type of cells.
Osteoid osteoma
It is a benign tumor of the bones that normally develops in long bones.
Depending on the type of fibrous component or cell component, we can divide this type of osteoma
into:
- Ivory osteoma (in Italian, “osteoma compatto”), in which lamellae are concentrically aligned
(dense bone48);
- Mature osteoma or Osteoma spongiosum (in Italian, “osteoma spugnoso”), in which the
trabecular bone is oriented in a disordered way and the dimension of the lamellae is irregular.
This tumor is often surrounded by an inflammatory reaction, hence it is difficult to make a differential
diagnosis from another type of reactive inflammatory process disease called hyperostosis, which is
also surrounded by an inflammatory reaction.
Osteoclastoma
It is also called giant cell tumor of bone.
Even though this tumor is classified amongst the benign tumors, it can be locally aggressive. In fact,
as the name suggests, the cancer cells that originate from the osteoclasts are giant and very active
(i.e. they have a high rate of proliferation).
This type of osteoclastoma can affect different types of bones, in particular: long bones, tendon
sheaths and joint capsules.
Leiomyoma
It is the benign tumor of the smooth muscle tissue.
It can easily develop in the uterus (figure n.8) so that, often, what is considered
to be a uterine fibroma is, in fact, a leiomyoma.
The type of tumor can be correctly diagnosed by the evaluation of a specific
staining of the fibrous tissue. These staining techniques include two types of
stains:
- Van Gieson; Figure 8
- Mallory's trichrome stain.
Therefore, it is possible to differentiate the uterine leiomyoma from the uterine fibroma by staining
cancer tissues with these types of stains, since the fibroma and the leiomyoma would stain
differently.
The fibers are oriented in a disordered manner, which is a similar characteristic to fibroma.
48
Written in the slides
211
Rhabdomyoma
This is the benign tumor of the striated muscles, in particular of the voluntary striated muscles and
of the cardiac muscles. Normally, this tumor can be found in different sites.
Cells usually proliferate in a disordered orientation as incomplete

and large muscle fibers in some parts of the tissue.
Generally, these cells are rich in glycogen. Figure 9
Mixed tumors
There are other types of tumors that are considered benign, but they can also develop and become
malignant.
These tumors originate especially during morphogenesis if there is a mistake in the differentiation of
the different types of tissues, since they arise as a combination of different types of mesenchymal
tissues.
Their name depends on the types of tissues from which they originate. For example:
- Fibrolipoma;
- Fibromyxoma;
- Fibrolipomyxoma;
- Fibroleiomyoma;
- Osteochondroma (figure n.10);
- Fibrorhabdomyoma.
Figure 10
Organoid tumors (angioma)49
These tumors develop in vessels in which endothelial cells proliferate in an exaggerated manner.
As a consequence, the vessels become dilated and their lumen is filled with plenty of blood as the
diameter is enlarged.
Depending on the type of vessel they develop in, these tumors are divided in hemangioma and
lymphangioma.
Hemangioma
The tumor cells originate from the endothelial cells of the blood vessels.
There are two types of hemangioma, depending on their localization: focal hemangiomas and deep
hemangiomas.
Focal Hemangioma (or Capillary Hemangioma or Simple Hemangioma, emangioma semplice in
Italian) are found only in the cutaneous and subcutaneous layers and in internal organs. According
to its morphology it can be further subdivided into:
- Flat Hemangioma (“emangioma piatto” in Italian), which is a superficial hemangioma, oriented
in the same direction as the surrounding tissue; it is also called salmon patch; it is not
persistent throughout life and can subside with time;
- Port Wine Stain hemangioma; the name is related to its color, which is similar to the one of
port wine (red); it is frequently found on the face and it is present at birth and remains persistent
throughout life, without regression.
49 An example of a cavernous angioma has been seen in the practical lesson

212
Deep Hemangioma (or cavernous hemangioma, “emangioma cavernoso” in Italian) can affect the
skin, internal organs and especially the liver50. It is also known as cavernous hemangioma because
it forms caves, similar to sacks due to the enlargement of the vessel. In fact, it forms vascular cavities
of different sizes with the vessel alternated with the fibrous tissues.
Figure n.11 clearly shows that the endothelial cells have
a strong proliferation, inducing the enlargement of the
vessels and therefore inducing the formation of caves
(kind of big cysts).
Figure 11
Lymphangioma
It is a tumor that originates from the endothelial cells of the lymphoid vessels that is very similar to
hemangioma.
Lymphangiomas have traditionally been classified into two subtypes capillary (or simple) and
cavernous lymphangiomas, based on their microscopic characteristics.
- Capillary lymphangiomas (or simple lymphangioma);
- Cavernous Lymphangioma.
Malignant tumors
Kaposi's sarcoma
It is considered a malignant tumor, although it is actually an intermediate tumor in between benign
and malignant.
It is a particular type of tumor which can be found in different types of diseases, conditions and
populations. Generally, it is virus-associated and is an aplasia that affects the mesenchymal and
endothelial cells.
Kaposi sarcoma is related to the alteration of perivasal cells. It appears like dermal irregular vascular
spaces that are surrounded by proliferating fused cells in perivasal sites.
It is important because it is found in the lymph nodes, for example in advanced stages of AIDS or in
particular populations, such as African people. According to this, it can be divided in four classes:
- Chronic type, endemic in Eastern Europe, which may occur in association with other
malignancies;
- African type, endemic in children, which is due especially to malnutrition and
immunosuppression in this population; in fact, it manifests with an aggressive systemic
lymphadenopathy;
- Associated with transplantation and AIDS in immunosuppressed patients51;
- Associated with AIDS (Acquired Immuno-Deficiency Syndrome).
50 The one seen in the slides during the practical lesson

51
Immunosuppression is an important cause of the development of this type of tumor
213
Figure n.12 shows that vessels are enlarged and the cells in the surrounding
tissue are fused together to envelope these enlarged vessels.
Figure 12
Overview of malignant tumors
Malignant tumors can be divided into blastic and anaplastic sarcomas.
In a blastic sarcoma it is possible to recognise the tissue origin because
they are relatively differentiated tumors; therefore, they are classified according to their histological
origin.
On the other hand, anaplastic sarcomas are highly undifferentiated, to the point that it is not
possible to recognise their tissue origin; therefore, they can only be classified according to the
features of the cells.
Therefore, all the malignant forms that correspond to the benign tumors mentioned above can be
classified as blastic sarcomas: the particular features that differentiate them from benign tumors
concern the orientation and volume of the cells and the disordered type of tissue that appears. For
instance, in fibrosarcoma collagen fibers are disorderly oriented, there is an abundant cellular
component, it is frequently a fibroma evolution from benign to malignant and therefore in the same
way
Fibrosarcoma Figure 13
This is the malignant version of the benign tumor fibroma. Collagen fibers are oriented
in a disorderly manner, there is an abundant cellular component and it frequently
develops as a malignant evolution of a fibroma. It can be located in any district of the
body, more frequently in the retroperitoneum, skin, myelin sheaths, tendon sheaths.52
Liposarcoma
It is a malignant tumor that derives from adipocytes, which show different

degrees of atypia and many karyokinesis i.e. division of a cell nucleus during
mitosis.
Xanthosarcoma Figure 14
Malignant evolution of xanthoma.
Myxosarcoma
Malignant evolution of myxoma.
Figure 15
Chondrosarcoma
The chondrosarcoma is a highly aggressive malignant tumor, since it is highly
invasive and metastasizing. It is formed by chondrocytes with high grade of
atypias and preferentially develops in young people.
52
214
Figure n.15 shows the level of mutations in this type of tumor: the volume of the cells is very large
compared to their normal size and there is the presence of spreading folds into the collagen part.
Osteosarcoma or osteogenic sarcoma

It is a malignant tumor derived from osteoblasts. It preferentially originates in young people. It is a
very aggressive tumor since there is a big pleiomorphism of the cancer cells.
Figure 16
Leiomyosarcoma
Leiomyosarcoma is a malignant tumor related to the smooth muscle tissue. It shows an extreme
differentiation among different fibrous cells as regarding the dimensions, volumes and orientation.
It shows strong cellular pleomorphism with hyperchromic nuclei and numerous irregular mitoses53.
Rhabdomyosarcoma Figure 17
Rhabdomyosarcoma preferentially develops in children and young
individuals. It presents myocytes with different types of atypias and it is often considered a
dysontogenic tumor, since it may have a dysontogenic origin, meaning that it affects other tissues
that, normally, have no muscles.
Mixed malignant tumors

Mixed tumors can have malignant forms as well; for instance:
- Fibroliposarcoma;
- Fibromyxosarcoma;
- Fibromyxochondrosarcoma;
- Fibrochondrosarcoma;
- Osteochondrosarcoma;
- Etc.
Angiosarcoma
Also at the level of organoid tissues there can be a malignant evolution of the angioma in which the
vessels are incomplete, enlarged, with different shapes, atypical and completely disordered. The
malignant tumor is called angiosarcoma.
Figure n.20 shows vessels in which it is possible to notice a destruction of the layer, which causes a
hemorrhage as it is evinced by the presence of red blood cells in the interstitial space.
53 Written in the slides

215
Figure 20
Endothelioma
It is the blastic sarcoma of the hemangioma and the lymphangioma, therefore they are called
hemangioendothelioma and lympho-angio-endothelioma.
It develops from endothelial cells of the blood and lymphatic vessels, showing significant formations
of protruding lines of cells in the lumen that can be narrowed.
Perithelioma
The perithelioma is related to the development of a tumor from the tunica adventitia.
Perithelioma and mesothelioma can develop from the different layers that cover the organs, which
are the pericardium, the peritoneum and the pleura.
Mesothelioma
Just as the perithelioma, it can develop in the layers that cover the different organs, such as the
pericardium, the peritoneum and the pleura.
It is a very aggressive tumor whose typical localization is the lung, in particular the pleura54.
It develops from the thin layer of tissue that covers

many of the internal organs (mesothelium). In the
pleura, peritoneum and pericardium. Characterized
by severe cell atypia with connective and/or epithelial
polymorphism (like-squamous cells and pseudo-
glands)55.
Anaplastic sarcomas Figure 21
Ewing Sarcoma
Ewing Sarcoma is an anaplastic sarcoma formed by small round cells.
It is a primary sarcoma of bone tissue (6-10%) which preferentially

develops in young individuals, from 10 to 15 years of age.
Figure 22
It can be considered to have a neural phenotype because it is considered
a primary neuro-endocrine tumor (PNET).
Figure 23
54
Two types of aggressive malignant tumors can develop in the lung: one comes from the epithelial
parenchymal lung cells, the other from the pleura (in which case it is called a mesothelioma)
55 Written in the slides
216
These tumor cells are found in the bone or in the soft tissue and form a
capsule delimiting a central fibrillar site. This particular formation of
fibrillar cells that are surrounded by small round cells are called Homer-
Wright rosettes (figure n.23).
It seems to be related to a translocation of an oncogene (EWS-FLI1) Figure 23

from chromosome 11 to chromosome 22 (figure n.24); this translocation
induces the formation of a fusion gene called Ewin gene, which has a potent
transcriptional activity to induce proliferation.
If it is localized, the Ewing sarcoma has a survival rate of 70%, whereas if it becomes
metastatic the survival rate is 30%.
Spindle Cell Sarcoma Figure 24

It is an anaplastic sarcoma, therefore it is characterized by different orientations, high anaplasia
and different types of cells, including also alternated fiber cells.
This particular type has spindle cells that are irregularly orientated, with poor cytoplasm and high
anaplasia, and which have a stroma containing pro-collagen fibers56.
Undifferentiated Pleomorphic Sarcoma

Figure 25
This is the most malignant type among the mesenchymal tumors and, as
suggested by the name, it is related to the pleiomorphism of its cells.
It is highly anaplastic and presents a maximal atypia of cells with abundant mitotic
activity.
We are absolutely not able to identify the origin of the cells.
HEMOLYMPHOPOIETIC TUMORS Figure 26

These tumors are very difficult to classify because the classification
keeps changing since hematologists are constantly finding new
types/isoforms, depending on the type of hematological cell that is
involved in the tumor.
The tumors that originate from the blood are related to the
hematopoiesis (figure n.27) and are therefore different depending on
the type of maturation of the cells: this is the reason why it is important
to know what are the main immature cells that are involved in this
process. These types of cells are in the bone marrow (figure n.26),
which is a rigid chamber where different cell populations exist; in fact, in
the bone marrow there are precursors of red blood cells, white blood cells and platelets and
adipocytes.
56
217
It is important to understand the symptoms of the patients that have these types of tumors, because,
if one of the different populations that arise from the bone marrow starts to amplify then it will alter
the ability of the other populations to proliferate, which will result in specific symptoms and signs.
Therefore, if for instance there is a proliferation of leukocytes, it is important to consider that
erythrocytes and platelets cannot proliferate in an adequate manner. Hence, in a patient affected by
leukemia (the malignant tumor of white cells), it is possible to see difficulty in the coagulation and a
reduced percentage of red blood cells (anemia). This is important for the diagnosis of different types
of tumors of the blood cells.
Figure 27
Depending on the population that expand in the line of maturation of the cells,
hemolymphopoietic tumors can be aggressive or not. If, for instance, there is a proliferation resulting
in a large number of megakaryoblasts (figure n.27), the corresponding tumor will be very aggressive.
If the proliferation concerns only one type of clone cells, in this case megakaryoblasts, it is said to
be an acute situation; if, instead of only one clone, different types of populations of the maturation
lineage are seen to be mutated, for example megakaryoblasts and megakaryocytes, this type of
tumor is said to be chronic, because different populations can develop into the different stages of
the maturation growing in an abnormal way.
Hence, depending on the type of population that the tumor comes from and the number of the
populations that are cancerous, in the case of the bone marrow the sarcoma can be divided into
acute or chronic. If it affects white cells, the tumor is called leukemia, which therefore can be
divided in acute and chronic leukemia: an acute leukemia is characterized by tumor cells in only one
population, a chronic leukemia is characterized by tumor cells present in multiple populations of the
lineage.
Leukemia
Acute leukemia can, in turn, be divided depending on the general lineage that is involved, so either
the lymphatic population or the myeloid population (i.e. granulocytes). Therefore, we can have acute
lymphatic (or lymphocytic or lymphoblastic) leukemia or acute myeloid leukemia.
Chronic leukemia as well can be divided into lymphocytic chronic leukemia and myeloid
chronic leukemia. In chronic myeloid leukemia, however, there is also the possibility that the
218
monocyte population can proliferate, although it is very rare compared to other types of myeloid
leukemia, resulting in what is known as monocytic leukemia.
Lymphocytic leukemia can be subdivided depending on the types of cells that can proliferate, so
we can have T cell leukemia or B cell leukemia.
Myeloid leukemia can be divided in two types: Ph+, Philadelphia positive leukemia, and Ph-,
Philadelphia negative leukemia. Chronic myeloid leukemia, moreover, consists of two different types:
the sporadic myeloid leukemia and the leukemia due to particularly important translocations.
Translocations
There can be many types of translocations, but the main one is the translocation from
chromosome 9 to 22 (figure n.28 e 29).
In this particular case, there is the translocation of the ABL
oncogene from chromosome 9 to the BCR gene on
chromosome 22. This mutation induces the formation of a
fused gene ABL-BCR which is considered a hybrid gene
coding for a tyrosine kinase; this kinase, however, is
considered a chimeric protein, since it doesn’t exist in nature,
and it has a high tyrosine activity and proliferation. Therefore, Figure 28
this translocation induces an increased rate of proliferation in
granulocytes, inducing chronic myeloid leukemia. This fused chimeric
gene is called Philadelphia and is a very small gene; this is why chronic
myeloid leukemia is classified as Ph+ or Ph-: if the patients have this gene
in the chromosomal map, then they are classified as Philadelphia
positive, otherwise they are classified as Philadelphia negative (sporadic leukemia).
There are other minor types of translocations which induce genes
Figure 29 involved in the induction of enzymatic pathways in proliferation.
There is another type of translocation, related to Burkitt lymphoma,

which involves the c-Myc oncogene. C-Myc is a protooncogene that
induces the passage from G1 to G2 and from G2 to S, which makes it
very important for the cell cycle. The gene coding for c-Myc is
translocated from chromosome 8 to chromosome 14, near the
genes that code for the immunoglobulins; therefore, this induces the
high proliferation of lymphocytes with high induction of
immunoglobulins.
Figure 30 Acute leukemia classification

Lymphoblastic leukemia can be acute or chronic; there are different
57
types of classification .
Acute lymphoblastic leukemia can be divided depending on the type of cells that are involved: B
cells, T cells, null cells or common cells. The types of acute lymphoblastic leukemias with the
highest incidence are T cell- and null cell- associated leukemias.
Acute myeloid leukemias are divided depending on the type of populations that are involved in this
type of population expansion. Therefore, depending on which cells provide the maximal contribution
to proliferation, leukemias are classified as:
57 In the lesson, only a simple classification is provided, more detailed and complex ones will be encountered in the
future
219
- M1, if it mainly involves myeloblasts;

- M2, if it mainly involves myeloblasts and myelocytes;
- M3, if it mainly involves myelocytes and promyelocytes;
- M4, if it mainly involves promyelocytes and monoblasts;
- M5, if it mainly involves monoblasts;
- M6, if it mainly involves erythroblasts and myeloid cells;
- M7, if it mainly involves megakaryoblasts.
Figure n.31 aims to show how many types of leukemias and how
many different types of classifications there are depending on the
type of cells, on the stage of the maturation of these cells and the
involvement of different types of cells.
Lymphoma
Figure 31
Lymphoma and leukemia are different: leukemia is a
cancer of the leukocytes, which include lymphocytes and
myelocytes, whereas lymphomas are malignant tumors of the
lymphoid tissues, i.e. those tissues that are involved in the activation of mature lymphocytes, such
as the spleen and the lymph nodes. In fact, normally the thymus induces the differentiation of a
particular type of immune cell, which then go to the lymphoid tissues (spleen and lymph nodes) to
become fully mature. In the lymph nodes there is a central part (medulla) and a peripheral part
(cortex) in which B cells and T cells are respectively found.
These types of tumors are malignant: all types of blood tumors are malignant. If asked what leukemia
is and what is a lymphoma, the answer would be: leukemia is a malignant tumor of blood cells (those
that originate from the bone marrow), in particular of the white blood cells (the name comes from the
Greek “leukos”, which means “white”), while lymphomas are malignant tumors that originate from
cells of the lymphoid tissues. Considering that in the lymph nodes there is a high percentage of
mature lymphocytes, which can develop and proliferate as well, it is possible to see these
differentiated cells circulating in the blood, as it happens in the leukemia, but they are different types
of tumors.
Lymphomas are primary tumors of the lymph nodes. Indeed, there is no possibility that lymph nodes
can become the site of a secondary tumor: they can be involved in the metastatization process since,
if they recognize them, they are able to momentarily stop the malignant cancer cells (although
sometimes they are able to escape), but a metastasis itself cannot grow in the lymph node.
Lymphomas are divided into two important types: Hodgkin lymphoma and non-Hodgkin
lymphoma.
Hodgkin Lymphoma
Hodgkin Lymphomas are subdivided into different categories depending on the type of cells that are
present in the lymphoid tissue and on their percentage compared to the lymphocytes; in particular,
there is a peculiar type of cell, called Reed-Sternberg cells (RSC), whose quantity compared to
lymphocytes’ is important for the classification.
Hodgkin lymphomas, therefore, are divided in:
- Nodular sclerosis, in which there is a middle quantity of RSCs and a middle quantity of mature
lymphocytes58;
58
Differently, the ones present in leukemia are immature lymphocytes
220
- Mixed cellularity, in which RSCs are present in a higher percentage compared to

lymphocytes;
- Lymphocyte rich, in which RSCs are present in a lower percentage compared to
lymphocytes;
- Lymphocyte depletion; in which RSCs are present in a much higher percentage compared
to lymphocytes, which are present in a very low number;
- Lymphocyte predominance; it can resemble the lymphocyte rich type, but it is a separate
category because RSCs have a different B cell immune phenotype compared to the classical
cell type cells.
Reed–Sternberg cells are very important for the

recognition of this type of lymphoma.
They are very large cells compared to the other cells
seen in figure n.32, which are lymphocytes; in fact,
they are very big and often show multiple nuclei
(usually two or three) and nucleoli surrounded by an
empty area. Because of this peculiar morphology,
they resemble the eyes of an owl and therefore they
are also known as owl’s eye cells or popcorn cells.
Non-Hodgkin Lymphoma
Non-Hodgkin lymphomas are classified according to
the types of cells that are involved, but since these
lymphomas are particularly undifferentiated, the
classification needs to be done according to the
shapes and volume of the cells.
There are three different degrees: low grade of malignancy, intermediate grade of malignancy and
high grade of malignancy.
In the low grade of malignancy, lymphocytes are small and in a high percentage, so this type of
lymphoma is called non-Hodgkin lymphoma at low grade of malignancy with small
lymphocytes. If lymphocytes and other cells are not well recognizable, except for their shapes and
volume, they are only called small cells; tumor cells can form islets, forming follicular small cells, in
which case the tumor will be a non-Hodgkin lymphoma at low grade of malignancy with
follicular small cells. Finally, another type can be mixed follicular, with small and large cells.
In the second degree, or intermediate degree of malignancy, the large cells are preponderant
(meaning that they are more than small cells); they can be follicular, diffused or mixed with small
cells. In this type, there is the involvement of large cells that are more aggressive than small ones,
both in follicular and diffused cells. Small cells are only diffused cells which are more aggressive
because it is impossible to recognize focused zones of tumor.
In the last one, the high-grade malignancy, there are in addition to blood cells also different types
of cells that have an immature form; it is possible to have an immunoblastic form and a
lymphoblastic form, in which we can see lymphoblasts (not lymphocytes, lymphoblasts).
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The higher malignancies are: Burkitt lymphoma and plasmacytoma or plasma cell myeloma.
Burkitt lymphoma
Burkitt lymphoma (mentioned also before) is a type of lymphoma related to

an important translocation of c-Myc oncogene between chromosome 8 and
14: mutation that induces the translocation of c-Myc near the genes that
codes for immunoglobulin.
It is a rare form of cancer predominantly

affecting young children in Central Africa, but
the disease has also been reported in other
areas. The form seen in Africa seems to be
associated with infection by the Epstein-Barr
virus.
WHO classification of lymphoid

neoplasia
WHO classification is now considered another type of classification that includes all types of blood
tumors, it can include leukemias or lymphomas. This type of classification follows the criteria of type
of cells that are involved:
- B cell neoplasia:
o Acute lymphoblastic leukemia;
o Chronic lymphocytic leukemia;
o Hairy cell leukemia;
o Myeloma or Plasmacytoma;
o Burkitt’s lymphoma;
- T and NK cell neoplasia:
o Acute lymphoblastic leukemia;
o NK cell aggressive leukemia;
o Mycosis fungoides/Sézary syndrome.
Only Hodgkin lymphoma follows a similar classification:
- Nodular sclerosis;
- Mixed cellularity;
- Lymphocyte-rich;
- Lymphocyte depletion;
- Lymphocyte predominance.
WHO classification of myeloid neoplasia

It can be classified according to the types of cells that are involved:
- Myeloproliferative disorders;
- Myeloproliferative/myelodysplastic disorders;
- Myelodysplastic syndrome.
These three are related to chronic leukemias.
- Acute myeloid leukemias:
o Acute myeloid leukemia;
o Myelomonocytic leukemia;
o Acute erythroid leukemia or Di Guglielmo syndrome: it is the only pathology related
to RBCs in which erythroblast precursors of erythrocytes are involved.
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EMT (EPITHELIAL TO MESENCHYMAL TRANSITION)

This lesson is about specific aspects of tumour invasion and metastasis. In particular, the main topic
is a process called EMT (Epithelial to mesenchymal transition).
Epithelial and mesenchymal cells: two of the main cell types in mammals
There are two main types of cells in mammals: epithelial and mesenchymal cells. Each of these
two types has different characteristics:
1. Epithelial cells: this type of cell has cohesive interactions among them. In fact, they are
connected to each other and they form a continuous cell layer. They have three membrane
domains: the apical, lateral and basal membrane domains. Besides that, they have tight
junctions between apical and lateral domains and an apico-basal polarized distribution of
organelles and cytoskeleton components. This type of cells is not able to move;
2. Mesenchymal cells: the interaction among these cells is loose or it doesn’t exist at all. They
are not able to form a continuous cell layer and there’s no clear apical and later membrane
domains. Besides that, they aren’t polarized, thus, there’s no apico-basal polarization of
cytoskeleton components and organelles. However, these cells can move, they are motile,
this is important because, thanks to this, they have invasive properties.
During normal development, certain cells can switch from an epithelial to a mesenchymal phenotype.
When it happens, this kind of process is defined as EMT (epithelial to mesenchymal transition). It
was initially described for normal cell differentiation during early development, thus, during
embryogenesis. This switch is important for the organization of specialized tissues and organ
systems.
EMT is a tightly regulated process, associated with molecular and cellular events. The result of these
changes in cell phenotype, triggered by EMT, is that cells reduce their adhesion with other cells and
they increase their motility.
In any case, EMT is a reversible process, in fact cells can undergo reciprocal MET (mesenchymal
to epithelial transition), thus, turn back and reacquire again the epithelial phenotype.
Figure n.1: epithelial cells (in blue) change their

phenotype to the one of mesenchymal cells (in
red) but this last one, when conditions around
are not good for the process (EMT), can return
to the epithelial phenotype.
Epithelial cells adhere to each other through
adherens junction, in which E-cadherin plays a
fundamental role. Desmosomes and, in
particular the protein called desmoplakin, are
important too. The polarization goes from Figure 1
apical to basal domain. When epithelial cells
change their phenotype in mesenchymal cells, the morphology is totally different: in fact,
mesenchymal cells are not adherent to each other and they aren’t polarized, E-cadherin is
sequestered in perinuclear vescicles and desmoplakin is internalized, so that they can’t allow cells
to form junctions between them.
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In normal conditions, epithelial cells are well ordered and form a continuous cell layer, they are in
contact with the basal membrane and show an apical-basal polarity determined by different protein
complexes that interact with the junctions. When cells change their phenotypes, the first step that is
observed is a change in cell-cell junctions, which is critical for EMT process.
At the beginning, there’s the dissolution of these junctions and a loss of polarity, but in particular a
Figure 2 decrease in the expression of E-cadherin can be
observed. This protein is very important in the connection
between cells, and as a consequence of its decreased
expression, the Scribble complex is not able to interact
with the lateral plasma membrane. Thus, the decreased
expression of Scribble and of E-cadherin reduces cells
adhesion and increases cells motility.
EMT has been studied during early embryonic
development. In fact, it has been observed that the first
MET occurs during pre-implantation while the first EMT
occurs during gastrulation.
Figure n.3:
Photomicrography of epithelial cells (b) and

mesenchymal cells (c): in which different
morphologies of the two cells can be seen.
Figure 3 Figure 4
(on the right)
Figure n.4: E-cadherin (d, e) is marked in red, a higher expression in epithelial cells compared to
mesenchymal cells can be noticed.
Vimentin (f, g), a specific marker for mesenchymal cells, in fact, is less expressed in epithelial cells
than in mesenchymal cells. It’s marked in green in figure n.4.
Besides gastrulation, EMT can occur also during neural crest formation, heart morphogenesis, and
wound healing. These are all physiological situations.
Recently, it has been observed that EMT can be involved also in the pathogenesis of fibrosis and in
epithelial tumours. Processes are similar but these latest two are pathological and not physiological
events.
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Side note: For invasiveness and the formation of metastasis, cancer cells must move and, to move,
epithelial cells have to change their phenotype to the mesenchymal one, thus increasing their
motility, therefore they can invade tissues and have metastatic properties.
This phenotype modification is important because tumour cells can leave their epithelial state and,
after crossing the basal membrane, they can enter the connective tissue, assuming a mesenchymal
state to interact in a more advantageous way with the surrounding connective cells.
To migrate, cancer cells must:

- activate genes for differentiation;
- reduce proliferation;
- activate anti-apoptotic mechanisms;
- alter cellular characteristics from epithelial to mesenchymal;
- down-regulate receptors for cell-to-cell attachment;
- up-regulate cell adhesion molecules to help cell movement;
- degrade cell-to-cell junctions;
- degrade ECM components to move in the connective tissue.
EMT differences
There are some important differences between physiological and pathological EMT processes.
During embryogenesis, EMT is controlled by specific cellular and molecular events which appear in
an ordered way. However, during malignant transformation, the order of events can be time-
independent and some of the passages can be bypassed.
These differences are consequences of specific oncogenic pathways induced by some growth
factors, enzymes, transcriptional factors or signalling.
EMT is a complex process and it’s very important for invasiveness and metastasis: this complexity
is due to the heterogeneity of the tumour; tumours are formed by different types of cells: at the
beginning, it is possible to talk about tumour homogeneity but then, during tumour progression, not
all cells can undergo the same events simultaneously. In fact, not all of them can change from the
epithelial to the mesenchymal phenotype at the same time and not all of them will. Besides that, not
all cells that have undergone EMT will successfully metastasize.
Arnaud’s Question: Regarding physiology EMT, transition between the two types of cells happens
at multipotent stem cells, not at fully differentiated cells, is it right?
Professor: Yes, it happens at totipotent stem cells.
Arnaud: Regarding wound healing, cells are not totipotent, pluripotent or multipotent when they start
to differentiate, if fully differentiated epithelial cells… (professor stops him)
Professor: Tumour cells are not totally differentiated. In anaplasia there’s a loss of differentiation,
cells are differentiated but tumour cells are not differentiated, malignant tumours have different
grades of differentiation, if you talk about non-differentiated tumours, the anaplastic tumours, it’s very
difficult to recognize different types of cells. In that case, genes involved in differentiation are lost.
Arnaud: Thus, fully differentiated cells lose their differentiation and return back to a progenitor state
and then they reach the mesenchymal line.
Professor: yes. (But I think she didn’t even listen to him anymore and starts talking about why it is
important for tumour cells to be able to move).
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EMT is regulated by genetic or epigenetic changes?

Cells change their phenotype because it’s important for moving, in fact, tumour cells have to move
to invade tissues, to create the first and the secondary tumour. The capacity of tumour cells to form
a metastasis is regulated by various oncogenes. The most important are SNAIL, TWIST and ZEB.
These oncogenes are able to encode transcriptional factors that are involved in the EMT process. In
EMT, cancer cells downregulate some epithelial markers like E-cadherin thanks to transcriptional
repressors, such as SNAIL and TWIST. In contrast, they upregulate certain mesenchymal markers
as vimentin and smooth muscle actin.
Besides that, all these genes involved in EMT are
regulated by various signals.
EMT is well studied in breast cancers because,
especially in this type, it was easier to observe that
tumour cells are able to cross basal membrane and
become invasive cells.
A critical molecular event of EMT is the

downregulation of E-cadherin (epithelial
cadherin). E-cadherin is a transmembrane
glycoprotein, important for cell adhesion, that can act as tumour suppressor too. In fact, when E-
cadherin is over expressed, it can inhibit the motility of the cells, thus, cell invasion and metastasis.
E-cadherin can be downregulated in tumours because of a defect in its function, or because it’s
repressed, degraded or because of gene mutations, hypermethylation of the promoter, post
transcriptional modification, etc.
E-cadherin genes can also be silenced, removed or deleted. It has been proved in in vitro experiment
that it is sufficient to reinsert the genes encoding for E-cadherin, in order to decrease the recidivism
of tumour cells.
Parallel to E-cadherin loss, there’s the increase in N-cadherin (neural cadherin), which can
increase cell death resistance: indeed, tumour cells may not die for apoptosis; moreover, it can
increase invasiveness. In general, N-cadherin favours the aggressiveness of the tumour.
Repression of E-cadherin gene expression may result from the activation of transcriptional
repressors: mainly SNAIL and TWIST.
Key transcriptional factors involved in EMT

SNAIL, TWIST and ZEB are the main important oncogenes for EMT; they can repress molecules
and proteins that are markers for epithelial cells, such as E-cadherin and desmoplakin, or they can
activate mesenchymal markers, such as N-cadherin, vimentin, etc.
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Figure 6
Other transcriptional factors involved in EMT

Besides these oncogenes, which are the most important, there are other transcription factors
involved in EMT:
- FGF is able to promote EMT in bladder carcinoma through upregulation of SNAIL2,
destabilization of desmosomes, expression of integrin and of metalloproteinases;
- HGF (hepatic growth factor) is able to convert epithelial cells into fibroblasts-like cells and to
induce expression of two types of SNAIL, 1 and 2;
- IGF1 receptor is able to decrease expression of E-cadherin and increase expression of N-
cadherin, vimentin and fibronectin;
- Human EGF (epidermal growth factor) receptor activation in epithelial cells can induce
tumours with EMT properties;
- VEGF (vascular endothelial growth factor) induces SNAIL expression in breast cancer cell.
Figure n.7: Passages of EMT process:

1. Primary epithelial tumour cells have to detach one from the other;
2. In order to do that (1), they have to change their phenotype and become mesenchymal cells:
EMT;
3. Once cells have changed their phenotype, they are able to move from primary epithelial
tumour cells and to develop the tumour mass;
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4. These cells can also reacquire the epithelial phenotype: MET. It happens especially when
the surrounding conditions are not suitable for EMT process, for example, when genes or
transcriptional factors specific for EMT are not present;
5. It’s important to form the
metastasis, the secondary
tumour.
Figure 7
The opposite process MET
is important for successful
metastasis
MET is possible because
EMT is a reversible process.
It’s an important event in order to develop a metastasis because after diffusion tumour cells can
reacquire the epithelial phenotype, leading to the formation of a secondary carcinoma with the same
phenotype of the primary tumour. In fact, the transition from epithelial to mesenchymal cell is
necessary only for moving, but once cells are set down, in order to form a metastasis, they must
return to the epithelial phenotype.
MET, initial step for tumour progression:
- Repression of mesenchymal genes, all mesenchymal markers must be down-regulated and
all epithelial markers must be increased;
- Activation of genes encoding for cell junction proteins: adhesion molecules have to be
expressed again.
Gene mutations, accumulation of mutations and genetic changes are all irreversible phenomena.
Why is EMT reversible if mutations are irreversible?
Hypothesis: since this process is due to epigenetic changes, it is reversible. The transition from
epithelial cells to mesenchymal cells cannot be explained by gene mutations because they are
irreversible. Thus, some epigenetic changes must be involved such as histone modifications, DNA
methylation.
Recently, some specific changes in microRNA have been studied and they seem to be involved in
this process.
miRNAs may play an important role in EMT and MET: it has been observed that the expression of
some specific miRNAs decreases during EMT with consequent increase in the levels of SNAIL,
TWIST and ZEB, favouring EMT progression. However, when the expression of these specific
miRNAs increases, there’s a reduction of EMT and an increase of MET.
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Figure 8
Summary: Tumour cells have to detach from primary tumour in order to move, to invade, to reach
metastatic routes and to spread in other areas of the body like tissues and organs. There, they have
to form the secondary tumour. For moving, they have to change their phenotype from the epithelial
to the mesenchymal one. However, when they reach the area where they want to develop the
secondary tumour, they must reacquire the epithelial phenotype. These steps are regulated by
various oncogenes, the main ones involved are SNAIL, TWIST and ZEB. These epigenetic changes
are important because, otherwise, gene mutations couldn’t justify the EMT process since it is
reversible. Thus, epigenetic changes can activate oncogenes that are able to repress epithelial
markers and increase mesenchymal markers during EMT. Eventually, after this transition, MET has
to occur for a successful metastasis.
INFLAMMATION AND CANCER

The second part of the lecture is about the association between inflammation and cancer. In fact,
inflammation is a critical process in cancer progression. It has been observed that at least in 15% of
cancers, tumour initiation is directly attributed to infection caused by different agents, such as
viruses, bacteria, parasites.
In 1863, Virchow hypothesized that the origin of cancer was at sites of chronic inflammation:
chronic inflammation can be caused by different biological agents such as viruses, bacteria, irritants
and as a consequence of tissue damages.
It has been observed that the tumour microenvironment plays a fundamental role in tumour initiation
and progression. The tumour microenvironment is occupied by inflammatory cells which produce
and release inflammatory mediators. This state, characterized by inflammation, can regulate cell
proliferation, migration and survival.
Figure n.9: it is now known that some cancers can arise from chronic inflammation condition, this
erases the great association between chronic inflammation and some types of tumours.
Some examples are to be found in figure n.9.
- Mesothelioma and lung carcinoma may develop following asbestosis and silicosis, chronic
inflammatory conditions of the lung;
- Chronic bronchitis may favour the development of lung cancer;
- Colorectal carcinoma is associated to all the chronic inflammations of the intestine; for
instance, the inflammatory bowel disease, Chron’s disease and chronic ulcerative colitis are
events characterized by chronic inflammation: these pathologies are the basis for the
development of colorectal cancer;
- Chronic pancreatitis is connected with pancreatic carcinoma;
- Skin inflammation is connected with melanoma;
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- Gastritis is connected with gastric adenocarcinoma; in this case, it has been recognized that
the Gram-negative bacterium Helicobacter pylori is the main responsible for gastritis and that
most of gastric59 cancers can develop following inflammations due to this bacterium;
- Hepatocellular carcinoma is associated with Hepatitis C infections and all conditions that can
lead to an inflammatory condition in the liver; actually, all hepatitises are connected with
hepatocellular carcinoma.
Figure 9
Inflammation can stimulate cancer development

Tumours microenvironment is constituted by inflammatory cells which release inflammatory
mediators in it.
Two particular situations must be distinguished:
1. In some tumours, inflammatory conditions may be the initiators for malignant transformation
(first there is the inflammation which then gives rise to the tumour);
2. In others, the tumour starts to develop in a situation without any kind of inflammation, but the
malignant transformation can later induce an inflammation condition. Thus, oncogenic
changes can lead to the development of an inflammatory microenvironment, which can itself
59
She says “gastritic” but she probably means “gastric”
230
favour tumour development (first there is a tumour which then favours the formation of an
inflammatory microenvironment).
Inflammatory cells can release inflammatory mediators, such as chemokines, cytokines,

transcription factors, ROS (reactive oxygen species) and RNS (reactive nitrogen species). All these
mediators are present since the first stages of tumour development; in particular, RNS and ROS can
interact with the DNA and can be responsible for different types of mutations, such as deletion and
rearrangements.
Inflammation is fundamental in stimulating tumour; in fact, chronic inflammation, infections and tissue
damages lead to the release of cytokines and chemokines, thus contributing to the development of
the tumour.
Figure n.10
Cancer cells interact with other cell types that are present in tumour microenvironment, for example,
with mesenchymal, lymphoid, mast cells, etc. Tumour cells are able to release cytokines and
chemokines, which are all cell types that can attract other inflammatory cells (lymphocytes, mast
cells, fibroblasts, monocytes, etc.) in the area where the tumour is growing. These inflammatory cells
are able to release cytokines and chemokines themselves.
Thus, there are different populations of cells that are able to release molecules in order to amplify
the inflammatory state. Then, there are the tumour cells that release cytokines and chemokines,
which attract inflammatory cells. However, inflammatory cells in the tumour environment also
produce and release cytokines, chemokines, proteolytic enzymes, such as metalloproteinases or
other mediators, and all these molecules released by inflammatory cells are mitogenic for neoplastic
cells.
This situation creates the strong relationship between inflammatory cells and cancer cells in the
tumour environment. All the factors released by inflammatory cells are important because they can
potentiate tumour growth, stimulate cell proliferation and angiogenesis, which is important since cells
need nutrition, induce fibroblasts migration and maturation and enable the metastatic spread of
tumour cells from the primary tumour via blood and lymphatic vessels.
Figure n.11
231
Signal pathways connecting inflammation and cancer

It’s known that the association between inflammation and cancer can
be present with two different possibilities. In the first one, inflammation
appears, and this leads to the development of tumour. In the second
one, the appearance of the tumour leads to an inflammation. These
two situations explain the two signal pathways that have been
proposed to explain the association between the two events.
1. Intrinsic pathway: neoplastic cells start to release
inflammatory mediators, thus generating an inflammatory
microenvironment fundamental for tumour progression. This
pathway is typical of all the tumours that start without an
inflammatory state. Breast cancer, for example, can develop
without an initial inflammation, that will instead develop later;
2. Extrinsic pathway: typical of tumours where there’s a pre-
existing inflammatory condition, for example in the pancreas, in
the intestine, in the colon and in the prostate, which functions
as a good substrate for tumour initiation. It’s characterised by
inflammatory cells that release cytokines, chemokines, growth
factors, etc.
The two pathways have a point in common because they both are able
to activate specific transcription factors which are important for tumour
development. These are NF-kB, STAT3 and HIF1-α (hypoxia-
inducible factor 1α). These transcription factors have different target
genes, among which there are genes encoding for inflammatory mediators. When transcription
factors interact with genes, they can regulate the production and release of cytokines, chemokines,
growth factors and metalloproteinases. The release of these molecules will activate inflammatory
cells which will release other inflammatory mediators. Thus, at the end, the inflammation will be
amplified because all the cells are able to create an inflammatory microenvironment. This
amplification is important for tumour development.
In fact, all these inflammatory events are also important for tumour cells proliferation, survival, EMT,
angiogenesis, lymphoangiogenesis, tumour-cell migration, invasion, metastasis, etc.
NF-kB
NF-kB is the most important transcription factor which regulates the inflammatory processes.
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It has been discovered in 1986 by Ranjan Sen and David Baltimore.

Figure n.13 represents the classical
pathway of the activation of NF-kB;
when this factor is activated, it is able to
translocate from the cytosol to the
nucleus. NF-kB has two subunits: p50
and p65. There is also an additional
subunit, Ikβ-α, which keeps NF-kB in an
inactivated form. In order to let its two
subunits reach the nucleus and interact
with gene targets, Ikβ-α must be
phosphorylated and degraded. The
activation of NF-kB is responsible for the
synthesis of many molecules: apoptosis
Figure 13
regulators, chemokines, cytokines,
growth factors, receptors and adhesion
molecules.
Besides that, NF-kB plays a key role in
inflammatory response and during cancer development. After its activation, due to bacteria,
viruses, inflammatory cytokines and necrotic cell products, NF-kB can lead to the secretion and
release of different factors such as cytokines, chemokines, growth factors and angiogenic factors.
All these molecules are able to activate NF-kB which is present in the malignant cells. After the
activation of the factor present in the tumour, this transcription factor plays an important role in genes
regulation, in the release of apoptosis inhibitors and of invasive proteases.
Its activation has been observed in different types of cancer, especially in cancers developed from
a state of chronic inflammation, such as colon carcinoma or hepatocellular carcinoma.
In pictures n.14 and 15, it can be noticed that NF-kB can directly or indirectly control inflammation.
Thus, inflammatory cells can activate tumour cells but also NF-kB of tumour cells can influence
inflammatory cells.
In summary, the activation of this transcription factor is important for tumour cell proliferation and
survival, EMT, invasion, cellular metabolism, metastasis and angiogenesis.
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Figure n.14 Figure n.15
Chronic inflammation crosstalk between cancer

cells and immune cells
NF-kB and STAT3 can be stimulated by inflammatory
cells through the release of cytokines, chemokines and
growth factors. They are important transcriptional factors
that can activate some genes in the nucleus of cancer
cells favouring cell proliferation, survival, EMT, invasion,
metastasis and angiogenesis. Then, following the
activation of tumour NF-kB, tumour cells are also able to
release chemokines and cytokines. These molecules
can then attract more inflammatory cells in the tumour
microenvironment, in this way they maintain and amplify
the inflammatory response.
Tumour-associated macrophages (TAMs) are key regulators of tumour-related inflammation

Tumour microenvironment is populated by many types of cells: dendritic cells, myeloid-derived
suppressor cells, neutrophils, mast cells, natural killer cells, natural killer T cells, T cells, B cells,
cancer-associated fibroblasts and endothelial cells. Besides these ones, there is a group of cells that
seems to play a fundamental role in the association between inflammation and cancer: TAMs
(tumour- associated macrophages). These TAMs are regulated by NF-kB and HIF. They are key
regulators of tumour related inflammation. At the beginning, they are normal macrophages with a
M1 phenotype, but then they change and become of the M2 type. When they are at their normal
phenotype, they release pro-inflammatory cytokines. Then, during the progression of the tumour, the
macrophages become of the M2 type and play an important role for tumour growth thanks to the
release of growth factors, such as the epithelial ones (EGFs). They are also necessary for cell
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survival, angiogenesis (through VEGFs), metastasis, immunosuppression and extracellular matrix

remodelling.
Figure n.16: During the tumour progression (in this case, colorectal carcinoma, from stage I to stage
IV), macrophage polarization and the switch from phenotype M1 to phenotype M2 are fundamental,
because when they change their phenotype and functions, this type of macrophages can contribute
to tumour progression and development. In fact, they will increase cell proliferation, survival,
metastasis, angiogenesis, etc.
Stage I Stage II Stage III Stage IV
Figure n.16
The emerging role of tumour-associated neutrophils (TANs) in tumour development and

growth
Recently, another group of cells that seems to play an important role in this association has been
studied: TANs (tumour-associated neutrophils). They are able to stimulate the release of different
factors involved in tumour progression in the tumour microenvironment. Thus, it seems that not only
macrophages but also neutrophils play an important role in the association between tumour and
inflammation.
Summary: Inflammation is a critical process in tumour development and progression.

There are two different situations:
1. Tumours can develop in a tissue without initial inflammation, for example breast cancer. In
this case, the tumour can release inflammatory mediators such as cytokines, chemokines,
growth factors and transcription factors. In this way, during tumour progression, an
inflammatory state will appear;
2. A chronic inflammatory state, for example at the level of the colon, prostate, pancreas and
skin, can be dangerous because over the years it can lead to cancer development.
In both cases, there is a strong connection between tumour cells and inflammatory cells, both are
able to release cytokines, chemokines etc. and eventually, there will be an amplification of
inflammation and inflammatory response which is important for the tumour. In this inflammatory
environment, it seems that macrophages and neutrophils are key regulators of the inflammatory
state.
Arnaud’s question: Thus, if there’s no inflammation, there’s no cancer?

Professor: Inflammation is a necessary condition but not sufficient for the development of cancer.
Nicolò’s question: Does inflammation only affect tumour in a positive way?

Professor: Yes. Inflammation is positive for cancerogenesis. If you look at a histological preparation,
you can see that in a tumour there is always an inflammation. It can be the starter or the consequence
of the tumour but it’s always present. it’s an important state for tumour development itself. For
example, in a patient with a tumour you can see that some inflammatory biomarkers are increased.
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For example, in a blood test: VES60 is the marker for velocity of sedimentation of red blood cells and
it will be increased in the case of a tumour, then there will be an increase of lymphocytes and white
cells.
Nicolò’s Q: In the early phase of the tumour formation, the inflammation and immune response has
a positive effect on the tumour?
Professor: When there’s inflammation, macrophages are able to decrease immune response.
There’s an immunodepression. The immune system works at the beginning of the development of
the tumour but then, when tumour cells appear, macrophages change their way of action.
(Arnaud’s sproloquio).
HEREDITY AND TUMOURS
Introduction
It is now well-known that genomic instability is at the origin of tumours and that all the genetic
alterations/aberrations are responsible for the increment of the many types of mutations which are
required/necessary for neoplastic cell transformation and for tumour progression/development.
Every day we are exposed to carcinogenic agents (all agents that are mutagenic) like chemicals
and biological and physical agents. A few lessons ago we talked about radiations, which can be
responsible for a series of cancers and tumours and can therefore be considered as physical agents
having mutagenic properties. Since we are exposed every day to these agents, most of the tumours
are of sporadic origin: up to 90%61 of the tumours are, in fact, sporadic tumours.
However, a small portion of tumours have another origin: they aren’t caused only by mutagenic
agents, but in some families the tumour is a hereditary trait due to a mutation that appears in the
germ line. This mutation usually affects tumours suppressor genes.
As already seen in previous lessons, there are two groups of genes, with an opposite function,
responsible for the genomic instability at the base of tumour progression: oncogenes and tumour
suppressor genes.
Oncogenes
Oncogenes, as the word itself suggests, are genes which induce the neoplastic transformation of
the cells and tumour development; they are, therefore, genes that favour the cancerogenesis.
They encode for growth factors, growth factor receptors, signal transducers, transcription factors,
cell cycle components, etc; their aim is to promote cell proliferation.
The coding error can be qualitative or quantitative:
- Quantitative because oncogenes can produce a greater amount of proteins, called
oncoproteins, whose role is to stimulate cell proliferation, or, in some cases, these proteins
cannot be synthesised at all;
- Qualitative because sometimes the oncoproteins are synthesised but they are not able to
work properly because they are both structurally and functionally abnormal proteins.
Concerning oncogenes, only one mutation in a single copy of the gene is sufficient to confer a
proliferative advantage, so it is enough to cause the transformation towards the neoplastic cell since
this cell starts to proliferate without any control. Therefore, oncogenes are said to have a dominant
effect.
Tumour suppressor genes
60 In English, erythrocyte sedimentation rate (ESR)

61 It says 95% in the slides
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Tumour suppressor genes have an opposite function: their role is to inhibit and contrast cell growth.
They control and block an uncontrolled growth, as it happens in tumours.
Unlike oncogenes, a single mutation on one copy of the gene is NOT sufficient. A second mutation
in the other allele is required (both copies of the gene are mutated) and only in this case, when two
mutations are present on the same gene, this type of gene cannot work properly. Therefore, tumour
suppressor genes are said to have a recessive effect.
Under normal conditions, it is clear that cell cycle regulation depends on the balance between these
two groups of genes, in particular, cell proliferation is regulated thanks to a balance between
protooncogenes62 and tumour suppressor genes: if protooncogenes stimulate cell proliferation,
tumour suppressor genes inhibit cell proliferation.
Mutations in oncogenes
Protooncogene become oncogenes when they are mutated.
The alteration/damage that appears on DNA can be point
mutations, causing the production of proteins that from a
structural and functional point of view are abnormal.
For instance, point mutations can appear on the oncogene
Ras, which plays an important role in the cell proliferation
process induced by growth factors (figure n.1). Ras stimulates
cell proliferation when it is activated, then it is inactivated to
stop cell proliferation thanks to hydrolysis of GTP. If Ras is
mutated, it permanently remains in the active form and will
continuously promote/stimulate cell proliferation because it is
unable to hydrolyse GTP and therefore it cannot pass in the
inactive form.
Another alteration that can appear on DNA is gene
Figure 1 amplification; this mutation causes the production of a larger
amount of oncoproteins.
For instance, gene Figure 2
amplification can affect the N-Myc oncogene (figure n.2). This
kind of gene amplification is found associated with human
neuroblastoma, where hundreds of copies of protooncogenes are
produced because of gene amplification.
Gene amplification can also affect other oncogenes such as erb-
B2, D cyclin, etc.
62
Protooncogenes are the parental genes of oncogenes. They are called protooncogenes when they are not mutated
and then, when a mutation appears, they are called oncogenes.
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[slide 8] Then there is gene rearrangement or chromosomal translocation, which involves for
example c-Myc and abl. In this type of mutation, the oncogene is moved from one chromosome to
another chromosome.
For instance, there is the translocation of the abl oncogene from
chromosome 9 to chromosome 22 which has been found in chronic
myelogenous leukaemia; instead, in Burkitt lymphoma the C-Myc
oncogene is translocated from chromosome 8 to chromosome 14.
Figure n.4 is a list of the main oncogenes that can be mutated either
because of point mutations or amplification or translocations. Each Figure 3
oncogene is associated with a type of tumour.
Figure 4
Mutations in tumour suppressor genes

Tumour suppressor genes have an opposite function compared to oncogenes, therefore they are
characterised by slightly different types of mutation.
Tumour suppressor genes can be altered because of point mutations. In this case, the protein that
is synthesised is abnormal, so it cannot work properly. As a result, there is a failure of growth
inhibition since the genes are not able to prevent an uncontrolled growth, as is seen during tumour
cell proliferation.
Another type of alteration which can be found in tumour suppressor genes is chromosomal
deletions, in which case the gene is totally lost (not mutated, it disappears). Therefore, of course, it
is not possible to synthesise factors and proteins that have a role in the inhibition of cell proliferation.
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Figure n.5 is a list of tumour suppressor genes. The main ones are NF-1 and 2, APC, Rb, p53,
BRCA-1 and 2. They are involved in the regulation of the cell cycle or in the repair of damaged DNA.
All of them are associated with some types of tumours which can be inherited from one parent.
Figure 5
Tumours with hereditary substrate

Individuals can inherit a mutation of one of the genes involved in carcinogenesis from their parents.
So, they inherit a mutated gene from one of the parents (father or mother) which represents a
predisposing factor to the neoplastic transformation: this is why it is possible to talk about
hereditary predisposition to a tumour, also referred to as family tumour. In fact, there are some
tumours that have a high incidence in a family because of a hereditary predisposition to develop this
type of tumours.
As said before, about 90% of tumours are sporadic tumours but at least 1-5% of tumours are
hereditary tumours.
Hereditary mutations mainly concern tumour suppressor genes, so what is possible to receive from
the father or from the mother is a mutated tumour suppressor gene. This means that we receive
only one copy of a gene that is mutated. However, as already said, tumour suppressor genes have
a recessive effect so only one mutation of one copy is not sufficient to favour tumour development,
both copies of the gene must be mutated: only in that case the protein synthesised by the tumour
suppressor gene is a protein that cannot work properly. Otherwise, there can be a deletion, in which
case the gene disappears. Rarely, although it is not impossible, individuals can inherit a mutated
oncogene, in which case, because it has a dominant effect, only one mutation on one copy of the
gene is sufficient to develop a tumour.
Of course, the mutation must be present in the germinal cells in order for it to be inherited.
There are different tumours with hereditary predisposition, only the most frequent are reported. They
can be divided in three groups:
- Hereditary neoplastic syndromes, which are characterised by an autosomal dominance;
they are uncommon tumours associated with phenotypic markers. In this group we can find
neurofibromatosis type I and II, familial adenomatous polyposis of the colon and
retinoblastoma;
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- Malignant family tumour, which have a very evident family incidence; they are, for instance,
carcinomas of the breast, ovarian cancers, gastrointestinal and uterine tumours and some
leukaemias;
- Autosomal recessive syndromes from DNA repair defects; they are tumours that can
develop because there isn’t the possibility to repair DNA when DNA is damaged. For instance,
they are xeroderma pigmentosum, hereditary non-polyposis colon cancer and ataxia
telangiectasia.
Group I – hereditary neoplastic syndromes

The tumour suppressor genes involved are NF-1, NF-2 and APC (see figure n.5). Mutations of these
tumour suppressor genes in the germinal cells are associated with benign tumours: at the beginning
they are responsible for the development of benign tumours which can later develop in malignant
tumour.
Neurofibromatosis type I
It is a benign tumour characterised by hundreds of neurofibromas that are evident on the skin;
figure n.6 shows how many neurofibromas are present on the superficial site of the skin (cutaneous
site), although they can also be present in the subcutaneous layer (in the deep sites). They are all
benign tumours of nerve tissue.
Affected individuals can have a specific hyperpigmentation, for example on the skin, the so-called
“coffee and milk spots” (figure n.7), or in the eyes (figure n.8).
Figure 6 Figure 7 Figure 8
Although they are benign tumours, some of them (3-5%) can develop in a malignant way, giving
neurofibrosarcomas63.
More frequently, tumours can develop from other tumors of neural origin, for example meningiomas,
optic nerve gliomas, etc. In children, there is also the risk to develop some kind of leukaemia.
In this type of tumours, the mutated gene is the NF-1 gene, a tumour
suppressor gene encoding for the protein neurofibromin 1. In normal Figure 9
conditions, the NF1 tumour suppressor gene acts through a negative
regulation of Ras signalling: the role of this gene is to inhibit Ras
signalling, thus blocking cell proliferation, since neurofibromin 1 is a
protein of the family of GTPases and the hydrolysis of GTP is
necessary to inactivate Ras. However, if the gene is mutated, the
protein is not synthesised, and therefore it is not able to stop Ras signalling, thus allowing cell
proliferation to go on. The uncontrolled cell proliferation is, in fact, responsible for the neurofibroma
formation (because there is an hyperproliferation of cells).
Neurofibromatosis type II
In this case, the gene involved is NF-2 another tumour suppressor gene64.
63 The word “sarcoma” indicates that these tumours are malignant.

64 We only talk about tumour suppressor genes because they are the main genes implicated in family tumours.
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Because of the mutation of the NF-2 gene, the protein merlin is not synthesised or synthesised in
an aberrant form. This protein is associated with cytoskeletal proteins and its role is to inhibit cell
proliferation. If merlin is not synthesised or is synthesised in an aberrant form, of course cell
proliferation cannot be inhibited. In this case there is the presence of neurinomas at the level of the
acoustic nerves and sometimes tumours at the level of the CNS can also develop.
Multiple family polyposis

Also called FAP (familial adenomatous polyposis).
It is an autosomal dominant disease involving the tumour suppressor gene APC, whose role is to
regulate the stability and the function of β-catenin. APC and B catenin are components of the WNT
signalling pathway.
Figure 10
In normal resting colonic epithelial cells (figure n.10 A), β-catenin forms a complex containing
APC proteins (derived from the APC gene). Thanks to this complex, β-catenin can be
degraded/eliminated and there is no cell proliferation65 because B catenin can stimulate cell
proliferation.
When normal colonic epithelial cells (figure n.10 B) are stimulated by the WNT signalling pathway
because proliferation is needed, the β-catenin doesn’t form the complex with the APC protein and
stays free; in this way β-catenin can translocate into the nucleus, bind with the DNA and then
stimulate the transcription factor TCF. This is the way in which β-catenin can stimulate cell
proliferation.
When APC is mutated or it is absent (figure n.10 C), β-catenin is again free (it is not possible for
it to form the complex with an abnormal APC) and it can translocate into the nucleus, bind the DNA
and induce the transcription factor, causing also in this case cell proliferation but without any
stimulation from the WNT signalling pathway. When the APC gene is mutated, it is not able anymore
to encode the protein APC and to control and degrade β-catenin, which is therefore able to
translocate into the nucleus and stimulate cell proliferation.
Individuals who are born with a mutated allele for the APC gene develop hundreds of thousands of
polyps in the intestine (mainly colon), usually around 40 years of age. Therefore, these individuals
have a high risk to develop colon carcinoma: the polyps are benign tumours which can develop into
65 Because β-catenin can stimulate cell proliferation, but if it is degraded the stimulus disappears
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malignant tumours, but only after the accumulation of other mutations (the mutation of the APC gene
alone is not sufficient, other mutations must accumulate to have the development of a carcinoma).
Figure n.11 shows a multiphasic process leading to colon carcinoma. To pass from a benign
Figure 11 tumour to a malignant tumour, to become a carcinoma, the
mutation of APC alone is not sufficient, other mutations must
be accumulated in the cell; for instance, they can be
mutations of Ras, of deleted colorectal carcinoma (DCC), of
p53, etc. only in this way there is accelerated progression
and the development of colon carcinoma.
Retinoblastoma
It is another type of hereditary tumour in which Rb is the
tumour suppressor gene involved (figure n.5 Nucleo)
When cells
Figure 12
are stimulated to divide, the Rb protein is inactivated
by phosphorylation because, in its
unphosphorylated form, it controls the cell cycle/cell
proliferation: it doesn’t permit the cells to pass from
G1 phase to S phase. For the cell to pass this control
point, Rb must be phosphorylated. Then at the
level of phase M, Rb is again dephosphorylated by
a phosphatase. Therefore, Rb in the
hypophosphorylated or active form stops cell
proliferation.
It is clear that Rb plays a fundamental important role in regulating
the G1/S checkpoint of cell cycle.
When Rb is hypophosphorylated it means that it is active and it
binds to E2F, a transcription factor, forming a complex which
binds DNA: in this case, they inhibit the transcription of genes
involved in the cell cycle (so in the cell proliferation) and therefore
the cells cannot pass from the phase G1 to the phase S and they
cannot divide.
When Rb is hyperphosphorylated it is not able to form the
complex with E2F, which is able to translocate in the nucleus and
bind to target genes which encode for all the factors that are able
to stimulate cell proliferation. In this case, the cells can pass from
the G1 phase to the S phase and so they can divide.
The Rb gene is important for a type of hereditary tumour called
Figure 13
retinoblastoma.
The retinoblastoma is an ocular cancer or a retinal
neuroepithelioma. This is a typical tumour which affects children, who also have a high risk to
develop osteosarcoma, the malignant tumour of the bones. There are two forms:
- Early form that can develop during the first months of life (within 15 months of life);
- Late form that develops later that develops between 2 and 5 years of age.
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Rb protein is important for the regulation of the cell cycle. Of course, if the gene Rb is deleted, the
protein Rb cannot be synthesised.
In figure n.14 you can compare a sporadic form of retinoblastoma with a familial form of
retinoblastoma with a hereditary predisposition. Figure 14
In the sporadic form, both Rb alleles undergo
somatic mutation, so the mutation will appear
in the somatic cells. A second mutation is also
needed because this is a tumour suppressor
gene, so both alleles must be mutated to have a
neoplastic proliferation of the retinal cells.
In the case of the familial form, a mutated copy
of Rb is inherited, for instance from the father,
so from the parent only one mutated allele of the
Rb gene will be received. This mutation will
appear in all somatic cells, but the child is
normal because it is only one mutation. Only
when a second mutation, for instance due to the
exposure to environmental agents, appears in
the somatic cell there is the neoplastic
proliferation of the retinal cells.
Syndrome of Li – Fraumeni
Another hereditary tumour is the so-called syndrome of Li-Fraumeni.
The gene involved is p53, a tumour suppressor gene. Therefore, the predisposition to develop a
tumour is due to the mutation of p53.
p53 is very important in the cell cycle regulation because the protein it encodes is able to block the
cell in G1 phase when DNA is damaged (if there is a damage at the level of DNA, the proliferation
must be blocked to have time to repair the damage). If p53 gene is mutated, it is not able to block
the cell cycle in the phase G1 and promote DNA reparation.
Figure 15 Normally p53 is activated when DNA is damaged by one of
the mutagenic agents present in the environment (figure
n.15). If it is necessary to repair the DNA or if there is
hypoxia, the cell cycle is stopped at the G1 phase thanks
to the p53 protein. When the cell cycle stops, we have
different systems able to repair the DNA damage. If there
is a successful repair, then the cells can continue the cell
cycle and from the G1 pass to the S phase. In the case the
DNA repair fails, p53 triggers either apoptosis (so the cells
will be destroyed) or senescence (the cell is old and
eliminated directly). Only if there is a successful repair of
the DNA the cell will start again to proliferate.
In cells with loss or mutations of p53, the protein is not able
to block the cell in G1 phase and to leave the time to repair
DNA, therefore all the mutations and alterations present in
the DNA will accumulate and will be transmitted to the other
cells (figure n.15).
The individuals with the Li-Fraumeni syndrome have a
defect/mutation at the level of the p53 gene and they can
develop sarcomas, carcinoma of the breast, leukaemia, brain tumours and adrenal carcinoma.
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Group II – malignant family tumours

Other two types of tumour suppressor genes are BRCA-1 and BRCA-2.
They are tumour suppressor genes involved in DNA repair. The deletions/mutations of these two
genes are associated with breast and ovarian carcinomas, tumours with a very evident family
incidence.
The cancer of the breast is a typical family tumour. It has been discovered that the missing/mutated
genes are BRCA-1 and BRCA-2. In fact, they are mutated in at least 80% of the family carcinoma of
the breast.
These genes are important for DNA repair processes, so if these genes are missing or mutated, the
DNA cannot be repaired, and the damage will be present (cannot be removed).
Group III – autosomal recessive syndromes from DNA repair defects

In this group are found the xeroderma pigmentosum, the
ataxia telangiectasia and also a human colon cancer
without the presence of polyps.
Figure 16
Xeroderma pigmentosum66
The individuals who suffer from this disease are not able
to repair DNA when it is damaged, for instance by radiation, like UV lights. The system involved in
DNA reparation is the NER system. It is a complex system in which many enzymes are involved: it
is enough that one of these enzymes is altered because of a mutated gene and the system doesn’t
work properly, impairing successful DNA repair.
Affected people are very sensitive to the mutagenic effect of UV action; they can have
conjunctivitis, skin full of hyperpigmentation, hyperkeratosis and they easily develop skin
cancers like melanoma or spinocellular epitheliomas or basocellular epitheliomas.
Hereditary non-polyposis colon cancer
In the previously mentioned colon cancer, individuals developed thousands of polyps in the colon.
In this case, however, the cancer doesn’t derive from polyps because there are no polyps in the
colon. This type of malignant colon cancer is seen in about 15% of the cases, while the remaining
85% derives from the multiple polyposis.
The gene involved is MSH2, which is involved in another DNA repair system called mismatch repair
system.
The carcinoma can appear around 40 years of age after an asymptomatic adenoma (see figure
concerning the progression of colorectal cancer to see the passage from adenoma to carcinoma).
66 Already seen when talking about UV light radiation, see pathology lessons 8 (UV light) and 9 (XP)
244
Ataxia telangiectasia
This is a rare genetic disease; like the two above-mentioned diseases (XP and HNPCC), it is an
autosomal recessive disease.
In this case, the mutation is on the ATM gene, which encodes for a protein necessary for p53
phosphorylation67. If the gene is mutated or deleted, the protein is missing, p53 cannot be
phosphorylated and therefore, if not active, cannot block the cell cycle in the G1 phase, thus
preventing DNA repair.
As already said, ATM gene encodes for ATM protein kinase necessary for p53 phosphorylation.
Only when p53 is in its phosphorylated state it can activate the DNA
repairing system. In case of mutation of the ATM gene, of course the
cell cycle cannot be stopped in phase G1 and the DNA cannot be
repaired by the different systems that we have in our body (figure
n.17).
This syndrome has typical symptoms:
- Ataxia: the meaning of the word is that people have problems
in the movement; in fact, affected individuals show tremors
during the first year of life (so they cannot move properly) and Figure 17
loss of sense of position with the first steps (so they cannot
walk properly);
- Telangiectasia: this word means vasodilatation i.e. some small
vessels are dilatated, especially in the skin and eyes
(cutaneous and ocular telangiectasia);
- Cerebellar ataxia;
- Mental retardation;
- Thymic hypoplasia;
- Premature aging;
- Neoplasia predisposition;
- High radiosensitivity.
The symptoms are quite heterogeneous and are not specific, they are different types of clinical
symptoms.
Next lesson will be about cancer cachexia. As we should already know, terminal oncologic patients
have this depauperation of the body, they lose weight and are not able to eat, because of all the
toxins present in the circulations, like for instance cytokines, interleukins, TNFs, etc. which makes
them physically destroyed. This is cachexia, one of the aspects of malignant tumours.
TUMOR CACHEXIA
Introduction
Cachexia is a very old condition; it had been already recognized by Hippocrates, one of the most
famous ancient doctors, who defined it as a “general bad condition”. We know that several chronic
illnesses, such as chronic heart failure, infection, sepsis and in particular cancer very frequently
result in the onset of this condition. Cachexia is not a disease, but a syndrome68.
67Necessary to activate p53

68Disease: a condition of the living animal or plant body or of one of its parts that impairs normal functioning and is
typically manifested by distinguishing signs and symptoms
Syndrome: A combination of symptoms and signs that together represent a disease process
245
Why is cachexia important?

Cachexia is important since it significantly challenges the outcome of cancer patients. When
cachexia is present, patients suffer from several bad states: weakness, fatigue, anorexia, loss of
body weight, loss of muscle mass, loss of adipose mass… This results in reduced tolerance to
anticancer treatment, which has to be frequently interrupted or at least reduced in dosage,
challenging in some way the anticancer action of these drugs. On the other side, cachexia
significantly reduces the chances of patient survival, both because it reduces the possibility to cure
cancer but also per se. Example: The loss of muscle mass eventually leads to respiratory failure and
heart problems, anticipating patient’s death.
Some tumors are more

strongly associated with
cachexia than others e.g.
tumors related to the GI
apparatus since they impinge
on patient food intake, lung
cancer… As you can see,
apart from the type of tumor,
the percentage of patients
presenting body weight loss is
significantly high.
There is a very strong
association between body
weight loss and patient
survival/response to treatment. For this is reason it is fundamental to find out a way to understand if
a cancer patient will eventually develop cachexia, is already cachectic or is still in good condition. A
few years ago, a group of doctors defined a staging method for cachectic patients, identifying three
different points:
- Pre-cachexia: the patient physical conditions are normal even if she/he has cancer;
- Cachexia stage: more than 5% loss of body weight, BMI less than 20% and other
characteristics;
- Refractory cachexia: there is no room for treatment of this syndrome.
Research is very active in trying to understand the mechanisms underlying cancer pre-cachexia in
order to pick up patients preferably in the pre-cachexia stage (or in the cachexia stage if that is not
possible), with the aim to set up therapeutic interventions and strategies able at least to delay the
246
progression towards refractory cachexia. First take-home message: it is mandatory to identify

patients in pre-cachexia or, if not possible, at least in cachexia - if a patient is identified in
refractory cachexia there is very little the clinician can do to help the patient to cope with the toxicity
of the treatment and cancer progression.
Body weight loss

Body weight loss is the first clinical sign of cachexia: when
patients go to the doctor and refer a sudden and important
body weight loss, the clinician must take it into account (unless
of course the patient has changed completely his/her life-style:
from sedentary to very active or from eating a lot to dieting). A
sudden decrease in body weight should always worry the
clinician.
How can we measure body weight?

Just the number of kilograms is not enough to define if the patient is overweight or underweight;
clinicians often use the BMI (Body Mass Index), which is a scale that puts height in relation to
weight. In this way we can define people as normoweight, overweight and underweight.
However, even people who are overweight or even obese can develop cachexia, therefore this scale
does not provide enough information to decide if the patient weight loss is related to cachexia: an
MD should take in consideration first the history of the patient and then the body composition i.e.
has he/she lost muscle, fat?
What clinicians really need are indications about body composition (% of muscle, fat...).
Taking only into account the BMI is not enough: the risk is to lose patients that are potentially going
towards cachexia, while our goal is to identify patients that could develop cachexia before they
become cachectic, in order to set up interventions (like nutritional advice) with the aim of helping the
patient’s organism to cope with the tumor for as much time as possible.
We have already said that the amount of body weight loss

is a predictor of survival. In this graph you can appreciate
the relation between body weight loss and BMI: what you
obtain is a sort of grade, from 0 to 4 – 4 being the worst -
which is related to the survival rate. The median survival
is progressively decreasing in parallel with the score you
obtain plotting the data.
How can I measure body composition?

Several devices are used.
247
A TC scan can easily quantify the amount of muscle, bone and fat. The same researches that
proposed the scheme above also defined the algorithm used to calculate the amount of loss of
muscle mass and eventually fat mass with these scans. Other devices are used: dex analysis,
hyperisciometry69, anthropometric measurements. Of course, a CT scan is more precise than
anthropometric measurements but even if every hospital has got a CT scan, it is not easy to require
one for each patient, therefore clinicians have to be aware of every possible way to get complete
information about body composition of the patient.
Cachexia is a multi-organ syndrome
Body weight loss and loss of muscle and fat are among the most prominent changes occurring in
cancer cachexia, but the situation is much more complicated than this: the tumor causes several
alterations in a sort of cascade. The tumor is leading reduction in food intake and metabolic
alterations (these depend on several humoral factors and have several effects); moreover, the
antitumor treatment worsens even more the metabolic derangements induced by the tumor.
The idea that the cachexia is a multi-organ symptom might seem obvious to us but is indeed quite
a new concept: in the last 15 years the
number of papers studying several aspects of
cancer cachexia increased constantly but
they were just concentrating on a specific
aspect: fat, muscle, mitochondria, effects of
chemotherapy. A comprehensive view is not
yet available. However, this is something that
has to be taken into consideration, both from
the research and the clinical point of view: the
tumor does not live on its own but in an
organism and is taking advantage of the
whole body’s metabolism for its own survival.
The tumor manipulates and modulates the
metabolism not only to obtain the
substrates for tumor growth but also to
produce metabolites that can alter
metabolism in distant tissues (causing
side effects). For example, changes occurring in the skeletal muscle affect the white adipose tissue,
69
The first two methods are impossible to understand from the recording, so I wrote down the closest thing,
but I couldn’t find anything on Internet. For the brave ones: 15:40 on the recording.
248
the brain, the brown adipose tissue, the heart and the liver. These anatomical districts reciprocally
impinge on the skeletal muscle tissue, resulting in muscle waste.
Humoral mediators
The hypothesis that inflammation is the driving force in the pathogenesis of cancer cachexia has
recently being established. About 30 years ago the first cachexia–inducing cytokine has been
discovered: TNF (Tumor Necrosis Factor). TNF has been discovered independently in two different
labs around 1975: in one lab it was studied in reference to its ability to induce necrosis in solid tumors
(from this the name tumor necrosis factor). The use of this cytokine was thought to be able to cure
tumors, through a sort of “physiological chemotherapy”. Completely independently, in another lab
researches investigated the biological effects of this molecule on healthy animals; the results
indicated that TNF leads to cachexia. Of course, after this discovery, the idea to use TNF as a
physiological chemotherapy was discarded, because the off targets effects of this molecule are really
dangerous for the organism.
From that point on, the knowledge about the involvement of inflammation in the pathogenesis of
cancer cachexia has grown and is now well accepted.
Inflammation plays a crucial role in cancer development: an inflammatory response is necessary for
cancer to be established; moreover, tumors are able to modulate the inflammatory response, shifting
it from pro-inflammatory to anti-inflammatory within the tumor, in order to create a sort of niche, in
which the tumor can grow without being hurt by external agents (macrophages, neutrophils…).
In addition to this role in cancer development, inflammation is also a driver of cachexia. Normally
pro-inflammatory cytokines are virtually undetectable in the circulation i.e. if you are healthy and you
want to measure the circulating level of TNF, interleukin-6 or γ-interferon in your circulation, you do
not see anything, because in physiological conditions we don’t need and we don’t want these
cytokines to be produced because they have dangerous side effects e.g. cachexia inducing effects
of these cytokines. On the other hand, in cancer conditions there is a persistent low-grade
inflammation that results in an increased level of these pro-inflammatory cytokines in the circulation,
with all the sides effects on liver metabolism, muscle, adipose tissue and brain.
The activity of these cytokines in distinct tissues is different:

- Brain: anorexia - food intake is reduced;
- Muscle and adipose tissue: activate catabolic pathways and inhibit anabolic processes;
- Liver: activation and production of acute-phase proteins.
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By contrast with the first tissues, the

liver is activated and therefore it needs
amino acids and energy in order to
produce reactants. This means that an
additional tissue is draining substrates
from the organism. Why “additional”?
Of course, the first tissue draining
substrates from the other districts is the
tumor itself, whose existence depends
on the mobilization of proteins, lipids,
carbohydrates in order to proliferate,
grow, produce new vessels,
metastasis…
Who produces these inflammatory
mediators? This has not been yet
clarified but what is accepted is that the
immune cells of the organism, which
react against the tumor, produce these mediators: activated inflammatory cells and immune cells
release these molecules. There is also evidence that shows that also the tumor itself is able to
produce these types of mediators. Different tumors release different molecules: some produce IL-6,
others γ-IFN; in addition, some produce also non-classical cytokines, mediators able to modulate
directly some aspects of metabolic balance, for example some tumors produce factors acting directly
on the brain to induce the anorectic behavior.
Cancer and malnutrition

Even in normal conditions, if a patient in not adequately nourished, the substrates he/she integrates
are limited. Also, depletion of specific foods (for example meat, or certain categories of food whose
taste is not appealing) can result in limitation of the substrates available for the organism.
Let’s take the example of ketogenic diets, which are frequently used to lose weight quickly: you don’t
introduce carbohydrates in your diet, so your body uses lipids, fats at first and, if it is not enough,
also proteins, resulting in hyperketosis. Consequently, alterations in the body metabolism occur.
Hyperketosis is also very common in malnutrition in cancer and it causes an additional stimulus to
mobilize lipids and proteins. Therefore, reduction of food intake due to alterations at the central level
(anorexia induced by action of tumor mediators at the brain level) is responsible for an additional
depletion of substrates.
Other factors influencing food intake are the side effects due to cancer treatment (nausea,
diarrhea, vomiting): these symptoms are not directly reducing food intake but reducing the availability
of fats since you are wasting substrates that are introduced. Another factor to take into account is
the physical site where the tumor is developing e.g. if the tumor develops in the pancreas, the
efficiency of this gland will not be optimal and thus food will not be digested efficiently; you may have
consequences that are not simply related to the tumor-induced alteration of metabolism but directly
due to the physical position of the tumor. Moreover, the psychological component of cancer often
impairs the ability of patients to maintain adequate food intake and a nourishing diet. This component
usually fluctuates: after the diagnosis, the patient will probably experience a hard time,
psychologically speaking, and won’t be able to maintain an adequate food intake; normally this
condition gets better with time: the patient gets used to the idea of living with an “alien”. Also, surgery
can influence the ability to get nourishment. In addition, not only food intake is challenged but also
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the ability to digest correctly, absorb and use the substrates that are introduced. In conclusion,
malnutrition in cancer is due to several factors: anorexia is just one component (one of the most
relevant of course) but there are several other components that must be considered.
Above this overall reduction of substrates availability

due to reduced food intake, malabsorption and so on,
another issue is that the organism of cancer patients is
set in a sort of wasting drive. Even if we have an
adequate number of substrates and we are eating
adequately, these substrates are not properly used,
they are wasted. The reasons of this waste are only
partially understood.
If we take measurement of glycaemia or circulating
amino acids not really at the end of the progression of
the disease in cancer patients, the parameters often
seem quite okay. This means that patients have the
right substrates in their blood; however, they are not able to use these substrates correctly since the
metabolism in peripheral tissues (mainly muscle and fat) is set towards catabolism, not anabolism,
and the inflammatory mediators significantly contribute to this kind of drive.
Energy metabolism
You already know that physiology is a matter “balance”: balance between
anabolism and catabolism, cell proliferation and cell death, protein/lipid
degradation and synthesis.
In physiological conditions energy intake is balanced by energy expenditure
(in our society the equilibrium is shifted more towards energy intake but
whatever) while in cachexia (not only in cancer cachexia, but also in
cachexia associated with several other chronic diseases) the balance is
shifted towards energy expenditure. Energy expenditure exceeds energy
intake - meaning energy derived from food.
We can take this concept of energy intake in terms of energy production

cell by cell, in a tissue, in the whole organism: every cell produces energy thanks to mitochondria.
Negative energy balance, shifted towards expenditure, has been explained in the last ten
years by alterations occurring at the mitochondria level. Even though the concept of negative
energy balance in cachexia is quite old, the mechanisms leading to this process were unknown until
few years ago (except for the reduced food intake, which is obvious).
Metabolic alterations
The occurrence of metabolic alterations at the level of several tissues ultimately leads to an
unbalanced energy crisis, which occurs in cachectic patient.
Take a look at the picture below: we can start from skeletal muscle or adipose tissue, the outcome
is the same.
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Skeletal muscle: we have an increased protein degradation (catabolic drive) that results in the
release of amino acids e.g. glutamine is used by the tumor in order to grow, while alanine is used by
the liver to produce glucose. In addition, factors that are produced by the muscle, the liver and the
tumor itself mobilize substrates from the adipose tissue, which go “feed” both the tumor and the liver,
in order to allow tumor proliferation and liver activation (this at least during pre-cachexia and
cachexia – in the refractory phase the metabolic activity of the liver is reduced).
Skeletal muscle
Why concentrating on skeletal muscle?
Skeletal muscle is required to maintain several physiological functions, such as breathing,

movement, heart pumping70 - these are all vital functions. Moreover, every movement of our life
depends on proper skeletal muscle functioning e.g. speaking, walking and so on.
Maintaining the skeletal muscle mass is the second primary goal of the clinician in taking care
of cancer patients.
When the skeletal muscle mass is reduced also its function (the ability to perform contraction) is
reduced; this phenomenon is caused by at least three factors:
- Increased activity of intracellular proteolytic systems: at the molecular level, we have
four proteolytic systems. At least two are over activated in the muscle tissue of cancer
patients: degradation depending of proteasome and degradation depending on
autophagy;
- Morphological alteration of mitochondria and dysfunction of these organelles: they
are unable to produce the right amount of ATP, even though they are morphologically
well-preserved;
- Occurrence of an impaired myogenic program: (quite recent, 10 years ago).
Myogenesis is NOT normally occurring in the adult organism, it is rather activated only
when damage occurs to the muscle. Conversely is constitutively active during
70
She said that the heart is a skeletal muscle even if it is behaving differently… not sure what this means
since we have seen that there are three kinds of muscles: skeletal, smooth and CARDIAC.
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embryogenesis and development. If we perform hard exercises during a gym session,

we produce micro-damage to the muscle fibers, which are able to activate the so-called
“regenerative program”. This process occurs also in cancer cachexia, but it is totally
unclear what is causing the “damage” to the fibers, enabling them to activate the
regenerative process. The general idea is that the program is activated as a sort of
compensatory response in order for the muscle to face the condition of catabolism: the
muscle is not able to cope with normal daily activities and activates the process as a sort
of salvage pathway. However, the regenerative response is unsuccessful: there is not a
completely fusion of muscle stem cells with the existing fibers, so the compensation for
the loss of muscle mass fails.
Even if this is not really obvious, we also have a

reduction in protein synthesis. However, there are
some data clearly indicating that at least at a certain
time during the progression of the disease, cancer
patients do not show reduced protein synthesis. Even
though the catabolism is over activated, the organism
(at least at some point) tries to strike back and
counteract the catabolic drive maintaining lipid and
protein synthesis rates, depending on the tumor and
tissue, in order to compensate for the loss of substrates.
What is driving the catabolic response?

We will concentrate on the
skeletal muscle.
In the picture you can observe
which pathways are disregulated
in cancer patients: IGF1
pathway, pathways acting
through the TNF receptor
family, on cytokine type I
family, on the myostatin family
of ligands... With the exception of
the IGF1 pathways (leading to
anabolism), which can be down
regulated, the others are
significantly upregulated. The
result is an increased expression of genes involved in the regulation of protein metabolism, in
particular genes that contribute to the protein degradation machinery.
The most disregulated pathways in muscular cachectic patients are:

- Ubiquitin-proteasome system;
- Autophagy pathways.
Genes coding for proteins involved in these pathways are over expressed in the muscles of cancer
patients; this is due to the overactivation of several signal transduction pathways that impinge on the
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expression for these genes. The result is the disruption of the sarcomere structure and function,
since myofibrillar and structural proteins are degraded.
Ubiquitin-proteasome system
It is a catabolic device localized in the cell cytoplasm, differently from the autophagy pathway that
works together with lysosomes. The proteasome is a sort of tunnel in which labeled proteins enter
and come out as peptides (di- or tripeptides). In order to enter this sort of tunnel, proteins must be
labeled by ubiquitin residues. Three types of enzymes are necessary in order to label proteins with
ubiquitin residues: in particular we will focus on the E3 ubiquitin ligases since several years ago
muscle-specific E3 ubiquitin ligases have been discovered: atrogin-1, MuRF1, MUSA 1 (discovered
in Padova) and others. The expression of these ubiquitin ligases is increased in the muscle of cancer
hosts and can be induced by the action of some pro-inflammatory mediators we mentioned before.
The expression levels of these proteins are frequently used as markers of disregulation of
protein degradation. However, this is not totally correct: in order to really know if in a muscle protein
degradation is above the physiological level, protein degradation rates should be measured in a
kinetic and dynamic way. Just “taking a picture” of what is happening in the muscle in that moment
is meaningless. Anyway, the systems available to measure protein degradation in vivo are too
invasive or costly to be routinely performed. In experimental settings with animals, radioactive
tracers, which cannot be used in human beings, can be used. With humans you should use
stabilizators that involve mass spectronomy and mass gas chromatography in order to get the
results. Nevertheless, the costs associated to these methods are really high.
Therefore, the use of markers related to the ubiquitin ligases Goals of a clinician when dealing
could be a way to simplify the identification of protein with cachexia (take-home msg)
catabolism. The idea is that increased expression of these  Identify cachectic patients
markers is a suggestion that the patient muscles are in a as early as possible
 Try to maintain muscle
condition of hyperdegradation of proteins. But even if these
mass as much as possible
markers could correctly determine the catabolic condition of  Hope to find biomarkers
patient, the only way to get the data would be through a muscle able to give a clear picture
biopsy. Therefore, those markers are only available when the of the state of progression
of cachexia
patient needs to undergo surgery (you cannot routinely
perform this evaluation in patients!). These markers can be used to have indications about the
muscles when the surgery is performed and in experimental settings but are useless to follow up
patients during the course of the disease and not useful at all in order to identify cachexia
very early (that is the first important goal of the clinicians when dealing with cachectic patients –
look at the box).
Research is actively working in finding easily collectable biomarkers, something we can pick up in
biological fluids e.g. blood, urine, saliva, useful to understand the state of progression of cachexia
since it is not easy at all to have a muscle biopsy unless the patient is undergoing surgery.
Few examples (graphs below)

Remember that research systems are not directly translatable to clinical practice but are still
necessary in order to at least screen the scenario and select things that should be checked also on
patients.
Data obtained in mice show that in association with muscle wasting we have an increased
expression of some of these markers. In tumor bearing mice we have an increased expression of
one of these markers, antrogin-1. Then, in the second graph, loss of muscle mass and increased
expression of two muscle specific ubitiquin ligases. This is also true in gastric cancer patients (data
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obtained thanks to muscle biopsy taken up during surgery): we do not have data on the level of
muscle specific ubiquitin ligases, but data on the level of ubiquitinated proteins which is an indirect
measurement of the cell degradative pathways and this is associated with increased activity of
proteasome, a sort of multicatalytic complex that has at least three different enzymatic activities that
are increased in this case.
It is also important to notice that alterations of the activity of the proteasome can also be detected in
patients that do not lose body weight. If you classify the patients of these studies according to body
weight loss, you can see that the increased enzymatic activity of proteasome can be detected in the
muscles of patients that do not lose body weight, so they are apparently in very good physical
conditions, but at the molecular level you can already see alterations. This is another strong
suggestion that clinicians need to identify patients with a condition that is preceding the one of
cachexia: if we are able (through biomarkers) to understand if patients in good clinical condition
already present molecular changes, we can try to modulate these alterations and at least delay
the progression of cachexia and maintain the anticancer regimen unchanged.
Autophagy system
The second degradation system which is over activated in cancer cachexia is the autophagic-
lysosomal system, that unlike
the proteolytic system which
is localized in the cytoplasm,
is segregated from the rest of
the cell: it depends on the
formation of membrane
vacuoles, which surround
part of the cytoplasm and can
include organelles or not.
The membrane (derived from
the ER) elongates and fuses
resulting in the formation of
the autophagosome. This
structure will then fuse with
the lysosome, forming the
phagolysosome, where the
cargo is degraded and the
substrates recycled.
The study of these systems
was very difficult in the past since the only way to investigate the disregulation of the antiphagocytic
system was through electron microscopy analysis and this was not an easy task (the preparation of
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the sample is quite a challenging process). About 20 years ago three proteins were discovered:
Beclin, LC3b and p62. Being strictly required in order to have autophagosome formation and fusion
with the lysosome, these molecules are considered markers of autophagy.
Experimental models show increased levels of these markers in cancer cachexia; the same happens
in the muscle tissue of cancer patients, even when they do not present body weight loss. This means
that molecular alterations in these patients are already present, therefore they will likely develop
cachexia.
Manipulation of proteolytic systems as treatment

Can we modulate these proteolytic systems to protect the organism against muscle wasting?
A proteasome inhibitor (bortezomib) is currently used in the treatment of multiple myeloma and
other hematological tumors; the commercial name is Velcade.
Since in the muscle of tumor-bearing animals we have an increase in proteasome activity and in the
expression of muscle specific ubiquitin ligase, we tried treating the animals with this proteasome
inhibitor in order to see if we can revert the atrophic condition of the muscle of animals with cancer
cachexia. Despite these molecules being active (we can measure the activity of the proteasome
system in research settings) no change is observed in terms of muscle wasting.
Conversely, results of inhibition of autophagy are less clear: some papers show a clear improvement
of muscle weight and muscle cross sectional area (you perform a transversal section of the muscle
and measure the fiber area which is reduced in case of muscle damage) when treated with AICAR
(an activator of AMPK) and tropomycin (an inhibitor of mTOR).
Consequently, inhibiting autophagy can be a tool
useful to protect against muscle wasting in cancer.
However, another study shows that the prolonged use of
these inhibitors is toxic. Moreover, inhibition of
autophagy when the animal is very late in the
progression of the condition, results in anticipation of the
death of the animal: normally animals are sacrificed after
15 days from tumor implantation (because of ethical
concern), while if they are treated with autophagy
inhibitors during the late stage of progression of
cachexia, they die a few days earlier. This happens
because we are inhibiting autophagy systemically, the
drug is given to the whole animal, not only in the muscle;
in addition, we are blocking a system that is providing substrates. The amino acids derived from the
degradation of muscle proteins can in fact be used for a sort of maintenance of the strictly vital
functions. Therefore, the goal should be to restore the basal autophagic degradation, while
excessive autophagy or impaired autophagy must be avoided, as they are very dangerous for
the organism and can negatively affect the survival chance. Inhibition of proteasome is not really
affecting the body homeostasis, the inhibition of autophagy is more critical: transient beneficial effect,
but if the inhibition is persistent, it results in death or is at least dangerous. In conclusion the
regulation of autophagy is critical regarding the outcome of the disease, while, in contrast, the
inhibition of proteasome is not really effective. In this view, the target of the research is to restore
the physiological balance of autophagy.
Furthermore, a very efficient way to regulate of autophagy regards the possibility of treatments that
act “upstream” of the proteolytic system itself. For example, animals treated with β-adrenergic
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agonists (used in the past as doping because they cause muscle hypertrophy) can result in reduction
of expression of markers of autophagy.
Protein synthesis
While protein degradation is always hyperactivated, protein synthesis can be differently regulated:
reduced, unchanged and sometimes it has even been shown to be above the physiological levels in
cancer patients. Of course, this also depends on the tools used to evaluate protein synthesis and on
the anatomical district evaluated e.g. just the muscle, the whole body and so on.
On the left there are some experimental systems that show
decreased muscle protein synthesis. However, these reductions are
often associated with molecular changes subjected to the
maintenance of protein synthesis. In simple words this means that
even if the markers of protein synthesis are decreased, molecular
markers of the pathways involved in protein synthesis are unchanged
or even increased: phosphorylated Akt or P7 are above the
physiological levels, hence the tissue is “set” towards anabolism, but
tissues are not able to increase the amounts of proteins because the
building blocks are missing, the tissue lacks the amino acids
necessary to synthesize peptides. The system is set toward protein
synthesis, but we do not have the amino acid necessary to perform it.
As a result, we can conclude that:
- We cannot use these markers as real indicators of what is
happening to protein synthesis inside the muscle;
- Despite the whole organism being set towards a metabolic
drive, the muscle is trying to compensate, it is setting its protein
synthetic machinery towards activation unsuccessfully, but still it is
trying to react.
We can observe the same phenomenon in human cancer patients. Ratio between phosphorylated
and unphosphorylated Akt in cancer patients is increased and the level of phosphorylated Akt is
high, even if it is not significant. The same happens to another marker of protein anabolism which is
this phosphorylated GSK3β. In patients with gastric cancer the level of this marker is increased. So,
what we see in the animal can be seen also in cancer patients.
This means that we still have the possibility to exploit the anabolic potential in cancer patients
in order to counteract the catabolic setting and avoid muscle wasting as much as possible;
furthermore, this anabolic drive can be used as element to correctly stage patients in pre-
cachexia, cachexia or refractory cachexia. Finding out the right way to interfere with the metabolic
alterations of the catabolic drive can result also in the exploitation of the anabolic potential trying to
help the muscle to counteract against catabolism.
Treatment of cachexia
What can we do to help patients during the journey from the onset to the progression of cachexia?
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From what is discussed you can understand that a specific system for treating cachexia is not
available - using just nutritional advice or just inhibitors of the proteasome or block autophagy or just
inducing myogenesis will not be enough. Fourth take-home message: since cachexia is a
multifactorial and a multi-organ syndrome, the treatment of cachexia must be a multimodal
treatment, a combined treatment that includes nutritional support, anti-inflammatory
treatment, anti-catabolic strategies and pro-anabolic strategies. The success of this approach
strictly depends on the moment in which it is applied to patients.
Another very important point is that the care of cancer

patient should always include the combination of
expertise among different clinicians: every single
patient should be really cared for by many specialists
(surgeon, nutritionist, oncologist, physiologist) in
order to set up a personalized approach and
treatment.
CANCER CACHEXIA
This is just to provide some biochemical basis related
to cachexia.
Cori Cycle
In contracting skeletal muscle, the formation and
release of lactate in the circulation lets the muscle generate ATP in
the absence of oxygen and shifts the burden of metabolizing lactate
from muscle to other organs. Excess lactate is taken up by the liver
and is converted first into pyruvate and then into glucose by the
gluconeogenic pathway. Contracting skeletal muscle supplies
lactate to the liver, which uses it to synthesize and release glucose.
These reactions constitute the Cori Cycle.
Cancer-associated cachexia is caused by the growing

Figure 1
tumor which requires a lot of energy (mainly glucose) and
Figure 2
building blocks.
Lipolysis (catabolism of lipids) is induced by the tumor. Adipose tissue mass is reduced owing to
the activation of lipases, which participate in the breakdown of triacylglycerols (TAGs), which
produce both non-essential fatty acids (NEFAs) and Glycerol. NEFAs are used as substrates by the
tumor. Glycerol is used as a substrate by gluconeogenesis in the liver. Something similar occurs in
anorexia, where there is a decrease in adipose tissue.
The tumor takes up glucose from the bloodstream. Glucose is transformed into pyruvate
(glycolysis) and then mainly to lactate (Lactic Fermentation, Aerobic Glycolysis or Warburg
Effect). Lactate is then released into the circulation and taken up by the liver that converts it back to
glucose (Gluconeogenesis). Glucose is then released in the bloodstream and taken up by the
forming tumor. A sort of “Cori Cycle” is thus established between the tumor (instead of skeletal
muscles) and the liver.
The glycolysis metabolism of the tumor is very inefficient, a lot of glucose is consumed and a little
energy (ATP) is produced but allows an efficient tumor growth providing the right amount of energy
and building blocks and a favorable acidic microenvironment.
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Due to the state of inflammation that the tumor establishes, the liver is also induced to produce
positive acute-phase proteins (e.g. C-reactive protein, mannose-binding protein, complement
factors), which serve different physiological functions within the immune system.
To synthesize these proteins amino acids are required, which come from muscle proteins derived
from proteasomal degradation. Therefore, muscle loss occurs.
Over-activation of the liver (gluconeogenesis and acute-phase protein production) leads to
hepatomegaly (enlarged liver).
In cancer-associated cachexia:
- Decrease in adipose mass;
- Decrease in skeletal muscle mass.
The tumor also produces some factors to increase substrate availability that leads to cachexia:
- Lipid Mobilizing Factors (LMF) leading to lipolysis;
- Proteolysis-Inducing Factor (PIF) leading to proteolysis.
Figure 3
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Here you have the transcript of what the professor said exactly while before there was Nicolò’s more
comprehensible version.
A process similar to the Cori cycle is used, instead of muscles, by tumours in cachexia.
A tumour needs glucose and it uses different sites from which it can take it from the adipose tissue:
in adipose tissue there is an increase in lipolysis71 (i.e. the catabolism of adipose tissue) to give free
fatty acids to generate glucose through the glucogenesis, therefore making this glucose free to be
used by tumour to enter into the glycolytic cycle, so tumour keeps from the adipose tissue through
lipolysis (so the catabolism), but it is not low lipogenesis, but it is lipolysis in this case and uses
glucose by the glycolytic cycle this glucose is transformed in pyruvate and lactate. Therefore, the
volume of the adipose tissue decreases (similarly to what happens in anorexia, in which there is a
decrease in the adipose tissue).
The molecules of lactate go in the circulation and reach the liver, which has to take it up and convert
it to glucose. The formation of glucose causes a loss of energy in the form of ATP for our body.
Therefore, when there is a tumour there is a loss of ATP during the glycolytic metabolism of the
tumour. It is a loss for the body because the energy is not used by the body itself by for the tumour.
Then, there is another loss for the body because the liver uses ATP to form again glucose, which
can in turn go in the circulation and reach again the tumour, so we have the loss of ATP. In the
meantime, the liver overproduces proteins called acute phase proteins, such as the c-reactive
protein, because there is a tumour and so the liver has to increase the inflammatory process. This
causes hepatomegaly since the liver has to work a lot both for the formation of the proteins of the
acute phase and for the production of new glucose from the lactate that arrives through the circulation
coming from the tumour. Therefore, as a result, there is a decrease in adipose tissue and an
increase in the volume of the liver (hepatomegaly).
However, the liver needs amino acids to produce inflammatory proteins. These amino acids come
from the muscles, since they are the main source of proteins. Therefore, there is a proteasomal
degradation of proteins that frees amino acids, which are then taken up by the liver and used to
synthesize acute phase inflammatory proteins. This cycle is very similar to the Cori cycle, but instead
of the muscle it is the tumour that uses the glucose.
This is the reason why in cancer cachexia there is not only a decrease of the adipose mass, but also
a decrease of the muscle mass.
It is therefore clear that the three metabolisms are all involved in cachexia.
There are different types of cytokines and inflammatory molecules, produced both by the tumour and
by the host. Some of them are very particular, like LMF (lipid mobilising factor) and PIF (protein
hydrolysis factor72), produced by the tumour, which releases one factor to induce lipolysis and the
other to induce proteolysis in the muscles. Therefore, in addition to the others (in particular the main
one is TNF-α), there are also some molecules that are synthesized by the tumour itself in order to
increase the lipolysis and the proteolysis.
71 She says lipogenesis, but later on she kinds of corrects herself saying it’s “lipolysis”
72
Further research hinted that the real name is “proteolysis-inducing factor”
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ETIOLOGY OF CANCER
There can be many types of causes of cancer: in a minority of cases cancers are due to inherited
genetic mutations, then environmental, lifestyle, or behavioral exposures play an important role
both in the promotion and progression of cancer development and, finally, also viruses play an
important role; as a matter of fact, oncogenes were first discovered in viruses.
Chemical carcinogenesis
It consists of cancers caused by chemical compounds; carcinogenesis is the history of cancer,
starting from the first mutation to the discovery of the mass in the body. During carcinogenesis,
chemicals play a very important role: chemical carcinogenesis is very important as study module to
understand tumor development.
Carcinogenesis can be divided in three phases:

- Initiation, that is the first oncogenic mutation and development of a potential neoplastic cell;
- Promotion, that is the selection of specific clones of mutated cells able to proliferate against
the surrounding defenses of the tissues;
- Progression, that is long or short depending on type of tumor, it is related to the acquisition
of further additional mutations that render the tumor more aggressive and eventually
metastatic.
These three steps can be assembled in two periods; initiation and promotion together constitute the
latent period of growth, during which the tumor cannot be macroscopically detected. During the
progression period the tumor can be detected by instrumentation, clinical evidences, symptoms,
etc.
These stages were studies for the first time in chemical carcinogenesis.
Initiation is the period in which a factor/molecule is able to induce a fixed mutation related to
proliferation: these molecules are thus considered initiating factors. Other chemicals are instead
involved in promotion, thus are able to favor proliferation of cells (i.e. initiated cells) which already
acquired a first oncogenic mutation (for example some factors that induce necrosis, especially in
parenchymal or epithelial tissue, favor tumor proliferation, as the tumor cells might develop
resistance and have a selective advantage with respect to the other cells of a tissue). These
compounds are called promoting factors. Some promoting factors are also involved in progression.
However, the roles of chemical carcinogens are mainly related to initiation and promotion.
Chemical carcinogens can be classified in:

- Direct-acting carcinogens, which can directly damage DNA and cause mutations (no
metabolic activation is required);
- Indirect-acting carcinogens or procarcinogens, which can also be ineffective when in a
particular chemical structure, they need to be metabolized by the body to form a product which
is a carcinogen able to act as a direct inducer of the mutations.
Remember: all xenobiotics (substances foreign to the body) that enter our body need to be
processed through the metabolizing systems by phase I and phase II enzymes:
- Phase I enzymes are metabolizing enzymes;
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- Phase II enzymes are detoxifying enzymes.
Figure 4
Indirect chemical carcinogens are metabolized by phase I enzymes (metabolizing enzymes), which
are mainly monooxygenases and hydroxylases. The products of these reactions are diol-
derivatives, epoxides and diol-epoxides (active carcinogens with important mutating/chelating
effects on DNA), these compounds are active carcinogens which bind and damage DNA causing
mutations. Active carcinogens can then be converted by the protective reactions of phase II
enzymes (detoxifying enzymes), which are mainly transferases, in particular
glucorunyltransferases, acetyltransferases, glutathione transferases, and epoxide hydroxylases.
These reactions convert active carcinogens into inactive derivatives: if, for instance, epoxide is
completely converted by the hydroxylase in an inactive derivative, there won’t be a mutation in the
DNA.
The ability of a chemical to induce the development of an initiated cell is dependent on the ratio
between phase I enzymes and phase II enzymes. If the ratio between phase I enzymes and phase
II enzymes is larger than 1, the phase II enzymes are unable to inactivate all the active chemicals
and therefore some active chemical will be able to alter DNA and induce the so-called DNA adducts,
therefore the equilibrium will be shifted toward the production of active carcinogens and the formation
of DNA adducts.
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There are many defenses against DNA

adducts: even if they are present, if there are
also enough possibilities to repair them with
DNA repairing enzymes, the chemical is
inactive. If the ratio between DNA adducts and
damage repair is more than 1 because, for
instance, there is a low quantity of repairing
enzymes, however, there will be genome
damage because there are no other
possibilities to repair the active chemicals.
The Ames Test is widely employed, for

Figure 5
instance in occupational medicine, to assess
whether if a chemical carcinogen can be
mutagenic or not. This test involves a plate in which there are some bacteria, more specifically
particular strains of Salmonella, unable to grow in the absence of histidine. In the presence of
mutations of the DNA, however, these strains can grow even in the absence of histidine.
To test if an active chemical is carcinogenic, so if it can induce mutations, the compound is put into
a plate containing Salmonella clones unable to grow in the absence of histidine, it is incubated and
after some time, if this chemical is mutagenic, into the plate different clones of Salmonella will have
grown, even in the absence of histidine. This means that this is an active carcinogen.
If it is indirect, it is necessary to add to the plate also an extract of liver; in fact, the liver is the tissue
in which all the of xenobiotics are metabolized by the drug metabolizing system, therefore the
homogenate put into the plate there are different type of enzymes, in particular the phase I enzymes.
If the tested chemical has an indirect action, it is converted by the enzymes of the liver homogenate
and again Salmonella can grow also in the absence of histidine.
In the case of non-mutagenic chemical, of course, no bacterial growth can be seen, so it is possible
to affirm that the chemical isn’t a carcinogen.
Figure 6
Chemical carcinogen can be classified according to their effects:
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- Genotoxic Chemicals: cause direct DNA damage inducing chemical DNA adducts which are
resistant to repair mechanisms. Genotoxic chemicals are initiating factors;
- Mitogens: molecules that bind to cell receptors and stimulate cell division without directly
affecting DNA. For example, in experimental models of skin cancerogenesis, mitogens that
bind Protein Kinase C inducing epithelial hyperplasia, are used. Mitogens can act as
promoting factors;
- Cytotoxic Chemicals: cause tissue damage and subsequent hyperplasia (proliferation) with
alternating cycles of regeneration and damage. These chemicals promote a state of stress and
thus inflammation that favours tumor development. Cytotoxic Chemicals are promoting
factors.73
Initiating factors
Initiating Factors, chemicals that are able to initiate the neoplasia process, cause first genetic
alterations necessary for carcinogenesis (cause indeed the first mutations) but not sufficient by
themselves to provoke alterations of proliferation (and thus neoplasia) because they need other
types of mutations. The initiated cells (exposed to initiating factors) are capable of developing a
neoplasia if subsequently exposed to other agents (promoting factors). Most of the initiating factors
are mutagens.
Initiating Factors are mainly organic compounds, such as:

- Polycyclic Aromatic Hydrocarbons;
- Alanine Derivatives (Aromatic Amines);
- Azocompounds;
- Alkylating Substances.
However, initiating factors can also be inorganic compounds, such as Arsenic:
- Arsenic (As) and inorganic arsenic compounds are found in arsenical insecticides, subjects
at risk of exposure are producers and users (e.g. winemakers), and arsenic minerals, in this
case miners are at risk subjects;
- Asbestos, depending on the diameter of the fibers. It can induce inflammation; therefore it can
be considered both an initiating and a promoting factor (chronic inflammation, necrosis, tissue
repair are all somehow involved in tumor proliferation).
These compounds are professional carcinogens and they can cause carcinomas after a long period
of latency (years).
Promoting factors
Promoting molecules are able to cause an increase in cell proliferation and are the drivers of the
promotion phase. Some promoting factors are able to induce necrosis, lysis of cells, for instance
detergents like dodecylbenzene and tweens, that are normally used in the household; in fact, there
is a limit in the quantity allowed to be in common detergents.
Examples of Promoting Factors:

- Croton Oil (Short-Chain Phorbol Esters e.g. Phorbol Myristate Acetate PMA, which activate
the signal transduction enzyme kinase C PKC);
- Mineral oils undergone thermal cracking;
- Dodecyl Benzene, key ingredient of common laundry detergents;
73
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- Tweens 40, 60, 80, non-ionic detergents commonly used, their use should be limited as they
have necrotic/cytolytic actions (the same is true for Dodecyl Benzene);
- Silica;
- Asbestos;
- Cobalt;
- Partial Hepatectomy;
-Hyperplasia74.
In experiment carcinogenesis two compounds are mainly Figure 7
used:
- Diethylnitrosamine (DEN), an initiating factor used to
induce hepatocellular carcinoma75;
- 2-acetylaminoflurorene (AAF), promoting factor.
These two compounds are used experimentally in
laboratories to induce hepatocellular carcinoma and study
the different steps of hepatocarcinogenesis, from initiated
hepatocytes to the formation foci (expansion phase of
initiated/resistant hepatocytes), then to nodules (evident
expansion of initiated hepatocytes) and eventually tumors.
Organic Carcinogens:
- Polycyclic Aromatic Hydrocarbons;
- Aniline Derivatives (Aromatic Amines), found in dyes and paints;
- Azocompounds, found in food dyes;
- Direct Alkylating Substances, for example the mustard gas (Iprit);
- Indirect Alkylating Substances, introduced with the diet or produced by bacterial flora, for
example diethyl- or dimethyl-nitrosamine.76
Polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons are procarcinogens, so they are indirect inducers of tumors.
They are in the pollution, in oil (diesel and gasoline) and in the smoke of both cigarettes and pipes.
Their action was firstly discovered in the first decades of the 1900s in England, where they were
thought to cause what was originally called the “chimney sweeper’s cancer” or “soot wart”,
discovered in workers that used carbon fossil; it was a squamous cell carcinoma of the scrotum. The
workers were not exactly clean, and this type of aromatic hydrocarbons have a topic action, that is
where they attach they act, so they remained in the lines of the scrotum inducing there the tumor.
Since they are also present in the smoke of the cigarettes and pipes, they are also responsible of
the so-called “pipe smoker cancer”77; it is a spinocellular carcinoma related to the high temperature
present near the lip, tongue and cheek when you smoke a pipe. This type of chemicals, in fact, are
formed by the high temperatures.
74
75
The only malignant tumor of the parenchyma of the liver
76
77
Different location compared to the cigarette smoker’s cancer
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Mainly found in:

- Cigarette smoke, 20 smoked cigarettes produced 400 mg of tar, the filter can retain at most 60%
of the tar;
- Air Pollution: the compounds are produced by petrol engines (benzopyrene) and oil or diesel
heating systems;
- In a lesser extent in roasted coffee and meat.
These compounds can be the cause of occupational tumors (tar, asphalt workers) and have a topic
action.
The basic form of these compounds is not a mutant,

therefore it is not able to induce cancer directly. The basic
forms are anthracene, phenanthrene and pyrene and
they have different sites in which they have a high activity
of electrons, one of the regions with high electronic activity
being the K region (figure n.8). When an additional
benzenic ring is added to the basic chemical to form
benzoanthracene, benzophenantrene and
Figure 8
benzopyrene (figure n.9), they become
carcinogenic; however, in order to be
effectively carcinogenic, they must first be
transformed by the drug metabolizing system,
in particular by aryl hydrocarbon oxygenase
and aryl hydrocarbon hydroxylases, P450-
dependent enzymes, in epoxide and diol-
epoxide (figure n.10). Figure 9
They are able to induce spinocellular carcinomas and lung cancer, that
can be spinocellular or microcitomas or small cell lung cancer, which is
the cigarette cancer. In the cigarettes we have millions of these different Figure 10
types of chemicals that induce cancer.
Aromatic amines - aniline derivatives

Organic compounds consisting of aromatic rings attached
to an amine. It is a broad class of compounds that
encompasses aniline derivatives (-NH2 as substituent
on aromatic ring). Aniline, the basic structure, is not
cancerogenic, but its derivatives, like β-naphthylamine
and 4-aminodiphenylbenzidine, are.
These compounds are used for the synthesis of dyes

(paints, not food dyes) and explosives, but also pesticides Figure 11
and pharmaceuticals.
They have been identified as the etiological agent of professional bladder tumors, more specifically
squamous tumors of the transitional epithelium. They are considered professional tumors
because they are mainly found in people working with this type of chemicals.
Mechanism of action
Aromatic amines are indirect-acing carcinogens (procarcinogens). For instance, β-Naphthylamine
is converted, in the liver, in the active carcinogenic compound, hydroxynaphthylamine, which has
266
a hydroxyl group (a very active nucleophile). This active compound is then converted by phase II
enzymes, in particular glucuronyltransferase or sulphatases, in an inactive compound in which
hydroxynaphthylamine is bound to a glucuronyl group or a sulphate group; therefore, these enzymes
induce an esterification, since esters are inactive compound. It is then filtered by the kidney, it
reaches the bladder and there it concentrates. In the bladder, however, there are bacteria which
have different types of enzymes of the esterase family, are able to cut the linkage between the
glucuronic acid or sulphuronic78 acid and the active molecule. Therefore, in the bladder this
compound is again free and active.
The human population can be divided in two population according to their ability to activate
acetyltransferases, important not only for the tumor, but also, for instance, to metabolize ethanol; the
two groups are:
- Fast acetylators (or strong acetylators): subjects capable of quick hepatic detoxification of
various types of chemicals (e.g. also the acetyl part of ethanol) because they can induce the
formation of the ester from the active aniline derivative through the link with an acetyl group
(acetylation). Bacteria don’t have acetylases, so it is impossible for them to cut the ester and
therefore the compound continues to be inactive also in the bladder. As a consequence, this
population is resistant to bladder tumor.
- Slow acetylators.
Other two derivatives of aromatic amines are the green magenta or moca, which are green or
yellow, and auramine, which is a fluorescent stain that we also use for instance in labs to stain.
Another derivative is acetylaminofluorene, which is a promoting factor that induces hepatocellular
carcinoma.
Other carcinogenic aromatic amines derivatives are:

- Diaminodiphenylmethane derivatives (figure n.12):
o 3,3’-dimethyl-diaminodiphenylmethane, or green
magenta;
o 3,3’-dichlorvos-diaminophenylmethane, or moca
(yellow coloration);
o Auramine, a common fluorescent stain used in
laboratories;
- Fluorene derivatives: Figure 12
o 2-acetaminofluorene (AAF, figure n.13),

originally used as an insecticide, now it is an
experimental carcinogen (promoting factor) used
in laboratories to induce bladder and liver tumors Figure 13
(e.g. hepatocellular carcinoma).
Azocompounds
This family of compounds includes the azodyes employed in food industry, since they used to color
the foods.
They are direct acting carcinogens. These compounds bear the basic functional group R-N=N-R’, in
which the R groups are usually aryl groups (aromatic rings), with many methyl groups. This chemical
78
She may mean “sulphuric” acid
267
structure renders the molecules highly active from the electronic point of view, making them able to
have an indirect nucleophilic action.
An important example is dimethylalminoazobenzene (DAB, figure

n.14), also called methyl yellow or “butter yellow”, because it was used
in the ‘70s as food additive in margarine (naturally white, to make it look
like butter), before its toxicity was recognized.
Different types of azodyes are called with a number preceded by an E Figure 14
(figure n.15). Some of them have been forbidden, such as yellow
E105, orange E111 and red E125.
Alkylating agents
These compounds are able to directly transfer alkyl groups
(alkylation) at specific positions of DNA:
- position 7 of Guanine;
- position 3 of Adenine;
- position 1 of Cytosine. Figure 15
Alkylation of polypeptide or nucleotide chains lead to the formation of
intermolecular bridges, which make chromosomes stick to each
other. Interchromosomal cross-linkages are the cause of the so-called “stickiness” of chromosomes.
Alkylating agents are also used as cancer chemotherapeutic drugs, the most common being
cyclophosphamide, because of their alkylating action.
N-Nitroso compounds are alkylating agents, and can be divided in two groups, nitrosamines and
nitrosamides.
Nitrosamines, unlike other alkylating agents, are indirect-acting carcinogens, activated by the
microsomal79 P450 system (phase I enzymes). One of them is DEN, which is used for the
experimental induction of carcinogenesis. Nitrosamines are found in industries, such as of the tire,
of the tanneries, in the gas of diesel, in the smoke of tobacco, in some salted foods like fish.
They are products of nitrates and nitrites that are generally used as preservers of foods (e.g. in the
“salame” and “insaccati”).
The activation reaction consists of a N-hydroxylation and a de-alkylation that yield the active
Diazomethane, a strong methylation agent.
Examples of dialkyl nitrosamines are:
- Dimethylnitrosamine (DMN);
- Diethylnitrosamine (DEN), used as experimental inducer of carcinogenesis.80
Nitrosamides are indirect-acting carcinogens. Some undergo non-enzymatic spontaneous

transformation to diazoalkanes, which represent true direct carcinogenesis.
79
Microsomes are vesicles of endoplasmic reticulum and attached ribosomes obtained by the centrifugation of
homogenized cells. When we talk about microsomal enzymes, we are talking about enzymes located in the endoplasmic
reticulum (the liver has the majority of the drug metabolizing system, but the DMS can also be found in other tissues)
80
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An example is N-nitroso-N-methylurea (NMU), which can lead to AT:GC transition. Used to induce
glioblastoma in experimental models.81
Sources of N-nitroso compounds:

- Air from tire industries;
- Air of tanneries;
- Exhaust gas from Diesel engines;
- Tobacco smoke;
- Smoked and salted foods (fish and meat);
- Fish and fish flour;
- And from the endogenous metabolism of Nitrates (used as food preservatives and fertilizers)
and Nitrites (used as food preservatives). Through nitrosation reaction they are transformed
in nitrosamines and nitrosamides.
Nitrates and nitrites and introduced through the diet. In the oral cavity, nitrates are reduced to
nitrites by bacterial reductase of salivary microflora. Nitrites are then converted intro nitrous acid
in a reaction accelerated by microflora and basic environment, or in conditions of high pH, for
example in the case of hypochlorhydria (low concentration of HCl in the stomach) due to gastritis.
Nitrous acid can be converted to nitrogen trioxide and water, in a reaction which depends on the
pH of the environment: the balance is toward the nitrous acid in case of high pH (basic environment,
for example in the case of stomach hypochlorhydria).
In the gastric cavity, amines and amides derived from the ingested food can react with the large
amount of nitrous acid present to yield nitrosamines and nitrosamides, carcinogenic compounds.
This reaction is called nitrosation.
Figure 16
Diet, in addition to the ones mentioned above, is another very important factor Figure 17
in carcinogenesis and has a huge impact on tumor progression.
Spontaneous alkylating agents are direct-acting carcinogens. They are:

- (Nitrogen) mustards gas, which are cytotoxic agents, also used as
chemotherapeutics, derived from the mustard gas. Mustard gas
81
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(mechlorethamine), which gets its name from its strong smell, was used as a blistering agent in
warfare;
- Epoxides, cyclic ethers with a three-atom ring, for example ethylene oxide and the compounds
that derive from the activation of polycyclic aromatic hydrocarbons. Can be found in epoxy resin
industries.
Other organic compounds with carcinogenic action

These substances are generally involved in the promotion of cancers.
Plastic substances, like Bakelite, cellophane, nylon, teflon, polystyrene, silicone, polyesters,
polyvinylchloride and vinyl chloride as starting monomer for the production of plastic substances.
Halogenated Hydrocarbons
Dichloro-diphenyl-trichloroethane (DDT), once used as an insecticide, introduced with diet, induces
experimental hepatomas in mice and rats.
Carbon Tetrachloride (CCl4) is a risk factor for its low decomposition; it was used in the past in
laundries, but not anymore, in a lot of perfumes with a very nice smell. Now it is used as experimental
carcinogen because of its high toxicity.
ETHANOL TOXICITY
Today we start talking about ethanol toxicity while next time we’ll start talking about hepatic steatosis,
specifically ethanol hepatic steatosis or ethanol fatty liver and also non-alcoholic fatty liver. Then we
will talk about cirrhosis, so we will see what happens in the liver for it to reach the histology of a
cirrhotic liver.
Now we talk about ethanol toxicity because one of the main causes of fatty liver, steatohepatitis and
cirrhosis is alcohol abuse: drinkers cause2 several damages to their organism when abusing alcohol,
so different organs can be damaged by alcohol abuse, among them the liver, which will be discussed
more in depth next week.
What is alcohol?
The term “alcohol” is used to refer to ethyl alcohol or ethanol which, as you should know, is a
monovalent, primary, saturated, liquid and colorless alcohol. Alcohol is formed as a result of
fermentation of some simple sugars, but it is also derived from the distillation of fermented must.
Ethanol is the main psychoactive component of alcoholic beverages, in fact it can directly affect the
brain: its main effects are on the CNS - not only on the brain of course but also on other organs - but
it alters the cognitive functions, so the capacity to be concentrated (focused), in control, it can affect
the behavior and so on. Another important point is that it is better not to take any kind of medicaments
or drugs like cocaine together with alcohol because this combination, alcohol plus
medicaments/drugs, has a major impact on the psycho-physical capacity of any individual.
How much alcohol is introduced when you consume alcoholic drinks?
How can we calculate how many grams of alcohol we introduce in our system when we drink wine,
beer or liquor? There is a specific calculation: we have to multiply the ml of alcoholic beverage
that we introduced, times the alcohol content, so the gradation (in Italian, gradazione alcolica) of
the beverage, times 0.8 that is the density of alcohol, all divided by 100.
For example, if we drink 500 ml of wine whose medium gradation is around 12%, how many grams
of alcohol are ingested? We do (500x12x0.8):100, so in this case we have introduced about 48
grams of alcohol.
If I drink a glass of wine with a medium gradation of 12% or 300 ml of beer with a gradation of 4.5%
or a small glass of liquor (40 ml) whose gradation is higher, usually around 40%, sometimes 50%,
the amount of alcohol that I have introduced in this case is around 12 grams.
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To metabolize one glass of alcohol our body needs at least two or three hours, to metabolize only
one glass of alcohol e.g. wine! The capacity or the speed of the metabolization of alcohol depends
on many variables that we will see later on.
World Health Organization (WHO) says that it is better not to drink too much, so it is better to
moderate the quantity of alcohol that we introduce in our system every day. They suggest that our
daily consumption is not higher than:
- For a man, 40-50 grams every day which means three glasses of wine with a medium
gradation of 12% or three cans of beer (4%) or one small glass and a half of liquor (40-50%);
- Women should drink less than men because they are more susceptible to alcohol’s effect
(we will see why), they can get drunk more easily than man. So, it is better if they don’t drink
more than 30-40 grams of alcohol which means two glasses of wine (12%), two cans of beer
(4%) and only one small glass of liquor (40-50%).
What happens if one goes out for a nice dinner with friends and drinks too much? What can happen
if the police man stops him? If a police man stops him and sees that he is drunk, there is a breath
alcohol test which he will make him do: he has to breath into a balloon, so that they can test the
concentration of alcohol in his blood. The limit is 0.5 for each liter of blood. If after the test, he
exceeds this limit, his driving license will be taken by the police man, so he won’t be able to drive.
Not to impair your “driving perception” you cannot have a higher (than 0.5) concentration of alcohol
in your blood.
Bianca’s question: before you said that there is a moderate and secure quantity of alcohol that people
can intake daily without doing any damage. What if this consumption is quantitatively the same but
is not on a daily basis because the individual never drinks and rather has just a one-time excess? Is
there a difference?
Prof.’s answer: If one doesn’t usually drink and goes out once with a friend and ends up drinking a
bottle of wine, of course what counts is the alcohol introduced in his system in THAT moment, so if
one drinks one bottle, the alcohol concentration in his blood will be higher. One can even drink a
bottle of wine throughout the entire day and his alcohol concentration will be less compared to
drinking one bottle in half an hour. So, it is much better to drink small doses of alcohol during the
day or the dinner – if one is in the restaurant for five hours – because his body will be able to
metabolize better the small doses. On the other hand, if one drinks one bottle of wine in short time,
the concentration of alcohol in his blood will rise immediately.
This limit, 0.5, is a very low one. For example, you see that to reach 0.5 gm/l of blood is sufficient to
drink two glasses and a half of wine. If you are outside for a nice dinner with your friends, I am sure
that you will drink more than two glasses and a half of wine – this of course if you like it; but this is
just to tell you that it is very easy to reach this limit.
The limit is very low, but some studies have shown that a small percentage of alcohol in our blood
is enough to already have some alterations at the level of our CNS, among which: less reflexes,
reduced visual capacity (visual field) and less ability to perceive stimuli so we cannot react as fast
as we do when we are not drunk. When one drinks a bottle of wine (usually 750 ml), he introduces
65 grams of alcohol. The table indicates how many grams of
ethanol/alcohol one can ingest during the day compared to his
body weight. A slim person is more susceptible to alcohol e.g.
if one’s body weight is 50 kg, he shouldn’t introduce more than
40 grams of alcohol. If one’s bigger and fatter (body weight is
90 kg), he is allowed to drink more because the alcohol is
equally distributed in his organism, in the tissues and
especially in adipose tissue where it accumulates. So, if one
is fat/bigger, he can drink more compared to a thin person.
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You see that the limit in Italy (probably in USA is less) is 0.5 gm/l of blood, which means that daily
consumption shouldn’t be more than 45 grams of alcohol. However, 45 gm of alcohol is NOT
recommended: it is not really that the WHO suggests to introduce 45 gm of alcohol daily; this is just
to say that a small/thin person cannot drink more than a big person because the effect that alcohol
can have on an individual can also change based on several variables.
Do not get confused with this table because there is written “daily consumption should not be more
than 45 grams of alcohol” while here they say something else. Why? If one goes out for a dinner and
doesn’t have to drive, he can drink one bottle of wine, what he cannot do is to drink a bottle of wine
every day! However, occasionally one can do it, why not? The issue is that one cannot drive
afterwards, and that his body might be badly affected by the drinking: we are all different, so our
susceptibility to alcohol’s effects can be different which means that if one is fat and big, he can stand
more alcohol than a thin or small person. If I (professor Leonarduzzi who is thin) drink one bottle, I
will not feel so well compared to you because you are taller/bigger than me and because you are a
man while I am a woman, so I am more susceptible to alcohol.
This, about the body weight, is a theory that suggests that if you are thin, you shouldn’t drink too
much, not more than those 40 grams, meaning 400 ml of wine that are equal to three-four glasses,
no more, even though it is not a big amount! For example, if you go to the Oktoberfest in Germany -
or nowadays you can find these parties with beer also in Italy - I am sure that you will drink more
than one pint of beer, one liter of beer. It means that if you are a big boy or woman, you can drink a
bit more than a small man or woman. This is the meaning of this table.
Going back to the quantity of alcohol that one can introduce during the day: a daily consumption of
more than 45 grams is not recommended, one can do it once a week or a month, but not every day
because if one drinks this much every day, he will have chronic damages to his body, especially at
the liver, heart and brain. A consumption of more than 100 grams daily is very dangerous. If the
concentration of alcohol in one’s blood is less than 50 mg for each 100 ml of blood, one can already
notice changed behavior of the subject e.g. some become happy and laugh all the time or can get
sad and start crying (sbronza triste). One can therefore change his behavior already when he
reaches a concentration of alcohol equal to 50 mg/100 ml of blood. If the concentration is instead
higher than 80-100 mg, one can start feeling sleepy, have less concentration and slower reactions.
If it is higher than 300 mg/100 ml of blood, one can enter into a coma. For higher concentrations
(>400 mg/100 ml of blood), one can die frequently of respiratory failure but also because the heart
can stop working.
This figure reports the alcoholic rates. To reach 0.4 gm/l of blood, it is enough to drink two glasses
of wine or one of beer or one small glass of liquor; to reach 0.5 gm/l of blood, it is enough to drink
two and a half glasses of wine or two small beers or two small glasses of liquor. It is very easy to
reach 0.8 gm/l of blood, it is enough to drink three and a half glasses of wine e.g. when I go out to
the restaurant for dinner, it is normal for me to drink three or four glasses
of wine (hai capito la Gabriella!), so I think that is not such a big amount.
Absorption of alcohol is total and extremely rapid
Absorption of alcohol is total: the whole quantity of alcohol will be
absorbed by our body and alcohol is absorbed very rapidly. The
absorption begins immediately after ingestion and can be completed after
15-40 minutes. I can already measure the alcohol in my blood just after
5 minutes from ingestion, that’s why if one drinks a bottle of wine at the
restaurant, his concentration of alcohol in blood will be immediately high.
Suggestion: when you drink, wait a little before going out and start driving
because if you wait a little bit, the alcohol will be metabolized and its
concentration in the blood can decrease. The presence of alcohol in your blood is detectable after 5
minutes from ingestion and reaches its maximum between 30 minutes and two hours, so the
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absorption of alcohol is very rapid. The speed of absorption of alcohol depends on the fact that the
ethyl alcohol spreads easily through the biological membranes: it can cross the biological
membranes of the cells at the level of the digestive tract, where its absorption occurs, and it is rapidly
absorbed at the level of the small intestine and oral cavity as well as stomach, colon and rectum.
Rapid diffusion and distribution of ethanol

Ethanol is also a small molecule and because of its size and solubility in water, it can travel very
fast in the blood and can be distributed at different levels of our body very quickly. It is present in
plasma, blood but also in extracellular fluids. The alcohol’s effect can be seen in the organs with
greater vascularization: immediately we will see its effect at the level of the CNS, then at the level of
liver, kidney, heart and only later at the level of the muscles because here the circulation is lower
compared to other organs i.e. the perfusion is slower; then the ethanol will be distributed at the level
of adipose tissue where it ultimately accumulates, which is why a fat person will be less susceptible
to alcohol’s effect compared to a thin person e.g. if I am fat and I drink alcohol, that alcohol will be
distributed also in my fat tissue where it accumulates but I will have less side effects caused by
alcohol.
Klaris’ question: what if two people have the same body weight but have different body
compositions?
Prof.’s answer: if two people have the same body weight, but one has more muscle and the other
more fats, of course the former will be greater affected by alcohol than the latter.
Factors influencing the metabolism, time and speed of ethanol absorption
The absorption and distribution of alcohol and its effects depend on several factors. We have a very
long list of factors that can influence the metabolism, the speed, the distribution and the speed of
absorption of alcohol:
- Ethanol concentration: e.g. if one drinks 1 liter of wine in five minutes, the concentration of
alcohol will be rapidly higher in his blood, the alcohol will be absorbed and distributed more
quickly in the body compared to drinking a small quantity of wine in a longer period. If one
drinks a bottle in five hours, it is much better than if he drinks it in half an hour, because at
the end the alcohol concentration in one’s blood is less when one drinks small doses
of alcohol in a long-time period;
- Blood flow at the site of absorption/Vascularization of the tissue: how much the tissue
of the organ is vascularized. If the blood flow is higher, the absorption of the alcohol will be
higher as well;
- Ingestion speed: if one drinks one glass altogether, in one time (shot, shot, shot!), the
absorption will be more rapid than drinking the same glass of wine in half an hour;
- Type of drink: beer and wine are absorbed more slowly than spirits/liquors like grappa,
whiskey and cognac.
Tugba’s question: why shouldn’t we mix the fermented drinks and non-fermented ones e.g. drinking
at the same time wine and vodka? Is it because of absorption or is it because of other mechanisms?
Prof.’s answer: it is not forbidden to do it. When you are adding alcohols, what is important is the
total amount, it is not that they are “fighting”, wine does not fight with beer or liquors, wine can be
already irritating your stomach, intestine and mucosa of the digestive tract while liquor is even more
aggressive. So together you have a synergistic action of these alcoholic drinks. There are no
chemical reactions involved.
[Tugba starts talking about the different types of alcoholic fermentations…]
In any case, there is no reaction between fermented and non-fermented drinks, it is better not to mix
them just because you are adding alcohols, therefore having a worse effect. Back to the list.
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- Food: it is much better to drink alcoholic beverages at a full stomach, because the effects at
the level of gastric and intestinal mucosa will be less destructive. It is better to have a full
stomach and avoid drinking when starving because when the stomach is empty, the
absorption of alcohol will be more rapid. Also, food can in some way inhibit or favor alcohol
absorption e.g. carbohydrates like pasta and bread can favor its absorption, while milk and
its derivatives and fats will reduce the speed of alcohol absorption;
- Gastric/Intestinal filling: as said, it is better to have a full stomach because it slows down
the absorption of alcohol;
- Stomach emptying or fasting: accelerates absorption;
- Gastritis: people with gastritis, having inflammatory responses at the level of gastric mucosa,
can easily absorb alcohol;
- Protein deficiency: alcohol is responsible for malnutrition because it can damage the
digestive tract’s mucosa so that vitamins, proteins and other important nutrients cannot be
properly absorbed;
- Sex (gender): women are more susceptible to alcohol damage because it has been found
that they can have less capacity to metabolize alcohol so the amount of alcohol which can
be metabolized at the gastric level is four times less compared to men’s. Men can metabolize
higher quantities of alcohol compared to women, because women have less quantity of the
most important enzyme responsible for alcohol metabolism which is alcohol
dehydrogenase (ADH). Men have double the quantity of ADH compered to women. So,
women get drunk more easily;
- Ethnic and genetic differences: some populations have less capacity to metabolize alcohol,
these individuals can develop more easily steatosis at the level of liver and cirrhosis e.g.
cirrhosis levels are higher for African-American drinkers compared to White-American
drinkers. There is something different at the genetic level, might be that they can produce
less enzymes, less ADH e.g. 50% of Asians have less activity of ADH enzyme;
- Age: old people are more susceptible to alcohol’s effects because their capacity to
metabolize alcohol is not as efficient due to the aging process. On the other hand, young
people and children should not drink because their system to metabolize alcohol is not fully
developed;
- Body weight: thin people are more susceptible than fat people;
- Body temperature: alcohol induces vasodilation; e.g. when you go skiing or go to the
mountains when it is quite cold, a friend might suggest you to drink a whiskey or grappa to
get warm, but it is not the right thing to do! This is because at first, when you drink, you feel
warmer, but alcohol will stimulate skin vasodilation. When we talked about hyperthermia, we
said that because of skin vasodilation you have a greater dissipation of heat so after drinking
you will feel colder than before because you will lose heat from your body;
- Exercise: use of alcohol-derived calories, 1 gram of alcohol is equal to 7 calories, it can
produce heat. So, on one hand it produces heat and on the other it induces skin vasodilation
causing heat dissipation;
- Menstrual cycle: women are also more susceptible because during the menstrual cycle, the
fluctuation of hormones such as estrogens or progesterone, can modulate/affect the activity
of ADH enzyme which is important for alcohol metabolism. Estrogen can increase gut
permeability as well.
Question: how can the body temperature affect alcohol’s absorption?

Answer: The list includes not only factors that can affect absorption and distribution, but also factors
that can be induced by alcohol’s absorption e.g. increased body temperature is an effect of drinking
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alcohol, while body temperature doesn’t really affect alcohol’s absorption. There are also factors that
can affect the metabolism, not only the speed of alcohol absorption.
If we are warm ourselves, we metabolize the alcohol more. We get warmer when drinking, because
we metabolize the alcohol that, as we said, produces 7 calories for each gram of alcohol introduced.
When we talked about hyperthermia, we said that one way to produce heat is the metabolism of
proteins, lipids and also alcohol, so drinkers cannot eat too much not only because their digestive
tract’s mucosa is damaged but also because they do not feel the need to eat since the alcohol is
metabolized to produce calories and therefore heat. It is not a good thing to use alcohol to produce
heat because there is the side effect of skin vasodilation.
Where is it absorbed?
In a small amount it is absorbed at the level of the stomach, mainly at the level of the small intestine
and then a small portion at the level of the large intestine. It is absorbed rapidly, and it can rapidly
spread through the biological membranes so that it can be rapidly distributed to all our body tissues
and fluids e.g. blood.
First of all, it can reach immediately the brain (CNS), then the liver, kidney, muscle and adipose
tissue.
Ethanol
When one drinks ethanol, if he drinks a lot, or drinks a liquor like vodka, whiskey, cognac, he can
burn his oral cavity causing irritation of oral cavity and esophageal mucosa. There is small absorption
at the stomach and mainly at the level of small intestine and, especially in chronic drinkers, it can
damage heart; chronic abuse of alcohol can damage brain, digestive tract, bones, heart and
muscles. Where is it metabolized? Partially, in a small quantity, it is metabolized at the level of the
stomach where alcohol dehydrogenase is present which is similar to the one present in the small
intestine. The product of ethanol metabolism is acetaldehyde which is an aldehyde, a very toxic
compound. However, 80-90% of alcohol is metabolized at the level of the liver in hepatocytes. Here
we have three systems that can metabolize alcohol.
The most important one is made by alcohol dehydrogenase (ADH), located in the cytosol of
hepatocytes. This enzyme transforms ethanol into acetaldehyde. In order to do so, ADH uses NAD+
as a substrate, yielding NADH + hydrogen + acetaldehyde.
This system however is able to metabolize just a certain quantity of ethanol, so if one drinks a lot,
another system can be involved in the metabolization process: the microsomal ethanol oxidizing
system (MEOS). This system works when the concentration of ethanol in blood is very high so that
MEOS can help ADH metabolizing the alcohol; it uses NADPH as a substrate yielding NADP+ +
water but in the end the final compound is always acetaldehyde. MEOS is involved not only when
drinking a lot, but also in chronic ethylism: M in MEOS stands for microsomal so it is located in the
ER and is therefore important in people who chronically drink ethanol/alcohol who constantly use
their ER leading to its hypertrophy. This MEOS pathway is activated because of the hypertrophy of
the ER.
The third system is catalase, located in the peroxisome, which can help in metabolizing alcohol,
even if no more than 5%, by using hydrogen peroxidase as a substrate. In this case we have water
production and always acetaldehyde, which is the final product obtained from alcohol metabolism.
Acetaldehyde is a toxic compound, as all other aldehydes, so it easily binds to lipids, proteins and
DNA forming adducts altering structure and function of the cells. For this reason, it must be removed,
and we must prevent the accumulation of this compound. How? At the level of mitochondria, some
also at the level of the cytosol, there is another enzyme called acetaldehyde dehydrogenase
(ALDH) which uses NAD+ as a substrate: it is able to transform acetaldehyde into acetic acid or
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acetate. Its role is to transform the toxic acetaldehyde, this is possible if one’s drinking a moderate
quantity of wine, while if one drinks a lot (chronic drinker), the organism is not able to eliminate the
entire amount of acetaldehyde that will be formed following alcohol metabolism. In this case,
acetaldehyde will accumulate in the organism causing a series of toxic effects. In normal situations,
it is transformed into acetic acid or acetate.
Acetic acid is then also transformed into acetyl-CoA which enters the cycle of Krebs where it is
transformed into carbon dioxide and water, or acetate can be a substrate for endogenous synthesis
of fatty acids. We will see why drinkers can develop hepatic steatosis i.e. how they can accumulate
lipids, especially triglycerides, in hepatocytes, because they can synthesize fatty acids in
hepatocytes starting from acetic acid that is produced in large quantities because they drink a lot.
How is it eliminated?
When drinking alcohol, the largest quantity will be metabolized but it can also be eliminated in an
unchanged formed, so without being metabolized. There is only a small quantity, 2-10% of the total
alcohol, which can be eliminated unchanged through urine, sweat and breath which is why one of
the tests to see if one can drive or not is a breath test: you have to blow into a balloon so that they
can check the concentration of alcohol in your blood. The limit for withdrawal of driving license is 0.5
mg/l of blood. A small quantity of alcohol can also be eliminated with saliva, tears, bile, gastric juice,
faeces and even breast milk, in fact during pregnancy women shouldn’t drink because alcohol can
have bad effects on the fetus, so the baby can be intoxicated by alcohol with subsequent neurological
disorders development. Alcohol can also reduce the production of oxytocin, the hormone responsible
for the production of milk, so it is better not to drink during pregnancy. Other ways to eliminate
ethanol: small amounts of ethanol can be eliminated after conjugation with glucuronic acid or sulfuric
acid.
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To summarize: we ingest ethanol which can be absorbed partially in stomach, mainly in the small
intestine and for a small portion in the large intestine as well. Total absorption takes 2-6 hours, after
which it is distributed throughout the entire body (tissues and fluids). Then most of it can be
metabolized at the level of the liver, but some of it can be eliminated in an unchanged form (as
ethanol) with breath. We have a peak: the maximum quantity of ethanol can be eliminated with breath
after 20 minutes from ingestion which is why it is better to chat a bit and wait one hour before going
out and drive since you have the peak of alcohol in your breath after 20 minutes. Peak in the plasma
occurs after 20 minutes as well. Ethanol can be eliminated also with urine, but in this case the
maximum peak occurs at two hours after ingestion;
it can be eliminated with sweat as well.
The concentration of alcohol can reach the maximum very quickly, after 20 minutes, at the level of
saliva, blood and breath, but with the urine we have the maximum after more or less two hours, so I
can eliminate alcohol through urine only later.
Both enzymes involved in alcohol metabolism meaning ADH and ALDH are NAD+-dependent
enzymes: they both use NAD+ as a substrate and release NADH. In normal conditions, the proportion
between NAD+ and NADH is 4:1, we have more NAD+ compared to NADH, but because I need to
metabolize a lot of alcohol, at the end I have an opposite situation. In the regular drinker, in an
individual who drinks every day, this ratio turns at 1:4, so there is less NAD+ and much more NADH,
because all NAD+ has been used for alcohol metabolism. The alteration of this ratio can have many
effects and provoke many metabolic changes at the level of glucidic, lipid and protein metabolism.
Effects on glucidic metabolism
Because of this change in the ratio between NAD+ and NADH, we can have activation of the glycogen
demolition process. Glycogen is our storage of energy, we can have glycogen demolition but there
is no glycogen synthesis (no reintegration), so we destroy all our energy storage. What happens if
glycogen is destroyed? Glucose increases in blood leading to hyperglycemia.
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The acid-base balance is also altered, I will have hyperlactacidemia and metabolic acidosis: the
quantity of ketone bodies will increase, so pH of the blood will decrease leading to severe metabolic
acidosis and so on.
Effects on lipid metabolism
There is a great quantity of acetic acid available, so the liver can produce more fatty acids and
triglycerides that can accumulate inside the hepatocytes’ cytosol, being responsible for steatosis.
There are also decreased β-oxidation of fatty acids in the mitochondria and increased cholesterol
synthesis. So, all the effects on lipid metabolism can promote the hepatic steatosis, the so-called
fatty liver: lipids cannot be removed from the liver and will therefore accumulate inside the
hepatocytes.
Effects on protein metabolism
These effects are more evident in the chronic situation. There is a decrease in catabolism of amino
acids, so there is a lower production of urea but at the same time an increased production of
ammonia, we will see, when talking about cirrhosis, what is the problem in having a great amount of
ammonia going around in our blood. There is a decreased production of proteins synthesized by
liver which will be damaged by the chronic intoxication of alcohol.
Damage from ethanol

What can ethanol damage? The abuse of alcohol can damage:
- Mitochondria and microsomal functions;
- Increase the activity of this specific enzyme system, cytochrome P450 leading to increased
production of any kind of toxic metabolites;
- Especially in chronic drinkers, alcohol dehydrogenase is not able to metabolize all the
alcohol, so MEOS must help and be active, but as a consequence of its activation, many
kinds of free radical, including ROS, may be produced. These radicals can react with cell
membranes causing their alterations e.g. alter the fluidity;
- Besides the production of free radicals, the concentration of antioxidants such as glutathione
and vitamin E will decrease.
Damage from acetaldehyde

Acetaldehyde is an aldehyde, so it is a toxic compound. At the beginning, the lesions induced by
acetaldehyde can be reversible, but later on all the damages will be irreversible with chronic alcohol
abuse.
- The production of this toxic metabolite can inhibit the oxidation of fatty acids at the level of
mitochondria promoting their accumulation in hepatocytes, thus contributing to hepatic
steatosis;
- Acetaldehyde can form adducts with proteins and lipids, and especially it can interact with
tubulin that is one of the components of the cytoskeleton causing damage in the cytoskeleton
of hepatocytes.
Digression: The lipids from hepatocytes can be secreted into the blood as lipoproteins. Those formed
in the liver are VLDL (very low-density lipoproteins). When we eat food rich in fat, these lipids will
ultimately arrive in the liver as chylomicrons (type of lipoproteins). Then also the liver can use these
lipids, some can be oxidized at the level of mitochondria to produce ATP, or the liver can form new
endogenous lipids – they are coming from food. Most of these lipids will be secreted as VLDL, then
they will go through the blood and transformed into other kinds of lipoproteins: at the end we will
have LDL, low-density lipoproteins. These lipoproteins will reach our cell tissues and will distribute
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cholesterol. When acetaldehyde interacts with tubulin, these lipoproteins cannot be secreted
because the cytoskeleton is damaged: lipids will accumulate in hepatocytes, which is one of the
reasons why drinkers develop fatty liver, hepatic steatosis.
- Inhibition of protein synthesis, including arrest synthesis and secretion of lipoproteins (from
the name, are formed by proteins and lipids). These people have malnutrition and low levels
of amino acids, so it is difficult for drinkers to form these lipoproteins which is why lipids will
accumulate inside the hepatocytes;
- Production of free radicals.
Acute effects of ethanol

As a consequence of acute intoxication from ethanol, we have mainly effects at the level of the CNS,
where alcohol can act as an inhibitory neurotransmitter i.e. it acts more or less as GABA. Alcohol
at the level of our nervous system can have a depressant effect, it can act as a weak anesthetic.
If I have an acute intoxication from alcohol, I can have some lesions at the level of gastric mucosa
and liver, but with an acute intoxication these are all reversible damages. So, if one drinks alcohol,
for one week he will have these acute effects, but then if he stops drinking, my liver will return normal
and my gastric mucosa will recover.
There are a lot of effects that I can have on my body when drinking wine:
- Alcoholic rate 0.1-0.2: this is a very very low concentration of alcohol in my body, initial
feeling of intoxication, weakness, not concentrated (sometimes it is good not to be so much
concentrated) and loss of inhibitory breaks (freni inibitori) and control e.g. laughing too much
and so on;
- Alcoholic rate 0.5-0.8: alcohol intoxication leading to mood changes, nausea, sleepiness,
emotional excitement that results in less judgment, slowed reflexes and vomiting. This is the
limit to avoid the withdrawal of the driving license in Italy;
- Alcoholic rate 3.1-4.0: loss of understanding capabilities, unconsciousness and
hallucinations, canceled reflexes, coma and possibility of death by suffocation from vomiting
(which started at range 0.5-0.8);
- Alcoholic rate over 4: respiratory problems, suffocation sensation resulting in slow
heartbeat, coma and possible death from cardiac arrest.
You can easily understand how our body reacts when we are drinking and all the effects that can
arise from alcohol abuse. However, in case of an acute intoxication, if one drinks a little bit, he can
survive, and the organs will not be damaged by the alcohol abuse. Still, it is better not to associate
alcohol with any kind of medicaments and drugs. Why? Because they can affect each other in three
ways:
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- Drugs may delay the elimination of alcohol: when I drink it is important to metabolize very
quickly the ethanol so that it can be eliminated but the drugs can delay the elimination of
alcohol, in particular of acetaldehydes, toxic metabolites, that can give some bad effects e.g.
heat (1 gm of alcohol = 7 calories), red face, nausea, headache, low blood pressure and
palpitations;
- Alcohol may delay the effect of certain
drugs: if one has to take an antibiotic, it will
not have a rapid effect because the alcohol
can delay the effect of certain drugs which
remain active for longer in the body leading
sometimes to drug overdose;
- Alcohol can increase the direct and side
effects of some drugs because they are
not removed, and the metabolites will
accumulate in our body.
In a normal situation, alcohol is metabolized at the level of the liver mainly thanks to ADH,
acetaldehyde can be formed and then transformed into acetic acid and removed through the Krebs’
cycle. Drugs are also metabolized at the level of the microsomes (MEOS) at the ER leading to many
kinds of metabolites that are then eliminated.
When drinking, the normal ways to metabolize alcohol are not sufficient: some of the alcohol will be
metabolized thanks to MEOS, but since alcohol is using this system to produce acetaldehyde, it
interferes with the drugs metabolism system: the drugs will stay longer in our body, will not be
metabolized as quickly leading sometimes even to drug overdose. Metabolites produced by drug
metabolism will accumulate in the body, particularly in the liver.
VIRUS AETIOLOGY - VIRUSES - ONCOGENE
Another important risk factor related to cancer development, in addition to chemicals, are viruses.
Viruses have a specific genome sequence that can be integrated in the host DNA and is able to
induce proliferation. In fact, the first types of DNA or RNA sequences to be called “oncogenes” were
viral sequences able to integrate into the cells. This invasion has for main purpose to induce
infection and permit virus proliferation.
Viruses have two different types of function:

- the infection process: viruses have to replicate and expand their population and they need
a cell as a host to do so. When the viruses have a host, they integrate into the DNA to induce
proliferation and the synthesis of several proteins important to replicate, build the capsid, the
virions and so on;
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- the simple integration of the virus into the host’s DNA; this is related to the type of inter-
exchanges between hosts and viruses and how the host cell is reactive against viruses. Once
the virus has integrated the host’s DNA, it will induce the alteration of the expression of
oncogenes, which can be both viral or of the host: for instance, viruses can induce the
activation of their own genes but also of some of the host’s genes that are close and able to
induce proliferation. Viruses are also able to delay the expression of some anti-oncogenes
to further induce proliferation, activate oncogenic expression and induce the deletion of anti-
oncogenes. To sum up, viruses can negatively modulate the normal regulation of the host’s
genes related to proliferation. Therefore, some DNA and RNA viruses are considered as
potentially oncogenic for our tissues.
DNA viruses are potentially oncogenic in those cells that are called “non-permissive cells”; in fact,
non-permissive cells only allow viruses to be integrated into the DNA but not to induce the complete
process of infection.
In this case, but also in the case of RNA viruses, the main viral genes involved in the cancerogenic
effect are the early genes, i.e. the genes involved in the replication process of the virus.
Arnaud’s question: If the cell was permissive, it would never be cancerogenic?

Professor: if the cell is permissive for DNA viruses, that cell is only able to induce the infection.
Arnaud: so we always prefer to proliferate rather than integrate the whole genome.
Prof: if it’s not permissive
Arnaud: Basically, the virus integrates the genome because it is not allowed to proliferate, so it has
to find another way to harm the organism
Professor: Yes, but only for DNA because RNA viruses are able to be oncogenic for both
permissive and non-permissive cells.
Carcinogenic activity of DNA viruses

DNA viruses can be involved in different steps of the carcinogenic process; viruses:
- Induce genetic instability;
- Activate proliferation to give cellular immortalization;
- Induce malignant transformation;
- Negatively control apoptosis;
- Modulate the cellular microenvironment; for instance, viruses can be involved in the
modulation of inflammation, like in the case of tumors, in which the modulation of inflammation
may induce proliferation but also promotion;
- Cause subversion of immuno-surveillance;
- Hit and run hypothesis of viral carcinogenesis (once the mutagenic effect is explicit, viral
sequences are no longer necessary). The same hypothesis is seen in retinoblastoma; the
retinoblastoma gene is an oncosuppressor, so for it to become oncogenic, both the alleles of
the gene must be deleted, only one mutation is not sufficient to develop the tumor. The ability
of retinoblastoma to induce a tumor is related to this hypothesis, which actually implies two
“hits”: the 1st hit consists in the mutation of one of the two alleles, while the 2nd hit is the mutation
of the other allele. In retinoblastoma, the 1st and 2nd hit happen at different times during
morphogenesis: the 1st hit happens in a pre-zygotic condition (during reproduction), the 2nd hit
in the post-zygotic period; therefore, the 1st hit is hereditary while the 2nd hit is a somatic
mutation. However, the 1st hit alone is not sufficient to induce a malignant tumor, a second
somatic mutation is needed: retinoblastoma only develops in children with both mutated
alleles, while in children with only one mutation (usually the hereditary one) different types of
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diseases related to the eye can develop. In viral carcinogenesis, the virus can induce the first
mutation, the “hit”, but then other mutations are needed for the tumor to develop: a second
mutation, for instance, in an anti-oncogene or other mutations in different genes, then able to
induce multiple other mutations;
- Viral cooperation in carcinogenesis.
Oncogenic-activity of RNA -viruses - retrovirus.

The oncogenic activity of RNA viruses affects both permissive and non-permissive cells because
of the presence of an enzyme called reverse transcriptase, able to integrate RNA viruses into the
DNA of all the host cells. Integration into the host is important also to induce an infection.
The way by which the RNA viruses are able to induce tumors and to be infectious is very similar.
Different genes, in particular early genes, are involved in the oncogenic activity of RNA viruses;
there are three main genes involved in the oncogenic activity:
- pol gene, related to the synthesis of the reverse transcriptase;
- gag gene, which transcribe specific viral proteins associated to the core;
- env gene, related to the proteins of the viral envelop (in fact, env stands for envelope).
Arnaud’s question: Reverse transcriptase is part of the structural protein or is it transcribed inside
the whole cells by the RNA?
Prof: The transcriptase is an enzyme and induces the change of RNA in DNA. Then DNA of the virus
is then responsible for the activation of these genes.
Arnaud: is it part of the structural protein of the virus or is it transcribed inside the cell by viral RNA?
Prof: Yes, it is a viral protein
Arnaud: so it is a structural protein
Prof: it is brought by the retrovirus because it needs to be transcribed to DNA.
Arnaud: Why can RNA be oncogenic in permissive and non-permissive cells, but not DNA?
Prof: Because RNA can be transcribed and so changed in DNA by the transcriptase.
Viruses with potential oncogenic properties in humans

There are different types of potentially oncogenic DNA viruses; the main ones are:
- Papilloma virus (HPV);
- Epstein-Barr virus (EBV);
- HHV-8 virus (Human Herpes Virus 8);
- Hepatitis B virus (HBV).
The main potentially oncogenic RNA viruses are:

- Hepatitis C virus (HCV);
- HTLV-1 (Human T lymphoma virus type 1).
DNA viruses
Human papilloma virus
Papilloma virus is involved in the formation of benign papillomas and warts, it can induce
squamous carcinoma of uterine cervix and anogenital region and, finally, it can also be associated
to oral cavity and laryngeal cancer.
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The different activities of the papilloma virus are due to the existence of different strains.
The most common strains that are considered at low risk of lesions are the strains 6 and 11;
HPV genome remains in the episomal state which means that it won’t be integrated into the host. In
this case there is a low risk of lesion and HPV can induce only warts or benign papillomas in general.
Three other strains, considered to be an intermediate risk for inducing cancer, are the 31, 33 and
35.
The two strains considered to be a high risk for cancer development are the 16 and 18; these two
strains are the ones responsible for the development of squamous carcinoma.
Different types of behavior can put at risk of the infection by HPV; the main factor is the number of
times of exposure to the virus. Therefore, sexual intercourse is a high risk of infection, especially
in the case when the first sexual intercourse happens before the age of 16. In addition, the number
of sexual partners will statistically increase the risk of exposure, and therefore the probability of
being infected by the virus.
The precancerous lesions are detected/diagnosed by the

Pap-test, a cytologic test named after the doctor that
invented it, doctor Papanicolaou. This test allows the
different volumes, shapes and characteristics of the
cells to be seen.
In case of HPV infection, the cells acquire a specific

morphology named koilocytosis (cells are called
koilocytes).
Koilocytes have specific characteristics (figure n.1): Figure 14
- Nucleus/cytoplasm ratio alterations;
- Binucleated cells;
- Perinuclear halo with optically empty spaces.
Figure n.2 is not a cytological but a histological test82 which shows a typical squamous malignant
tumor characterized by typical cells, similar to the spinous/squamous layer of the epithelial tissue.
Figure n.2 also shows the formation of squamous pearls due to the centered degeneration of tumor
cells.
Figure 2
Pap-test is used mainly to diagnose the carcinoma of the uterine cervix. However, it can also be
used for the diagnosis of other types of tumors related to the mucosa, or, more precisely, to all the
82
Therefore, it is related to the tissue, not the cells
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types of tissue from which cells can be withdrawn (such as the oral mucosa, for instance), since
usually we refer to exfoliating cells. For example, cells that are exfoliating from the bladder can be
extracted in the urine.
In fact, urine is used for the diagnosis of a type of professional squamous tumor. The professional
tumor from the bladder is induced by a type of carcinogenic chemicals, the aniline derivatives,
present in staining and colors; these chemicals are metabolized, arrive into the bladder where
bacteria are able to cut the compounds (which are effective compounds) and convert them into
tumoral compounds. Therefore, this type of Pap-test is used for the professional diagnosis of tumors
in the workers that are in close contact with staining, as a preventive diagnosis.
The pap-test results can be classified in five classes:
- Class I: absence of neoplastic cells;
- Class II: definitely non-neoplastic cellular atypias;
- Class III: suspected neoplastic cellular atypias;
- Class IV: likely malignant cellular atypias;
- Class V: highly malignant cells.
Class IV and V are characterized by the certainty of neoplasia, while if a cytologist observes a class
III, neoplasia is suspected. Instead, class II is not neoplasia, it is only related to inflammation and
infection.
There are also other types of tests, in this case histological and not cytological, which provide similar
information; namely, they are CIN and SIL.
CIN (cervical intraepithelial neoplasia) is characterized by only three classes:
- CIN I: slight dysplasia, similar to degree I of the Pap-test;
- CIN II: moderate dysplasia, similar of class II of the Pap-test;
- CIN III: it is considered neoplastic, so either a carcinoma in situ or a severe dysplasia, and it
includes classes III, IV and V of the Pap-test.
SIL (squamous intraepithelial lesions) can only give information about whether neoplasia is
present or not:
- Low grade lesions: absence of neoplasia (they include HPV alterations, so CIN I);
- High grade lesions: presence of neoplasia. They include moderate (CIN II) or severe
dysplasia (CIN III or carcinoma in situ).
The human papilloma virus can induce the formation of a tumor thanks to different types of proteins,
divided into early and late. The most important proteins are the early proteins, in particular E6 and
E7, primary transforming proteins able to transform and bind two important antioncogenes, blocking
their activity. In particular, E6 binds to p53 while E7 binds to RB.
The normal mechanism of infection of HPV is related mainly to the presence of different
microlesions in the mucosa, required for the virus to insert into the basal layer.
Statistically speaking, the risk of infection is related to the number of microlesions and to the
number of opportunities, i.e. to the number of exposures to the virus, during the reproductive age:
the more microlesions there are, the higher the risk of infection is.
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The viral genome is integrated in the cells of the basal layer of the epithelium, exposed through
the microlesions, and, over the course of time, it continues to differentiate and to produce proteins
that will complete the assembly of the
virions, able to induce the infection of the
apical part, from where they will be able to
infect other tissues. During these
different steps, some of the viruses
remain into the DNA of the cells and
continue to induce mutations affecting the
proliferative process.
Slide 17 Figure 3
HPVs infect keratinocytes in the basal layer
of the epithelium that becomes exposed through
microwounds. ‘early genes’ (E) expression.
- The viral genomes are replicated in synchrony with cellular DNA replication;
- One daughter cell migrates away from the basal layer and undergoes differentiation;
- Differentiation of HPV-positive cells induces the productive phase of the viral life cycle, which
requires cellular DNA synthesis machinery;
- The expression of E6 and E7 deregulates cell cycle control;
- The late-phase L1 and L2 proteins encapsidate newly synthesized viral genomes and virions
are shed.
There are two vaccines related to the HPV83.
Two other types of DNA viruses that are able to be oncogenic are EBV and HHV-8.
Epstein-barr virus (ebv)

EBV is an endemic virus. In fact, it is able to induce different types of disease depending on the
population it infects; in particular, this virus is responsible for the African form of Burkitt’s
lymphoma, for the B-cell lymphoma, predominantly seen in immunodeficient individuals like
transplanted patients or AIDS patients, for some cases of Hodgkin’s disease and, in particular, for
the nasopharyngeal carcinoma in the Asian population/east population and mononucleosis in
Europe.
Therefore, depending on the type of strain but also on the individual response to the virus there is
the development of very different types of diseases: if the infection happens in someone with
European origins there will be a higher probability to develop mononucleosis while if it happens in
someone with African origins, a lymphoma will be more likely to develop.
EBV alone does not have a direct oncogenic action, but it simply acts as a polyclonal mitogen of B
cells, favoring the occurrence of translocations84. The main translocation this virus can induce is the
t(8; 14), which occurs between chromosome 8, which contains c-Myc, and chromosome 14, which
contains the genes encoding for the immunoglobulin heavy chains.
EBV latent genes
83
Already discussed in the microbiology course, so they will not be treated during this lesson
84
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Like for HPV, in EBV as well some early genes are

involved in the carcinogenic effect. In particular, there
are EBERs, EBNA-1, EBNA-2, LMP-1, LMP-2,
which are considered as antigens; in particular, the
latent membrane proteins (LMP) are exposed
normally at the surface of the virion, therefore the
immune system normally recognizes them and so the
virus can be killed. Of course, if these proteins are not Figure 4
exposed on the membrane, these cells can escape the immunologic surveillance: this is the case of
Burkitt’s lymphoma which is an anaplastic tumor in which there is no expression of three proteins
related to the early genes of the virion that are not expressed (figure n.4); nasopharyngeal, a blastic
tumor, has only one of these genes not exposed; the same happens for T lymphoma, in which only
one gene of this list of genes is not expressed (figure n.4). In lymphoproliferative diseases, like
mononucleosis, all the proteins are exposed and, in fact, in this case there is an inflammatory
reaction against the infection.
ETHANOL TOXICITY (pt.2)85

Chronic effects of ethanol
It has been observed that since alcoholics chronically abuse of alcohol, they are able to develop a
tolerance towards high concentrations of alcohol in the blood. In fact, it is possible to see effects at
the level of the CNS when the concentration of alcohol in the blood is higher than 100-200 mg/100mL
of blood.
The mechanism involved in the promotion of tolerance in chronic alcoholics is still unknown, although
some hypotheses have been made: probably, there is an adaptation of the cells of the CNS; then,
it has also been observed that the composition of lipids changes, which could play a role in alcohol
tolerance; furthermore, the MEOS (microsomal ethanol oxidizing system) is activated, probably even
hyperactivated, another possible explanation to this tolerance.
There are other two aspects concerning chronic alcoholics, addiction and the abstinence
syndrome.
Alcohol is a kind of drug, so alcoholics develop an addiction to it and once they start drinking, they
are not able to stop: they drink every day and increase the quantity of alcohol they introduce with
time. This addiction seems to be due to certain changes happening in the central nervous system,
more specifically, changes in the amount of some neurotransmitters like GABA (gamma-amino-
butyric acid) and dopamine, which seem to play an important role in this addiction.
If chronic drinkers stop drinking for more than 24h, they start to show strange behaviors, collectively
known with the name delirium tremens or abstinence syndrome; this syndrome consists of clinical
symptoms like trembling, hallucinations, nausea and vomiting.
Diseases associated with chronic alcoholism

A chronic abuse of alcohol can promote a lot of damages in different organs of the body, of which
the liver is the most susceptible one. The most frequent clinical complication of alcoholism is the
alcoholic fatty liver disease.
85 prof. Leonarduzzi
286
Liver
At the beginning, the alcoholic fatty liver disease starts from a condition of hepatomegaly: the
liver gets bigger because its volume is increased. Consequently, there is an accumulation of lipids
inside the cytosol of hepatocytes: indeed, alcoholic fatty liver is also called steatosis or liver
steatosis or hepatic steatosis (there are different terminologies). Fatty liver disease is still a
reversible state: stopping the drinking is enough to reverse the situation, the liver goes back to its
normal size and the accumulation of lipids will disappear.
However, if the drinking doesn’t stop, there is a further evolution of the condition. An inflammatory
response can appear in the liver, due to the presence of necrosis of hepatocytes, causing a reactive
flogosis86 known as acute alcoholic hepatitis, which is still a reversible situation/state.
Carrying on with the drinking, after a situation of alcoholic hepatis, fibrosis will develop and, finally,
as the end point of liver damage, cirrhosis. Fibrosis and cirrhosis are irreversible conditions.
Moreover, cirrhosis is also included in the group of situations called precancerosis, because, since
there is a chronic inflammation, there is a higher risk to develop hepatocellular carcinoma.
Figure 5
Gastrointestinal tract
Also the GI tract can be damaged by alcohol abuse; in particular, the toxic effects of alcohol have
an impact on the mucosa of the esophagus and of the stomach and they arise because of the
gastric high concentration of HCl (hydrochloric acid), which, as a consequence, will lower the pH.
Alcoholics therefore can be affected by chronic gastritis, characterized by massive blood bleeding
and gastric ulcer. However, they can also present esophagitis, in which one of the main clinical
symptoms is the esophageal reflux, a lowering of pH caused by the hypersecretion of gastric acids;
also in the case of an esophagitis there is a chronical inflammation, which means that also this
situation is a precancerous state that can evolve into an esophageal carcinoma. In a condition
known as Barrett’s esophagus there is the substitution of the stratified squamous epithelium by
cylindrical epithelium and muciparous cells, typical of the gastrointestinal epithelium, and so this is
a precancerous state too.
Peptic ulcers can also develop in the stomach and in the duodenum.
In addition, different damages at the level of the mucosa of the small intestine can appear, for
example villi damage, decreased production/secretion of the digestive enzymes, steatorrhea and
alteration of the absorption of nutrients; if the absorption of nutrients is not done properly, it leads to
a deficiency of vitamins, proteins etc. This usually means that chronic drinkers present malnutrition.
Immune system
86
Another way to say “inflammation”
287
The immune system can also be affected by chronic abuse of alcohol. Alcoholics are more prone to
infections especially at the level of the lungs.
Pancreas
The pancreas is also very susceptible to the toxic effects of chronic abuse of alcohol. Acute and
chronic pancreatitis can develop.
Skeletal muscle
Alcoholic myopathy can also develop. There is a muscle weakness characterized sometimes by
muscle fibrosis and diffuse fibroses etc. Rarely, muscle necrosis can also be observed.
Bones
Bone can also be damaged by this chronic abuse. The mechanisms concerning these damages are
not known. However, it has been observed that especially women are often affected by osteoporosis.
Another characteristic is the aseptic necrosis of the femoral head.
Heart
The heart can be damaged. The abuse of alcohol can induce the dilation of all four cavities of the
heart, which means that because of this dilatation a failure of the heart is more likely to happen.
Sometimes steatosis can also be present, meaning accumulation of lipids and especially
triglycerides in the muscle fibers. Finally, sometimes fatal and sudden arrhythmias can occur. So,
the heart cannot work properly causing bad effects.
Nervous system
Nervous system can also be affected by the chronic abuse of alcohol. It can be affected at different
levels and induce several diseases/syndromes. To sum up the effects of chronic alcoholism on the
nervous system we can say that there is a cortical atrophy and all cerebral diseases are induced
by malnutrition. As said before, the absorption of nutrients doesn’t occur correctly at the level of the
intestine leading to a deficiency in vitamins, especially vitamins of the B family, but also of other
nutrients. An alcoholic can develop mental confusion, be affected by ataxia, have brain instability,
lack of coordination etc. Alcohol abuse has many repercussions on the CNS.
Blood
Blood can also be affected by these toxic effects. For example, because of the malnutrition, the
subject can develop hemolytic anemia. There can be an increase of volume of the red blood cells
due to the deficiency of vitamin B12, for example. There can also be megaloblastic anemia. Or also
transient thrombocytopenia, in this case alcohol can interfere with the aggregation of the platelets
and therefore, there is a higher risk of hemorrhages. Finally, alcohol can also induce hypertension.
Fetal alcohol syndrome

Women during pregnancy are advised not to drink alcohol. Especially during the first three months
because it is the period with higher risk, this because alcohol can induce abnormality in the
development of the fetus which may lead to: growth retardation, microcephaly, facial dysmorphisms,
neurological dysfunctions. It is better to avoid drinking alcohol, or limit drinking, during pregnancy
especially during the first three months because the fetus can show different malformations.
Endocrine system
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The endocrine system can also be affected.

In male chronic drinkers it is possible to observe a sort of “feminization”. They can develop
gynecomastia and body hairs are reduced because of the alteration of estrogen metabolism. In
addition, there can also be testis atrophy.
Female chronic drinkers can develop a gonadal insufficiency and present anovulatory cycles.
To sum up, because of this chronic abuse there
Figure 6 is an inflammatory condition at the level of
several organs (oral cavity, esophagus,
stomach, liver, intestine, etc.). This inflammatory
condition increases the incidence of cancer
because the organs are put all in a precancerous
state.
In figure n.6 are present all the damages that the
abuse of alcohol can induce. Especially
damages can occur in the CNS, heart, liver,
pancreas, intestine, stomach.
Alcohol and drug metabolism

As said in the precedent lesson, it is better not
to take any medicament while consuming
alcohol because drugs and alcohol can affect each
other; this, however, in case of an acute situation. If for
example, I go to a dinner and I take few glasses of wine,
it is better not to take any anti-inflammatory or other
medicaments in contemporary/at the same time
because they can affect each other.
In acute conditions, drugs can delay the elimination of
Figure 7 alcohol or for example, alcohol may delay the effect of
certain drugs, but also it can increase the direct and/or side effects of some drugs.
In figure n.7 is presented the normal situation in which alcohol is metabolized at the level of the liver
thanks to the alcohol dehydrogenase, the enzyme present in the cytosol of the hepatocytes.
Drugs, or any type of medicaments, are metabolized at the level of microsomes.
As a final product of ethanol metabolism there is production of acetaldehyde which is a toxic
compound. As a final product of drugs’ metabolism there is the production of different types of
metabolites that will be eliminated thanks to the function of the liver which is able to eliminate all the
toxic metabolites/compounds.
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During acute intoxication of alcohol, because of the high concentration of alcohol in the blood, the
normal system is not able to eliminate the alcohol totally, so part of the alcohol won’t be metabolized
thanks to the MEOS (microsomal ethanol oxidizing system) found in the microsomes, but rather
through the same system that is normally used by drugs for their normal metabolism.
In the case of chronic intoxication there is a competition between the two systems.
Chronic drinkers are not able to metabolize all the alcohol only through alcohol dehydrogenase
Figure 8
because of the very high concentrations of alcohol in blood. Therefore, there is the need to
metabolize alcohol through the MEOS system (microsomal ethanol oxidizing system), so there is
interference with the normal pathway of drug metabolism. The MEOS will be hyperactivated i.e.
hypertrophy of the microsomal system.
If an alcoholic stops drinking and only absorbs later a small quantity of alcohol, this small quantity
can be metabolized thanks to the normal system (= alcohol dehydrogenase). The microsomes
however will remain hyperactivated and hyper-stimulated. This means that when a chronic alcohol
abuser, that has stopped drinking, takes some drugs, the result will be the production of a lot of toxic
metabolites. This could lead to an accumulation of metabolites at the level of the liver. Moreover,
MEOS is working more than necessary because it remains hyperactivated as a consequence of the
past chronic abuse of alcohol.
Biological markers of alcohol abuse

There are some biological markers of alcohol abuse.
A simple way to see if someone is an alcoholic is to analyze the concentration of alcohol in
someone’s body fluids. For example, in urine, blood or in breath (e.g. driving alcohol test). The
volume of the red blood cells is another biological marker e.g. in chronic drinkers the volume of the
cells is bigger than normal due to deficiency of vitamin B12 subsequently to malnutrition. A third
marker is the development of thrombocytopenia which is the reduction of platelet aggregation. These
persons have a higher risk to develop hemorrhages.
There are several enzymes, which are markers of cell necrosis, whose levels can be analyzed in the
blood. As said before, because of the abuse of alcohol, liver is one of the organs that is the most
easily damaged by alcohol’s toxic effect.
After developing fatty liver, there can be inflammation, so, necrosis of hepatocytes which is marked
by the production of these enzymes in the blood:
- GGT (γ-glutamyl-transpeptidase): index of hepatic necrosis, enzyme present in cells membranes;
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- AST and ALT (aspartate transaminase and alanine transaminase): typical enzymes analyzed
in blood to have the proof of hepatic necrosis.
Another marker is EGT (ethylglucoronide) and can be found in urines.

Most of ingested alcohol is metabolized at the level of liver. However, a small quantity can be
excreted in an unchanged way (as alcohol, without changing its chemical composition) in the urine,
breath or sweats for example.
Some of it can be eliminated after conjugation with glucuronic acid or sulfuric acid. In the urine this
complex, alcohol plus glucuronic acid, can be found because it is more stable compared to alcohol
alone.
Finally, the last biological marker are acetaldehyde adducts. Acetaldehyde can easily bind to
proteins and lipids so that these adducts, acetaldehyde and lipids or proteins, are found easily in the
tissues.
Alcoholic abusers develop a tolerance of the toxic effects of alcohol, but after some time alcohol
damages their CNS, liver and so on; these damages, especially at the level of the liver, will cause
the malfunction of the mechanisms of alcohol metabolism which may lead to alcohol intolerance and
the inability to contrast the effects induced by alcohol abuse.
LIVER
The normal liver is a quite big organ, its weight in a
normal adult is 1.4-1.6 kg.
Its circulation is complex, in fact, there is a dual blood
supply:
1. Portal vein which provides almost 60-70% of
hepatic blood flow;
2. Hepatic artery which provides 30-40% of
hepatic blood flow.
These vases divide in many branches inside the liver.
It’s composed by two anterior lobes, right and left, and
one posterior87, the caudate lobe. Each lobe is divided
into lobules with a diameter of 1-2 mm. Lobules are the
structural and functional units of the liver. They have a
hexagonal structure, in the middle of which there is the
terminal hepatic vein, which is also called central
vein, while at the corners there are the portal tracts. In
these tracts there are the bile duct, one branch of the
hepatic artery and one of the portal vein. Hepatocytes
have a typical, characteristic distribution inside the
lobules, they are organized into anastomosing sheets.
They extend from the terminal hepatic vein in direction
of the portal tracts88. These cells look like the “spots”89
of the bicycle because they extend from the central area
87
Actually, we know from Anatomy that there are two smaller lobes located on the visceral surface of the liver, the
caudate and the quadrate.
88
On the slides the opposite is written
89
I.e. radianti
291
in order to reach the periphery. From an anatomical point of view, two models of the liver can be
recognized:
1. Lobular model: in this model the terminal hepatic vein or central vein is in the middle of the
lobule, while the portal tracts are at the periphery. Two areas can be distinguished:
- The periportal, which is around the portal tracts;
- The centrilobular, which is around the central vein.
2. Acinar model: the lobule is divided in three zones. Zone 3 is near the central vein, zone 1
and 2 are near the portal tracts, where there’s the blood supply i.e. where we find artery,
veins and bile ducts.
Liver functions
Liver is an important organ and it has many functions:
- Metabolic functions: control of lipids for glycolic and protein metabolism;
- Synthesis functions: synthesis of proteins present in the plasma, factors
involved in coagulation, so fatty acids, cholesterol, phospholipids and various
structural and enzymatic proteins;
- Reserve functions: it can act as a storage for iron, copper, glycogen,
triglycerides, vitamins;
- Catabolic functions: it plays an important role in catabolism of endogenous
substances, such as hormones and proteins. Detoxification function i.e. it is
involved in all the processes whose aim is to eliminate toxic products, bacteria.
Moreover, ammonia can be eliminated through the liver. In fact, ammonia’s
accumulation can promote many damages in our body and has to be removed;
- Excretorial functions: the main excretory product of the liver is bile;
- Endocrine functions: it is important for the catabolism of various hormones.
When the liver is damaged either because of primitive pathologies or secondary pathologies, a liver
failure may happen, and it may not be able to perform all its functions. Thus, it may not be able to
work in a proper way.
Liver alteration may involve the whole organism and lead to other pathologies:
- Primitive pathologies;
- Secondary pathologies.
Primitive pathologies may involve:
1. Hereditary pathologies;
2. Malformative pathologies;
3. Tumor pathologies;
4. Vascular pathologies;
5. Traumatic pathologies;
6. Inflammatory pathologies;
7. Autoimmune pathologies;
8. Gallstones: the presence of gallstones at the level of biliary ducts.
9. Degenerative diseases: steatosis, for example, is characterised by the accumulation of fats
inside the liver. In general, degenerative diseases are characterised by the presence inside
hepatocytes of something that shouldn’t accumulate: in this case, we talk about accumulatio
of fats, but could be also of iron, copper, agar products;
10. Fibrosis: cirrhosis which is characterised by fibrosis, necrosis of hepatocytes, regeneration
of hepatocytes etc.
Steatosis
This state shows morphologically evident accumulation of fats (mainly triglycerides) inside
hepatocytes, this accumulation is also chemically demonstrable. The topic of the lesson is the
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liver, but this accumulation of fats can occur also in other organs. Thus, steatosis occurs when there’s
accumulation of fats in organs that normally store fats in minimal quantities and not histologically
revealed. There are many histochemical techniques that are used to demonstrate that lipids are
accumulating inside hepatocytes. Different staining can be used:
- Osmium tetroxide: lipids will assume a black coloration;
- Sudan III: lipids will assume an orange coloration;
- Black Sudan: lipids will assume a black coloration;
- Blue Nile sulphate:
o Triglycerides are stained in red;
o Cholesterol is stained in pink;
o Free fatty acids (not esterified) are stained in blue.
- Oil red: free fatty acids will be stained in red.
Figure 1
Figure n.1: haematoxylin eosin fixation. It’s a normal histological
technique to show the presence of lipids inside hepatocytes, all
these white bubbles represent the fats which are accumulated
inside hepatocytes.
Figure n.2: Staining with Sudan III. It shows a darker coloration for
the staining of lipids.
Main organs affected by steatosis Figure 2

Liver
Liver is one of the main organs affected by steatosis.
This situation is characterised by:
- Increased triglycerides intake;
- Increased endogenous synthesis;
- Decreased fat utilization for energy purposes;
- Decreased blood secretion.
There are two types of steatosis:
1. Macrovescivular;
2. Microvescicular.
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The macrovescicular form is characterised by the accumulation of lipids at the level of the
cytosome, hepatocytes are extended, and the nucleus is pushed at the level of the membrane. Thus,
only one big drop of fats (triglycerides) pushes the nucleus near the membrane at the periphery of
the cell.
Figure n.3 shows a structure which may Figure 3
resemble a ring: the finger is represented by the big drop of
lipids that pushes the nucleus close to the
membrane, usually this form is associated with a
chronic but still reversible disorder. However, this
situation can easily evolve into steatohepatitis,
meaning inflammation and necrosis of
hepatocytes. Following steatohepatitis, cirrhosis
may occur.
The microvascicular form is characterised by the

presence of numerous droplets of lipids inside the
cytoplasm around the nucleus, which is not pushed
to the periphery of the cell, but rather stays in the
middle. Usually this type of steatosis is more
dangerous and associated to liver failure i.e. the
liver has difficulties in carrying on all its functions.
Myocardium
Lipids can accumulate in the myocardial fibres
when there is a deficient oxidation of fatty acids.
Main causes are:
- Hypoxia from anaemia or stasis;
- Insufficient heart circulation;
- Toxic damages.
Kidneys
Lipids can accumulate also in kidneys when there’s a deficient oxidation of fatty acids which occurs
at the level of mitochondria i.e. β-oxidation. In this case, there’s a reduction in the supply of oxygen.
Main causes are:
- Hypoxia;
- Choline deficiency.
Skeletal muscle
Accumulation of lipids can also be found at the level of skeletal muscle i.e. lipid myopathies.
Fate of fatty acids in the tissues

In normal tissues, fatty acids are used for energy purpose, to produce ATP thanks to β-oxidation of
fatty acids at the level of mitochondria. However, they can also be used for the turnover of cells of
the membrane, thus, they can be included in biological structures.
Fate of fatty acids in the liver

In the liver, fatty acids are used for many purposes. They are used to produce energy, for the
inclusion in biological structures, but also for inclusion in order to form glycoproteins. The liver is
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able to remove lipids in the form of glycoproteins, the main glycoprotein which is produced by the
liver is VLDL (very low-density lipoproteins).
Lipid metabolism in the liver

1. Liver can receive non-esterified free fatty acids (NEFA), so free fatty acids, which are
released in the plasma and travel through the blood in order to reach the liver. They are
released in the plasma after hydrolysis of triglycerides which are present in the adipose
tissue: lipolysis of triglycerides that are present in our adipose tissue which constitutes our
reserve/storage of energy. These free fatty acids may reach the liver thanks to the diet too
(exogenous lipids): food as butter and cheese contain many lipids that will reach the liver as
chylomicrons, another type of lipoproteins;
2. Endogenous synthesis of free fatty acids starting from acetyl-coenzyme A;
3. Some of these free fatty acids may be oxidised at the level of mitochondria through β-
oxidation in order to produce ATP;
4. All the fatty acids that are not used directly by the hepatocytes will be removed in the form of
lipoproteins, these lipids will bind to a specific type of protein which is called apoprotein.
Lipids together with apoproteins then form lipoproteins that will go outside the liver. The main
types of lipoproteins which are made up at the level of the liver are VLDL. Besides that, in a
small quantity, the liver is also able to produce HDL (high density lipoproteins). Thus,
triglycerides, phospholipids and cholesterol can be removed from the liver through the
production of lipoproteins and then be distributed to all tissues for the normal turnover of our
cell membranes, for the production of other hormones, for energy etc.
Causes of excessive hepatic accumulation of triglycerides→ hepatic steatosis

1. Increased intake of lipids: in this case the liver is not damaged but there are bigger
quantities of free fatty acids that will reach the liver either because there’s an increase in
lipolysis, for an hyperlipidic diet or a repeated lipid diet at short intervals and a higher number
of chylomicrons. Lipolysis of adipose tissue can also be stimulated by some substances as
theine and caffeine or by some hormones such as ACTH, cortisone, catecholamines as they
activate lipase which is able to hydrolyse triglycerides, thus obtaining glycerol and non-
esterified fatty acids which will be released in the blood and from there the liver;
2. Increased endogenous synthesis: it occurs when there’s an increase in precursors for the
synthesis of these substances, for example, when there’s bigger quantity of acetic acid i.e.
more substrates available for the formation of lipids;
3. Accumulation of fatty acids can also be promoted by their oxidation because when there’s
a reduction of β-oxidation at the level of mitochondria, this will lead to a reduction of energy;
4. Reduction of excretion: lipids can be removed from the liver in order to go in the blood as
lipoproteins but before that, apoproteins need to get together with lipids. It can happen that
the synthesis of these proteins may be inhibited and stopped for different causes: malnutrition
(e.g. drinkers), less absorption of proteins, less amino acids or less glycoproteins can be
difficult to produce lipoproteins also if there’s a deficiency of phospholipids due to malnutrition
due to the fact that they are necessary for the binding of triglycerides and apoproteins;
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5. Damages at the level of microtubules: lipoproteins cannot migrate properly outside of the
liver. For example, in drinkers, acetaldehyde, which is the toxic compound released from
ethanol metabolism, can bind to tubulin which is one of the most important components of
microtubules. In this case, even if lipoproteins are formed correctly, they cannot go outside
from hepatocytes.
To summarize (figure n.5): Lipids accumulate inside the hepatocytes because there is an increase
of NEFA intake, increase in free fatty acids cycle. We can have an increase in endogenous synthesis
Figure 5
of free fatty acids because of an increased quantity of precursors such as acetic acid, when there’s
less oxidation of fatty acids at the level of mitochondria, when there’s a decrease in phospholipids
synthesis due to malnutrition or choline deficiency and if the synthesis of phospholipids in general is
reduced. In fact, they are important for the binding between triglycerides and apoproteins. There can
also be an increased intake of triglycerides because of the diet. Indeed, if a person has a diet which
is very poor in proteins, a so-called hypoproteic diet, there will be difficulties in forming lipoproteins.
On one hand, phospholipids are needed because they help triglycerides to bind to apoproteins, on
the other hand, apoproteins can be missed because there aren’t enough amino acids coming from
the diet or protein synthesis is less, so there isn’t the possibility to form lipoproteins. However, a
person may be able to produce lipoproteins, but they may not be able to be excreted due to some
problems at the level of microtubules.
Causes of hepatic steatosis:

- Alcohol abuse: the type is macrovescicular steatosis i.e. there’s a big drop of
lipids that pushes away the nucleus, so macrovesicles are formed;
- Drugs can promote the accumulation of lipids inside hepatocytes, in this case we
can have both macro and microvescicles;
- Metabolic syndrome may present in the macrovescicular form. Patients may be
affected by hyperinsulinism, obesity, diabetes, dyslipidaemia and
hyperadrenocorticism;
- Malnutrition, malabsorption: having the right quantity of amino acids, lipids and
proteins is very important. During fasting, for example, or after chronic intestinal
inflammatory diseases, we can observe the accumulation of lipids in
macrovescicular form. In fact, when the intestinal mucosa is damaged, there can
be problems in absorption e.g. patients with celiac disease, cachexia and fasting-
ileal-by-pass can also be affected by steatosis at the level of the liver;
- In the case of some infections: such as Hepatitis C Virus (HCV) or some
endotoxins.
Alcoholic liver disease

296
Alcoholic liver disease is one of the main causes of hepatic steatosis90. It is induced by excessive
consumption of alcohol. In fact, 80-95% of alcohol is metabolized at the level of the liver:
acetaldehyde is produced, and it is converted to acetic acid, which is one of the precursor for the
synthesis of free fatty acids.
When someone drinks a lot and for a long time, the liver is damaged.
1. Hepatocellular steatosis: the liver is bigger than normal for hepatomegaly. If the patient
keeps drinking, hepatocellular steatosis may evolve to the next step;
2. Acute alcoholic steato-hepatitis.
They are both reversible. In the first step the liver is big but can still function quite well (no liver
failure); instead in the second case, there’s already a minimal or acute liver failure, leading to
anorexia, loss of weight, some troubles concerning the production and excretion of bile. In this
situation, increased level of specific enzymes which are markers of hepatocytes necrosis such as
aminotransferase and alkaline phosphatase can be found in the blood. There may appear also an
increase in leukocytes, especially neutrophils, since they are the indicators of inflammation and
necrosis.
3. Steatofibrosis which can lead to cirrhosis: these are irreversible conditions. Thus, even if
the patient stops drinking, the liver will not return normal;
4. For many of the patients that develop cirrhosis, there is a higher risk of development of
hepatocellular carcinoma.
Figure n.6: the picture shows the interrelationship
among hepatic steatosis, alcoholic hepatitis and
alcoholic cirrhosis.
At the beginning, there’s a normal liver which is
normally exposed to some toxins, such as alcohol. In
this case, there’s the possibility to develop a fatty liver,
thus steatosis. From a normal liver, if the exposition is
severe, we will directly go to a situation of
steatohepatitis with necrosis and inflammation. These
two conditions are still reversible. In fact, if the patient
stops drinking or removes the cause of these liver
damages, the liver can return in a brief time to a normal situation. If there’s a severe exposition, a
fatty liver can develop steatohepatitis. From a condition of steatohepatitis, the liver can return back
to steatosis in rare situations in which there’s the stop of the exposure to toxic compounds, however
if steatosis and steatohepatitis develop into cirrhosis, this is an irreversible situation.
Hepatic steatosis
- Lipid droplets accumulate at the beginning in the cytosol of hepatocytes: around
zone 3, where there’s the central vein and around zone 2 and 1 (Acinar model);
- The liver becomes larger and bigger so the weight increases. It reaches 4-6 kg, it
becomes soft, yellow and greasy. It’s still a reversible situation.
Causes responsible for accumulation of lipids inside of hepatocytes even in chronic alcoholics:
- Increased intake;
- Less oxidation at the level of mitochondria (β-oxidation);
- Increase in triglycerides synthesis;
- Decrease in synthesis of lipoproteins;
90
We will see the differences between alcoholic hepatic steatosis and non-alcoholic hepatic steatosis.
297
- Decrease in excretion of lipoproteins.
Alcoholic steatosis and steatofibrosis

In the figure, the white bowls are fatty droplets which accumulate at the
beginning around the terminal hepatic vein, then around zone 2 and the
portal tracts. Thus, accumulation starts at the centre of the lobules.
Fibrotic tissues are stained in blue and they are appearing at the level
of a simple fatty liver.
Alcoholic steatoepathy (reactive inflammation)

- Some of the hepatocytes, before dying, start to
become bigger because there’s an hydropic degeneration: inside hepatocytes,
there’s not only the accumulation of lipids but also of water and other proteins.
These hepatocytes undergo balloonin, they become quite big and swallow. At
the end the cells of the membrane can break, and hepatocytes are destroyed;
- Other important characteristics are the Mallory-Denk bodies, which are
inclusions typical of steatohepatitis induced by alcohol;
- Besides necrosis and inflammation, fibrosis can also be observed. Thus, there’s
a deposition of collagen which starts around the terminal hepatic vein and then
can reach the portal tracts.
Cirrhosis
If the individual carries on with drinking, there will be the accumulation of the damage, leading to
cirrhosis which can evolve into hepatocellular carcinoma.
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Figure 8
Figure n. 8: it shows the morphology of a cirrhotic liver due to alcoholic cirrhosis. It is characterised
by cirrhotic lobules and thick bands of collagen among the hepatocytes lobules. It is an irreversible
condition; thus, the morphology of the liver won’t return in a normal condition. However, after a year
of abstinence from drinking, even if the normal morphology won’t be reacquired, necrosis and
inflammation will reduce. Moreover, fibrotic tissues and scars will be thinner. These reductions are
possible because, after one year, all the effects of necrosis and inflammation are stopped.
Non-alcoholic hepatic fatty liver disease (NAFLD)→ steatosis not induced by alcohol
There are three types of non-alcoholic fatty liver diseases:
1. A simple condition which is characterised by accumulation of triglycerides inside hepatocytes,
this condition is the “condizione benigna”. It is also called Non-Alcoholic Fatty Liver (NAFL)
because it is a simple situation characterized by accumulation of triglycerides inside
hepatocytes;
2. From a normal condition the so-called Non-Alcoholic Fatty Liver Disease (NAFLD) may
develop. It is a simple condition, not a disease, but with an increase of aminotransaminases
in the blood which are markers of necrosis; thus, it means that there’s inflammation and
hepatocytes are dying;
3. Non-Alcoholic Steatohepatitis (NASH) is characterised by the presence of real necrosis
and inflammation at the level of the liver.
From NAFLD patients can remain stable or they can develop a NASH. From this situation there can
also be a regression concerning fibrosis, or a progression concerning it.
Eventually, some of these patients can develop cirrhosis and, from it, a hepatocellular carcinoma.
299
NAFLD are all the types of steatosis not induced by alcohol. Sometimes, fatty liver can contribute to
other liver diseases, for example, virus infections, such as HCV and HBV infection.
NAFLD is also indicated as a multifactorial metabolic disease or syndrome because it is caused
by the interactions of many factors: accumulation, inflammation, necrosis, cirrhosis, cancer etc.
It has been observed that this disorder can have ethnic and genetic differences. In fact, it is more
diffuse among Hispanics, Africans, Americans and Caucasians.
Since it’s a multifactorial metabolic syndrome, many factors can be found among the causes of
steatohepatitis, cirrhosis and then cancer:
1. Primary factors;
2. Associated factors that can interact with primary factors.
Primary factors
- This metabolic syndrome can occur in people affected by type 2 diabetes
mellitus which is characterised by insulin resistance. Insulin is produced by
pancreatic cells, but it cannot interact with the target cells because receptors are
missing so there is a resistance between insulin and target cells. Reduced insulin
sensitivity (insulin resistance) promotes activity of lipase enzyme that hydrolyses
triglycerides present in adipose tissue. This leads to an increase of lipolysis and,
as a consequence, more free fatty acids will be released in the plasma and will
enter inside the liver;
- Hyperinsulinemia: insulin is produced in large quantity, but presence of high
levels of insulin can inhibit β-oxidation of fatty acids inside mitochondria. The
synthesis of apoprotein is important for synthesis of VLDL will be reduced as well
i.e. reduction of Apo B-100 that is a specific apoprotein used to form the VLDL
leading to reduction of VLDL secretion;
- Hyperinsulinemia + gluconeogenesis: they lead to an increase in endogenous
hepatic synthesis of fatty acids.
Associated factors
- A specific obesity: at the level of abdominal area there is the accumulation of
visceral adipose tissue. The limit values for women are 84 cm, while for men 104
cm. They are not obese in the whole body, but rather only in the abdominal area
(belly). These people may have also:
o Hypertension;
o Hypertriglyceridemia: high blood levels of triglycerides;
o Hypercholesterolemia: high blood level of cholesterol;
o Hypocholesterolaemia: low level of high density lipoproteins (HDL);
o Hypoalbuminemia: low level of albumin, which is a plasma protein.
Difference between HDL and VLDL: all the lipids which are not used for membrane turnover or for
the production of energy, thanks to HDL, can go back to the liver in order to be removed or reused.
In contrast, VLDL and LDL can bring lipoproteins rich in cholesterol to all the tissues.
VLDL carries the “bad cholesterol”, while HDL carries the “good cholesterol”. In fact, it’s positive to
have high levels of HDL which is able to remove excess of cholesterol, but it’s not positive to have
high levels of VLDL since it’s one of the main causes of atherosclerosis. Indeed, cholesterol diffuses
to the periphery of the body to reach all the tissues and may deposit inside the wall of our arteries
(atherosclerotic plaque formation).
The important thing is to have a balance between these two types of lipoproteins.
It has been hypothesised that NAFLD is induced by two hits:
1. Insulin resistance, which is responsible for hepatic steatosis;
300
2. Hepatocellular oxidative injury which causes lipid peroxidation, hepatocytes necrosis and
therefore inflammation.
The first hit is insulin resistance, thus, individuals with diabetes mellitus (type 2), with
hyperinsulinemia (high levels of insulin, they are able to release it but the insulin is not able to “talk”
to other cells and then glucose cannot be used for the normal metabolism), hypertriglyceridemia,
obesity, lower levels of VLDL, accumulation of fats inside hepatocytes, increased intake of non-
esterified fatty acids, higher hydrolysis of triglycerides from adipose tissues and higher intake of lipids
through diet. This leads to development of steatosis.
The second hit is oxidative stress, lipid peroxidation and inflammation; as a consequence of oxidative
stress, free radicals and reactive species are produced and there’s oxidation of lipids. In these
individuals, there may be also a genetic predisposition to develop this kind of syndrome. After the
oxidation of membrane lipids, necrosis of cells, inflammation, activation of Nuclear Factor-kB (NF-
kB), which is one of the main transcriptional factors in inflammation response and leads to the release
of cytokines, may occur. Oxidative stress can also damage mitochondria leading to a reduction in β-
oxidation of fatty acids. Kuppfer cells, which are macrophages in the liver, can release different kinds
of cytokines and factors such as TGFβ, that is the principle factor able to stimulate fibrogenesis i.e.
release of collagen by Ito cells, there is also an antioxidant deficit.
Eventually, all these interactions between the two hits will lead to necrosis of hepatocytes,
inflammation and synthesis of collagen by Ito cells, fibrosis will appear as the last fact of NASH and
may lead to cirrhosis. Cirrhosis with all its complications may cause hepatocellular carcinoma.
Figure n.11: it shows a non-alcoholic fatty liver disease. Fatty droplets at the level of hepatocytes
are clearly visible, both small and large. Fibrosis is present with thin scars which start to appear
around terminal hepatic vein.
301
Figure n.11
Figure n.12: in this picture all the histological characteristics of NASH are reported:
- Steatosis: macrovescicular, lipids push nucleus close to the membrane;
- Steatohepatitis: inflammation leads to hepatic degeneration. There’s
accumulation of fats, water and other proteins that leads to an hydropic
degeneration. Hepatocytes are also called balloon because they appear as big
cells, there’s membrane rupture too and appearance of Mallory-Denk bodies,
which are inclusions in hepatocytes;
- Fibrosis;
- Cirrhosis.
Figure 12
CIRRHOSIS OF THE LIVER
Introduction on chronic hepatitis
Cirrhosis is a degenerative disease of the liver, quite often the final histological condition of a
progressive chronic liver failure. With “chronic hepatitis” we mean all the situations characterised
by necrosis and inflammation over a long period of time, usually when the damages are present for
more than 6 months. In all or most of the cases, a chronic hepatitis (steatohepatitis or inflammation)
can evolve in cirrhosis.
Among the causes of chronic hepatitis there are:
- The persistence of viral hepatitis, mainly by HCV (hepatitis C virus) or HBV (hepatitis B virus);
- The alcoholic but also the non-alcoholic disease;
- The use of some drugs;
- Some systemic diseases, such as Wilson’s disease, a condition characterised by the
accumulation of copper (therefore, not fat in this case) in the cytosol of hepatocytes;
- Some diseases of autoimmune origin, for example lupus erythematosus;
302
- Some unknown causes, as in all situations.
In figure n.1 it is possible to note the difference,

from a morphological point of view, between an
acute and chronic hepatitis: in the case of acute
hepatitis, the inflammatory response is
reduced, so there is little portal infiltration of
mononuclear cells, while in the case of chronic
hepatitis the inflammatory response is stronger
and, in the area of the portal tract, there are an
important portal infiltrate, necrosis and
formation of fibrotic tissue. The presence of the
fibrotic tissue, in particular, is a specific marker
of chronic hepatitis.
We can distinguish two types of chronic
Figure 1
hepatitis.
The first one is known as persistent chronic hepatitis (figure n.2); in this case, there isn’t a
progression, inflammation and necrosis are minimal and more or less around the area of the portal
tract.
The second one is known as active chronic hepatitis (figure n.3); in this case necrosis and
inflammation are consistent and all the inflammatory cells, in particular lymphocytes but also
macrophages and neutrophils, from the portal tract can be also present in all the areas of the lobules.
Figure 2 Figure 3
This situation is, of course, more dangerous than the persistent chronic hepatitis, and can easily
evolve in cirrhosis and sometimes also in carcinoma.
Definition of cirrhosis
Cirrhosis is a diffused hepatic fibrosclerosis accompanied by irregular and insufficient hepatic
hyperplasia, so insufficient regeneration of hepatocytes.
303
In fact, an acute hepatitis and especially a chronic hepatitis (in particular, if it is an active chronic
hepatitis), are characterised by the presence of necrosis, meaning that the hepatocytes are dead
and there is inflammation. Of course, the liver tries to recover from this chronic regressive process
characterised by persistent necrosis of hepatocytes and persistent inflammation; so, the healthy
hepatocytes start proliferating91 to replace the dead ones in a process called hepatocellular
regeneration, which however is not sufficient: as explained more in detail later, the newly formed
hepatocytes are not organised and distributed to form a normal lobule;
Normally, hepatocytes are disposed in an organised fashion inside the lobules, forming a structure
similar to the spokes of a bicycle, starting from the central vein and moving towards the periphery
(figure n.4).
In liver regeneration, however,
hepatocytes will not distribute in this
organised way but will form the so-
called regenerative nodules or
pseudo-lobules, which are round
formations of hepatocytes, clearly
seen in figure n.5, which shows a
cirrhotic liver characterised by the
presence of regenerative nodules
Figure 4
already at the level of the surface of the
liver and also inside. The nodules are
surrounded by thick or thin bands of
scar and, as can be guessed from figure n.5, the morphology Figure 5
of the liver is totally changed, meaning that the liver is not able to carry out its
function, which results in liver failure.
As already mentioned, hepatocytes can proliferate, so it’s easy to substitute the lost volume but not
the normal morphology of the liver. Figure n.6
shows a regenerative nodule in which the central
vein is missing; as the name suggests, this is a
pseudo-lobule, so something similar to the lobe
but not the right one: besides the central vein (or
terminal hepatic vein), also the portal tracts are
missing92. All around the nodule there is fibrotic
tissue and then inflammation (all the small violet
spots in figure n.6 are inflammatory cells).
As easily imagined, the circulation is altered, and
the fibrotic tissue presses the hepatocytes, so the
structure of the liver is totally altered. Figure 6
Main causes of liver cirrhosis

The main causes of liver cirrhosis are:
- Alcohol abuse is the main cause: at least 60-70% of all cases of cirrhosis are due to alcohol
abuse;
- Chronic hepatitis induced by viral infection, especially HCV and HBV, or by some drugs or
some toxins;
- The non-alcoholic steatohepatitis, also called metabolic syndrome;
91
Hepatocytes are stable cells, therefore they don’t proliferate in a normal liver, but, if needed, they can start to
92
Portal tracts should be in the periphery while the central vein should be in the middle
304
- Some diseases of the biliary tract that can evolve;

- Some hereditary diseases, in which case there may be the accumulation in the cytosol of
hepatocytes of substances different than fats, like for instance iron in a hereditary disease
called hemochromatosis, copper in Wilson’s disease and an enzyme called α1-antitrypsin
which causes an abnormal protein synthesis. The dangerous effects of the accumulation of
these substances are similar to those due to the accumulation of fats;
- some unknown causes.
Viral infection
Only hepatitis B and C viruses (HCV and HBV) are responsible for chronic hepatitis, the hepatitis
A virus (HAV) is not involved in chronic hepatitis since the infection, in this case, never takes a
chronic course but is only acute, and the disease confers permanent immunity.
Concerning HBV, figure n.7 shows the possible courses of infection: first, there is the infection, from
which most of the patients can recover; however, sometimes the viral infection develops into an
acute infection: in most of the cases the patients recover, but in a small percentage of cases the
patients develop a chronic hepatitis. The individuals who develop a chronic hepatitis mainly act as
carriers of the virus but some of them, a small portion (10-30%), can evolve into cirrhosis. Finally, a
small number of these patients can develop carcinoma.
Figure 7
Pathogenesis of cirrhosis
Cirrhosis is a slow progressive process, it is a chronic disease that requires a very long period of
time before arriving to the typical histological picture.
305
Cirrhosis starts with a situation of fatty degeneration of hepatocytes, which is the accumulation of
fats in the cytosol of hepatocytes or, in the case of Wilson’s disease or haemochromatosis, it can
start with the accumulation of copper or iron; more generally, cirrhosis begins when something starts
Figure 8 to accumulate in the hepatocytes and disturbs them, causing their death. Steatosis starts mainly in
zone 3 and 2 of the lobule and, as already said, is still a reversible
situation/condition.
Then, because of fat, iron, copper or α1-antitrypsin accumulation
or because of any kind of toxin, hepatocytes start to die, which
causes necrosis and, as a consequence, an inflammatory
response characterised by the presence of a high number of
Kupffer cells93, neutrophils and lymphocytes, which start to
produce all the cytokines and other molecules necessary during
the inflammatory response. Colliquative necrosis is a type of
necrosis in which there is a total destruction of the cells,
membranes, proteins, etc. Figure n.8 shows hepatocellular
necrosis starting at the level of zone 3. In the upper part of figure n.8 there is an area where there is
normal tissue, normal hepatocytes and the central vein, while near the arrow there is an area of
extended necrosis.
After necrosis and inflammation, many hepatocytes are dead, the healthy ones start to proliferate,
because specific growth factors are released. However, the proliferation of hepatocytes, also
referred to as compensatory hyperplasia, is insufficient to substitute all the dead cells. As a result,
there is the formation of regenerative nodules or pseudo-lobules of different sizes, small or big,
and then at the same time fibrosis; the fibrotic tissue starts to appear thanks to the proliferation of
specific cells responsible for the synthesis and secretion of all the components of the ECM,
especially of collagen type I and III.
The final result is the loss of the normal architecture/morphology of the liver, which is occupied by
the regenerative nodules surrounded by fibrotic tissue and so by the dense bands of scar; thick
fibrosepta and regenerative nodules are finally responsible for the alteration of the hepatic
circulation.
If the hepatic circulation is heavily altered, the hepatocytes will not receive enough blood, oxygen
and nutrients; therefore, because of the fibrosis and of the consequently altered circulation, the new
hepatocytes and other hepatocytes will die too, because on one hand the healthy hepatocytes try to
substitute the dead ones but on the other the healthy hepatocytes are not able to receive blood,
nutrients and oxygen, which creates a state of hypoxia and so they are prone to die too.
93
The macrophages of the liver
306
Figure 9
To summarise, for instance in the case of alcohol abuse, the hepatocytes are damaged and start to
die; the degenerative process can start from a fatty liver but sometimes it can go directly from the
etiological agent to the necrosis of the hepatocytes, although usually fatty liver is the first step
followed by necrosis of hepatocytes and inflammation (figure n.9). Because of the inflammatory
response, there is the activation of Kupffer’s cells, which start to release all the mediators typical of
the inflammatory response, in particular various cytokines and growth factors (like TNFα and TGFβ,
figure n.9) able to attract other inflammatory cells in the area where the tissue is damaged; moreover,
these cells release TGFβ, the principle profibrogenic growth factor able to stimulate the so called Ito
cells or stellate cells. Stellate cells are transformed in myofibroblastic-like cells, so even if they are
not fibroblastic cells, they start to behave like them and start to synthesise and to secrete the
components of the ECM, especially collagen; therefore, as a consequence of the activation of these
cells, there is the formation of fibrotic tissue that can compromise the blood circulation inside the
liver: the presence of fibrotic tissue plus the regenerative nodules confer the typical histological
morphology of a cirrhotic liver.
These new nodules can have different sizes (big or small) based on which cirrhosis can be divided
into different types:
- Micronodular cirrhosis is characterised by the presence of small regenerative nodules and
a thin fibrotic tissue (the scar), so the connective tissue surrounding the nodules is quite thin.
This type is typical of alcoholic cirrhosis, primary and secondary biliary fibrosis, of
hemochromatosis94, or of Wilson’s disease cirrhosis;
- Macronodular cirrhosis, characterised by the presence of bigger nodules and a very thick
dense fibrotic tissue around the nodules. This type is often associated with active chronic
hepatitis, typical of viral infections;
- Mixed cirrhosis where both types regenerative nodules can be present.
94
Accumulation of iron
307
Figure n.10 shows how the liver looks when there is

the alteration of the normal architecture; in this case,
it is an example of alcoholic cirrhosis and it is possible
to see all the regenerative nodules, some big and
some small, surrounded by fibrous tissue (specifically,
in figure n.10 is the part stained in blue located all
around the nodules). In a situation like this, when the
liver is already cirrhotic, the accumulation of fats
inside the hepatocytes cannot be seen any longer.
Figure n.11 shows an example of macronodular
cirrhosis from a macroscopic point of view. Not only Figure 10
inside but also looking at the surface of the liver, it is
possible to see that the surface is not smooth, there are
several “ugly” nodules on the surface surrounded by fibrotic
tissue and inflammatory cells.
Consequences of liver cirrhosis
Since its architecture is totally altered, the liver is not able to
Figure 11
work in a proper way, eventually resulting in hepatic failure when the liver parenchyma is totally
destroyed.
Moreover, also all the blood circulation is altered, which is bad for the hepatocytes that cannot
survive without enough blood; therefore, necrosis, inflammation, regeneration and fibrosis95 keep
going on and, since it is not an acute damage but a chronic one, they persist for months or years as
a slow and progressive process.
95
Some moments later she says “fibrogenesis”
308
Another consequence of liver cirrhosis is the rise of the so-called hepatic jaundice or icterus; in
this case, the affected people’s skin starts to become yellow because their liver cannot work properly
creating, among other effects, troubles in formation of the bile.
Other problems and consequences depend on the affected person’s gender (so if the affected
individual is a man or a woman); for instance, in men some
spider angiomas, which are dilatated vessels, can appear
on the skin (figure n.12). Other possible consequences are
the gynecomastia, a condition in which men start to have
breasts, and palmar erythema, which is a local
vasodilatation at the level of the hands.
Women, on their part, can be affected by sterility or
oligomenorrhea.
Patients affected by cirrhosis can have troubles in the
coagulation process since the liver is responsible for the
synthesis of some of the factors involved in blood
coagulation, for instance there may be a deficiency in the
synthesis of vitamin K-dependent and independent
factors.
A serious risk for affected people is to develop bacterial
infections.
Figure 12
The risk of developing a hepatocellular carcinoma is
increased because cirrhosis is a condition
characterised by chronic damage and so it can be
considered a precancerosis.
Portal hypertension
As a consequence of portal hypertension, there may be an
accumulation of fluids inside the abdominal area which
creates a non-inflammatory oedema called ascites. Other consequences may be the creation of
collateral circulations called venous shunts, a congestive splenomegaly, i.e. the spleen becomes
bigger and quickly destroys the red blood cells (rbc) and, finally, a hepatic encephalopathy.
Portal hypertension arises when the blood is not able to reach the liver through the portal vein96, a
big and important vessel which carries most of the blood drained from the GI tract, pancreas and
spleen.
In normal conditions, the pressure in the portal vein is around 5-10 mmHg; it is possible to talk about
portal hypertension when the pressure is higher than 15 mmHg.
Hypertension can be due to different causes97:
- Pre-hepatic causes comprise, for instance, an obstructive thrombosis, in which a thrombus
can appear at the level of the portal vein blocking the blood flow and thus increasing the
pressure inside the vein, or structural abnormalities, in which the portal vein has an abnormal
structure;
- Intra-hepatic causes comprise cancer, liver cirrhosis, fibrosis, etc.
96
“Vena porta” because this vein drives the blood into the liver
97
The blood flow can be stopped before arriving to the liver, because there is something stopping it in the liver or it can
be blocked outside the liver
309
- Post-hepatic causes comprise, for instance, an obstructive thrombosis of the portal vein,
a sclerosis, a fibrosis or other structural abnormalities, so all the situations in which the
blood cannot flow in a correct way.
Figure 13
The principal cause of the hypertension is cirrhosis (a condition in which, indeed, as already
mentioned, the blood circulation is severely altered).
Clinical complications
Ascites
One of the main complications is the presence of an abnormal amount of fluid in the peritoneal cavity
which results in an extremely enlarged belly (seen in figure n.12); this situation is known as ascites
and the fluid that accumulates is called transudate98. It is important to note that the fluid accumulates
not because of inflammation but because of an increase in pressure inside the portal vein: the wall
of the vessels is normal, there isn’t any increase in the permeability, the liquid goes outside because
of the high pressure.
In our normal vessels there are two distinct types of pressure, the hydrostatic and oncotic pressures:
the hydrostatic pressure depends on the quantity of water and liquid, the oncotic pressure
depends on the presence of plasma proteins; in a physiological condition there is balance between
these two pressures.
In the case of a cirrhotic patient, the blood has difficulties in entering and going through the liver
because it is stopped at the level of the vein; this results in a bigger amount of blood that causes the
hydrostatic pressure to increase. However, the oncotic pressure decreases because the liver cannot
synthesise enough plasma proteins like albumin, globulin, fibrinogen and so on. If the oncotic
pressure is decreased, however, the two pressures are not in equilibrium and the fluid goes out
because of the high hydrostatic pressure. In fact, the fluid in the peritoneal cavity doesn’t contain
many plasma proteins besides a small amount of albumin, since it is the smallest of the plasma
proteins and so it can go outside, or cells99.
The fluid in excess can be removed by a medical doctor who must be sure to do it in a very aseptic
way because, otherwise, it is very easy to have infections since it is a very good ground for bacterial
growth; the presence of this fluid in the cavity as well can be a very good ground for bacterial
infection. This is a dangerous situation because, if the patient gets this type of infection, bacteria
can reach the blood and be distributed to the whole organism, causing death because of septic
shock.
98
It is the opposite of exudate, an accumulation of fluid present in the oedema of inflammatory origin; on the contrary,
transudate is defined as an oedema of non-inflammatory origin
99
Once again, the opposite of what is found in exudate
310
Of course, if water goes out of the vessels and

accumulates at the level of the belly, there is a
decrease in the blood volume; the kidneys recognise
the decrease in blood volume and will try to reabsorb
water and not eliminate it with the urine: systems are
activated to induce the absorption of sodium and so
retention of water (figure n.14). In this way, the blood
volume is restored to the normal value but then,
however, water will go out again creating a circle
(“un gatto che si mangia la coda100”) which results in
an increased accumulation of fluid in the abdominal
cavity.
Portosystemic shunts
Another clinical complication due to portal
Figure 14
hypertension is the formation of portosystemic shunts.
The blood from the vena porta cannot reach the liver in
a proper way because there is some trouble in or outside the liver. For instance, if you are in a
motorway and there is a traffic jam, what do you do? When you can, you go out and take another
route. Similarly, if the blood cannot go through the liver, other ways will be created because
otherwise, if the blood stops and stays inside, the vein can break; the only solution to get rid of the
blood is to form other connections to bypass the liver.
Nevo’s Question: are we talking about secretion of VEGF or just anastomose?
Prof: anastomoses are other vessels; you think that there is a release of VEGF, ah ok, yes, why not?
Figure n.15 shows a cirrhotic liver in which the
blood from the vena porta cannot go through;
therefore, collateral branches/connections, for
instance with the vena cava, are needed to
bypass the liver, remove the blood from the
portal vein and decrease the portal pressure.
However, the formation of new shunts has
some side effects.
One of the most dangerous and important side
effects which may even result in death of the Figure 15
patient is the formation of esophagogastric
varices (figure n.15); in this condition, the
oesophageal vein will dilatate to try to decrease
the pressure at the level of the portal vein since
the veins of the oesophagus are connected with the vena cava. However, food passing through the
oesophagus can mechanically cause the breakage of the hyperdilatated vessels: in fact, the main
risk related to this condition is for the patient to die because of a heavy haemorrhage resulting in
haemorrhagic shock101 (a lot of blood is lost, bp drops down and the patient collapses).
At the level of the rectum haemorrhoids can form; they can be painful, and they can bleed so often
these dilated veins need to be removed.
100
“A cat eating its tail” in English
101
Another type of shock that can result in the death of the patient: the first type was septic shock due to a bacterial
infection in the peritoneal cavity, the second one is haemorrhagic shock due to the breakage of esophagogastric varices
311
A less severe side effect is the formation of the so-called caput medusae around the umbilical area
(figure n.12); these are hyperdilatated veins that can be observed on the surface of the skin. The
name comes from the shape of the hair of a mythological creature, medusa.
Hepatic encephalopathy
The last very dangerous complication due to portal hypertension is the so-called hepatic
encephalopathy. This condition represents a very severe risk for the patient, who might go into
coma and die.
Since blood from the portal vein is not able to go through a cirrhotic liver, new anastomoses or
collateral branches will be created. However, as told before, the portal vein drains the blood from the
GI tract, where, especially in the intestine, a great amount of toxic compounds like ammonia is
produced as a consequence of the digestion of food.
One of the functions of the liver is to remove all the toxic metabolites/compounds; however, if the
blood is not able to go through the liver or if the liver stops working, the toxic compounds, of which
ammonia is the most dangerous one, will stay in the systemic circulation. In normal conditions,
ammonia is transformed into urea and then released thanks to the urine; if it stays in the systemic
circulation, it can reach the brain and accumulate there causing the so-called hepatic
encephalopathy.
The hepatic encephalopathy is a disorder of neurotransmission at the level of the CNS.
Depending on the gravity of the disorder, different degrees can be recognised:
- 1st degree is characterised by some disturb in the wake and sleep rhythm, depression or
euphoria (the patients are too happy), light confusion (the patients cannot concentrate very
well);
- 2nd degree is characterised by an accentuation of the symptoms with the appearance of
sleepiness (the patients cannot react, they only want to sleep), slow movements, confusion,
disorientation, etc.
- 3rd degree is characterised by a further accentuation of the symptoms, like deep sleepiness,
inconsistent speech (the patients start speaking in an inconsistent way), loss of the control of
the sphincters (patients have issues in retaining urine and faeces), hypertonia, etc;
- 4th degree is characterised by a deep coma, abnormal breath, muscular hypotonia (patients
cannot move or walk).
Hepatic encephalopathy exists in two forms:

- Acute form: the progression through the degrees is very rapid and death can occur in a few
days or weeks;
- Chronic form: progression from the 1st degree to the 4th degree is slower.
When the liver failure is very important, especially when the patient is at the stage 3 or 4 and clinical
symptoms are at their higher manifestation, the only solution is liver transplantation. Before arriving
to the liver transplantation, a solution is to reduce or avoid the presence of big amounts of toxic
substances in the blood; a way to achieve this is to change diet, for example reducing the protein
content (hypoproteic diet) to produce less ammonia and less toxic products; another way is to
empty the intestine of affected people putting them under laxative treatment: this will remove the
faeces from the intestine, not giving toxic compounds the time to be absorbed by the intestinal
mucosa and to reach the blood. Of course, quickly emptying the intestine creates the risk that the
necessary nutrients are not absorbed, so it is important to pay attention to maintain the correct
balance.
312

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Medicine & Surgery – MedinTo Università degli Studi di Torino

Academic Year 2019/2020

CELLULAR ADAPTATIONS TO STRESS

Cellular responses to stress and noxious stimuli

General understanding of cellular adaptations

Bizzozzero’s classification of cell types

Cell injury and cell death

In normal conditions cells are in a state of homeostasis

If the cell is unable to adapt to the stress it undergoes injury.

There are two different types of cell

Cell injury is characterized by:

The difference between hypertrophy and pseudo-hypertrophy

castone”) because of the following reasons:

- Hypertrophy of smooth muscle cells of the uterus during

If you were to compare, under a microscope, the

- Compensatory hypertrophy in paired organs.

- Hypertrophy of endocrine glands on which the adenohypophysis

The mechanisms by which cells become hypertrophic

What happens in a hypertrophic cell?

Protein degradation by the proteasome (inhibited in hypertrophic cells!)

Why is ubiquitination important?

E.g. The NFkB inhibitor, called IKB, is degraded after

Activities of proteasomes in given diseases

Autophagy (inhibited in hypertrophic cells!)

- Compensatory hyperplasia for liver regeneration

When a liver is donated from a dead individual, one can

Liver regeneration is composed of two main components:

- Compensatory marrow hyperplasia

The bone marrow is able to undergo rapid hyperplasia in response to a

- Lymphocytic hyperplasia in immune defense.

- Regenerative hyperplasia of the epidermis (wound repair, Psoriasis)

- Hyperplasia of the parathyroid glands as a consequence of decreased calcium.

- Gynecomastia due to increased estrogen levels.

- Hyperplasia due to chronic damage.

- Loss of innervation (denervation atrophy).

This individual had cardiovascular dementia as the loss of

- Loss of endocrine stimulation

Physiologic atrophy caused by a reduced functional demand

- Involution of embryonic structures after birth as seen with the cells in

Physiologic atrophy caused by reduced stimulation by hormones

Squamous metaplasia in the trachea of smokers

Barrett’s esophagus (Columnar metaplasia)

Also, in the case of

Mechanisms that occur in skeletal muscle

Role of Satellite Cells in muscle fiber growth

In the case of myocardial hypertrophy, mechanical stretch is detected by mechanical sensors

CELL DEATH PROCESSES

Causes of pathologic cell death (Necrosis or Apoptosis) are:

Morphological changes in necrotic cells

There are different types of necrosis:

Two examples showed in the pictures:

Biochemical mechanisms of cell death induced by ischemia

- Interruption of blood supply decreases delivery of

The colliquative tissue is invaded and removed by macrophages

The necrotic area in

Fat Necrosis (Steatonecrosis)

Apoptosis in pathological conditions can be stimulated or inhibited. Pathological states in which

- Accumulation of misfolded proteins. Improperly folded proteins may arise because of

The antioxidant system in humans