Proceedings of the National Academy of Sciences, 1984
Aminomalonic acid (Ama) has been isolated from proteins of Escherichia coli and human atheroscler... more Aminomalonic acid (Ama) has been isolated from proteins of Escherichia coli and human atherosclerotic plaque. The presence of Ama has important biological implications because the malonic acid moiety potentially imparts calcium binding properties to protein. Ama was obtained by anaerobic alkaline hydrolysis and identified by chromatographic behavior, quantitative acid-mediated decarboxylation to glycine, and unambiguous gas chromatographic/mass spectral detection. The chromatographic, chemical, and mass spectral properties of naturally occurring Ama were identical to those of the synthetic compound. Amino acid analysis and GC/mass spectrometry also revealed the presence of beta-carboxyaspartic acid and gamma-carboxyglutamic acid in the base hydrolysate of human atherosclerotic plaque. The ratio of Ama to beta-carboxyaspartic acid to gamma-carboxyglutamic acid was 20:1:10, and the quantity of Ama per 1,000 glycine residues was 0.2. Ama is a relatively unstable, minor amino acid in co...
ISSATISFACTION with currently marketed devices for the treatment of hydrocephalus has provided th... more ISSATISFACTION with currently marketed devices for the treatment of hydrocephalus has provided the impetus for the development of a new shunting system. Problems attendant to shunt placement are many, 2,3,s,1~ consisting primarily of: 1) erratic performance or blocking of the device in vivo, especially in the presence of particulate contamination or unusually elevated protein levels of the cerebrospinal fluid; 2) a significant incidence of infectious complications, conceivably related to nonautoclavable devices with metallic components or plastic devices with complex recessing; 5,9 and 3) the appreciable expense of the shunting device itself. The latter factor limits the availability of the operative procedure to a restricted population of patients within a favored social setting. Our objective has been to develop an inexpensive device of simple yet rugged construction, devoid of metallic parts, capable of passing fluid of high viscosity or particulate contamination without obstruction. This report deals with a description of a new shunting system, "'the Denver shunt"* designed to meet these specifications, and our initial surgical experiences with this device in 57 patients at the University of Colorado Medical Center over an 18-month period. Description and Flow Characteristics The bodies of the Denver shunt are molded and vulcanized in one piece from Silastic.t The proximal valve of the shunt is a simple slit contained within the molded part and analogous to a venous valve. The distal
Certain tumor cells have a remarkable viability despite the absence of glucose or oxygen, even in... more Certain tumor cells have a remarkable viability despite the absence of glucose or oxygen, even in the presence of cyanide (23). The ascites tumor cell, an extreme example, can remain viable for 24 hr without glucose or oxygen (18, 22). This unusual property has important implications for the in vivo existence of tumors and deserves intensive investigation. This is a quantitative study of changes in the metabolites of glycolysis in a series of intracranial tumors during pro longed anaerobic incubation. No opportunity was given during the incubation for gain or loss of metabolites from the environment. By measuring the levels of adenosine triphosphate (ATP), P-creatine, and certain substrates of the glycolytic system at intervals, an attempt has been made (a) to determine the rate of energy expenditure, (b) to identify the source or sources of high energy phosphate bond generation, and (c) to measure the length of time that significant levels of ATP are maintained under the status of complete ischemia. The investigation has defined more problems than it has resolved, but it does indicate that brain tumors as a group have some very unusual metabolic properties which merit full-scale investigation. MATERIALS AND METHODS SOURCE OF MATERIAL Thirteen human tumors and 3 experimentally induced tumors were studied. The tumors were arbitrarily divided 1 Supported by grant 2B-5221 from the American Cancer So ciety. 2 Research fellow under a training grant in Neuropharmacology of the National Institute of Neurological Diseases and Blindness of the USPHS.
Cell cultures were prepared from nine human brain tumors. Fibrin plate assays showed plasminogen-... more Cell cultures were prepared from nine human brain tumors. Fibrin plate assays showed plasminogen-dependent fibrinolytic activity in lysates and in material released by these neoplastic cells but not in those from normal adult human white matter. Antibodies against human urokinase caused catalytic inhibition of the urokinase and of the plasminogen activator from WI-38 cells, simian virus 40-transformed WI-38 cells, human prostatic cells, and human ovarian carcinoma cells. However, the anti-urokinase immunoglobulin G did not inhibit the plasminogen activator activity of any of the human brain tumor preparations. These studies indicate that the plasminogen activator produced by human brain tumor cells is antigenically different from the plasminogen activator of other human normal and neoplastic cells.
The function of the amyloid precursor protein (APP) in brain health remains unclear. This study e... more The function of the amyloid precursor protein (APP) in brain health remains unclear. This study elucidated a novel cytoprotective signaling pathway initiated by the APP transcriptionally active intracellular domain (AICD) in response to 27-hydroxycholesterol (27OHC), an oxidized cholesterol metabolite associated with neurodegeneration. The cellular response to 27OHC was hormetic, such that low, but not high, doses promoted AICD transactivation of microtubule associated serine/threonine kinase family member 4 (MAST4). MAST4 in turn phosphorylated and inhibited FOXO1-dependent transcriptional repression of rhotekin 2 (RTKN2), an oxysterol stress responder, to optimize cell survival. A palmitate-rich diet, which increases serum 27OHC, or APP ablation, abrogated this response in vivo. Further, this pathway was downregulated in human Alzheimer's Disease (AD) brains but not in frontotemporal dementia brains. These results unveil MAST4 as functional kinase of FOXO1 in a 27OHC AICD-driv...
The role of iron metabolism in Alzheimer's disease (AD) is well documented. Regulation of the pro... more The role of iron metabolism in Alzheimer's disease (AD) is well documented. Regulation of the proteins that maintain cellular iron metabolism is mediated by two cytoplasmic RNA-binding proteins, the Iron Regulatory Proteins (IRP1 and IRP2), that function through post-transcriptional interactions with RNA stem loop structures called iron-responsive elements. As the primary mediator of iron homeostasis in neuronal cells, IRP2 is a strong candidate for polymorphisms that could impact AD pathogenesis. Thus, we performed a pilot study to assess polymorphisms in the gene encoding IRP2 (IREB2) on clinically well-characterized, post-mortem samples (50 AD and 50 controls). DNA sequence analysis of the IREB2 gene region revealed 14 polymorphisms. Two (rs2656070 and rs13180) showed statistically significant skewing of allelic and genotypic distributions between AD patients and controls. In silico analyses revealed that rs2656070 lies within a probable promoter and disrupts the binding sites of at least two known transcription factors. Though silent and likely not functionally relevant, rs13180 is in complete LD with rs2656070 (D' > 0.999), creating an IREB2-haplotype that is significantly associated with AD. Confirmation of this association in a larger cohort of cases and controls would further support the role of iron regulation in the pathogenesis of this catastrophic and increasingly common neurodegenerative disorder.
Chitosan has great potential as a pharmaceutical excipient. In this study, chitosan flake was mic... more Chitosan has great potential as a pharmaceutical excipient. In this study, chitosan flake was micronized using cryo-ball and cryo-jet milling and subsequently sterilized with nitrogen plasma. Micronized chitosan was characterized by laser diffraction, scanning electron microscopy (SEM), conductometric titration, viscometry, loss on drying, FTIR, and limulus amebocyte lysate (LAL) assays. Cryo-jet milling produced mean particle size of 16.05μm, 44% smaller than cryo-ball milling. Cryomilled chitosan demonstrated increased hygroscopicity, but reduced molecular weight and degree of deacetylation (DD). SEM imaging showed highly irregular shapes. FTIR showed changes consistent with reduced DD and an unexplained shift at 1100cm(-1). Plasma treated chitosan was sterile with <2.5EU/g after low-pressure plasma and <1.3EU/g after atmospheric pressure plasma treatment. Plasma treatment decreased the reduced viscosity of chitosan flake and powder, with a greater effect on powder. In concl...
Base of Skull Surgery Exquisite surgical demands are imposed by manipulations within the anatomic... more Base of Skull Surgery Exquisite surgical demands are imposed by manipulations within the anatomic confines of the skull base. Vital structures must be precisely and confidently dissected, then rejoined under conditions of limited exposure. These circumstances can make conventional needle and suture approximation cumbersome and dangerous, particularly for attenuated dura, venous channels, and arteries. Thus, skull base reconstructions merit trials of alternative, nonsuture methods for tissue approximation.
Because of the growing impact of late onset cognitive loss, considerable effort has been directed... more Because of the growing impact of late onset cognitive loss, considerable effort has been directed toward the development of improved diagnostic techniques for Alzheimer's disease (AD) that may pave the way for earlier (and more effective) therapeutic efforts. Serum-based biomarkers are the least expensive and invasive modality for screening and routine monitoring. We systematically reviewed the literature to assemble a list of serum biomarkers relevant to AD. In parallel, we conducted a proteomic LC-MS/MS analysis of serum collected from neurologically normal subjects and subjects with mild cognitive impairment (MCI) and early AD (n = 6 in all). Complement C3 and alpha-2-macroglobulin were identified from both the literature review and our proteomic screen for further validation. For these two candidates, ELISA was performed on serum collected from a small independent cohort of subjects for longitudinal analysis. Serum was serially collected from neurologically normal subjects (...
Accumulation of amyloid-β (Aβ) peptide and the hyperphosphorylation of tau protein are major hall... more Accumulation of amyloid-β (Aβ) peptide and the hyperphosphorylation of tau protein are major hallmarks of Alzheimer's disease (AD). The causes of AD are not well known but a number of environmental and dietary factors are suggested to increase the risk of developing AD. Additionally, altered metabolism of iron may have a role in the pathogenesis of AD. We have previously demonstrated that cholesterol-enriched diet causes AD-like pathology with iron deposition in rabbit brain. However, the extent to which chelation of iron protects against this pathology has not been determined. In this study, we administered the iron chelator deferiprone in drinking water to rabbits fed with a 2% cholesterol diet for 12 weeks. We found that deferiprone (both at 10 and 50 mg/kg/day) significantly decreased levels of Aβ40 and Aβ42 as well as BACE1, the enzyme that initiates cleavage of amyloid-β protein precursor to yield Aβ. Deferiprone also reduced the cholesterol diet-induced increase in phosph...
Alzheimer's disease (AD) brain is marked by severe neuronal death which has been partly attri... more Alzheimer's disease (AD) brain is marked by severe neuronal death which has been partly attributed to increased oxidative stress. The pathophysiology accounting for this free radical injury is not well-delineated at this point, but one hypothesis is that a derangement in transition metal metabolism contributes to the process. We tested the hypothesis that peripheral derangement of transition metal metabolism is present early in the dementing process. We analyzed non-heme iron and copper levels in serum from subjects with normal cognition, mild cognitive impairment, and early stage senile dementia and followed these subjects over 5 years. An increase in the ratio of serum copper to non-heme iron levels predicted which subjects with mild cognitive impairment would progress to dementia versus those that would remain cognitively stable. This increase did not correlate with changes in expression of iron regulatory protein 2 or selected downstream targets in peripheral lymphocytes. A ...
Cerebral amyloid angiopathy (CAA) is a vascular lesion associated with Alzheimer's disease (A... more Cerebral amyloid angiopathy (CAA) is a vascular lesion associated with Alzheimer's disease (AD) present in up to 95% of AD patients and produces MRI-detectable microbleeds in many of these patients. It is possible that CAA-related microbleeding is a source of pathological iron in the AD brain. Because the homeostasis of copper, iron, and zinc are so intimately linked, we determined whether CAA contributes to changes in the brain levels of these metals. We obtained brain tissue from AD patients with severe CAA to compare to AD patients without evidence of vascular amyloid-β. Patients with severe CAA had significantly higher non-heme iron levels. Histologically, iron was deposited in the walls of large CAA-affected vessels. Zinc levels were significantly elevated in grey matter in both the CAA and non-CAA AD tissue, but no vascular staining was noted in CAA cases. Copper levels were decreased in both CAA and non-CAA AD tissues and copper was found to be prominently deposited on th...
One of the remaining challenges in Alzheimer's disease (AD) research is the establishment of ... more One of the remaining challenges in Alzheimer's disease (AD) research is the establishment of biomarkers for early disease detection. As part of a prospective study spanning a period of five years, we have collected serial serum samples from cognitively normal, mild cognitively impaired (MCI), and mild AD participants, including same patient samples before and after cognitive decline. Using mass spectrometry we identified several promising leads for biomarker development, such as prosaposin, phospholipase D1, biliverdin reductase B, and S100 calcium binding protein A7. Selected candidate markers were verified using reverse phase protein microarray assays. Of 15 protein/protein abundance ratios that were significantly altered in sera from subjects with mild AD compared to Normal or MCI subjects, 14 were composed of ratios containing heme oxygenase-1, biliverdin reductase A, or biliverdin reductase B. Moreover, an increase in the protein abundance ratio of matrix metallopeptidase 9...
A new iron sensitive MR sequence (susceptibility weighted imaging-SWI) enabling the simultaneous ... more A new iron sensitive MR sequence (susceptibility weighted imaging-SWI) enabling the simultaneous quantitation of regional brain iron levels and brain microbleeds (BMB) has been acquired serially to study dementia. Cohorts of mildly cognitively impaired (MCI) elderly (n=73) and cognitively normal participants (n=33) have been serially evaluated for up to 50 months. SWI phase values (putative iron levels) in 14 brain regions were measured and the number of brain microbleeds (BMB) were counted for each SWI study. SWI phase values showed a left putaminal mean increase of iron (decrease of phase values) over the study duration in 27 participants who progressed to dementia compared to Normals (p=0.035) and stable MCI (p=0.01). BMB were detected in 9 of 26 (38%) of MCI participants who progressed to dementia and are a significant risk factor for cognitive failure in MCI participants (risk ratio = 2.06 (95% confidence interval 1.37-3.12)). SWI is useful to measure regional iron changes and presence of BMB, both of which may be important MR based biomarkers for neurodegenerative diseases.
Dysfunctional homeostasis of transition metals is believed to play a role in the pathogenesis of ... more Dysfunctional homeostasis of transition metals is believed to play a role in the pathogenesis of Alzheimer's disease (AD). Although questioned by some, brain copper, zinc, and particularly iron overload are widely accepted features of AD which have led to the hypothesis that oxidative stress generated from aberrant homeostasis of these transition metals might be a pathogenic mechanism behind AD. This meta-analysis compiled and critically assessed available quantitative data on brain iron, zinc and copper levels in AD patients compared to aged controls. The results were very heterogeneous. A series of heavily cited articles from one laboratory reported a large increase in iron in AD neocortex compared to age-matched controls (p<0.0001) while seven laboratories failed to reproduce these findings reporting no significant difference between the groups (p=0.76). A more than threefold citation bias was found to favor outlier studies reporting increases in iron and this bias was particularly prominent among narrative review articles. Additionally, while zinc was not significantly changed in the neocortex (p=0.29), copper was significantly depleted in AD (p=0.0003). In light of these findings, it will be important to re-evaluate the hypothesis that transition metal overload accounts for oxidative injury noted in AD.
High-resolution susceptibility weighted MR imaging at high field strength provides excellent depi... more High-resolution susceptibility weighted MR imaging at high field strength provides excellent depiction of venous structures, blood products, and iron deposits, making it a promising complementary imaging modality for cerebral cavernous malformations (CCMs). Although already introduced in 1997 and being constantly improved, susceptibility weighted imaging is not yet routine in clinical neuroimaging protocols for CCMs. In this article, the authors review the recent literature dealing with clinical and scientific susceptibility weighted imaging of CCMs to summarize its prospects and drawbacks and provide their first experience with its use in ultra–high field (7-T) MR imaging.
Accumulating evidence implicates a role for altered iron and copper metabolism in the pathogenesi... more Accumulating evidence implicates a role for altered iron and copper metabolism in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). However, imbalances in the levels of the various forms of iron at different stages of AD have not been examined. In this pilot study we extracted and measured the levels of loosely bound, non-heme and total iron and copper in the frontal cortex and hippocampus of patients with mild-moderate AD (n = 3), severe AD (n = 8) and dementia with Lewy bodies (DLB, n = 6), using graphite furnace atomic absorption spectrometry (GFAAS). Additionally, the expression of iron regulatory protein 2 (IRP2) was examined in relation to the pathological hallmarks of AD and DLB, amyloid plaques, neurofibrillary tangles (NFT), and Lewy bodies, by immunohistochemistry. We found significantly decreased loosely bound iron in the hippocampal white matter of mild-moderate and severe AD patients and a trend towards increased non-heme iron in the hippocampal gray matter of severe AD patients. Furthermore, decreased levels of total copper were seen in severe AD and DLB frontal cortex compared to controls, suggesting an imbalance in brain metal levels in both AD and DLB. The decrease in loosely bound iron in mildmoderate AD patients may be associated with myelin breakdown seen in the beginning stages of AD and implicates that iron dysregulation is an early event in AD pathogenesis.
Alterations in the peripheral immune system are associated with dementia and the neuropathology o... more Alterations in the peripheral immune system are associated with dementia and the neuropathology of Alzheimer's disease, but have yet to be studied early in the disease process. To test the hypothesis that the balance of immune cell phenotypes is disrupted in the early progression of memory deterioration, patients with mild cognitive impairment (MCI) and healthy elderly controls were examined for the distribution of subpopulations of leukocytes (lymphocytes, granulocytes, and monocytes) and lymphocyte subtypes (helper/inducer and suppressor/cytotoxic T lymphocytes and B lymphocytes) in blood. MCI subjects had a significantly higher percentage of total lymphocytes and a lower percentage of granulocytes compared to elderly controls. Furthermore, the expression of cell surface amyloid precursor protein (APP) and intracellular amyloid-β peptide (Aβ) in lymphocytes and monocytes were determined. We found lymphocyte APP expression to be significantly increased in MCI subjects compared to controls. Our data indicate that changes in immunological parameters may be detected early in MCI, and an alteration of the immune response may precede clinical AD.
Proceedings of the National Academy of Sciences, 1984
Aminomalonic acid (Ama) has been isolated from proteins of Escherichia coli and human atheroscler... more Aminomalonic acid (Ama) has been isolated from proteins of Escherichia coli and human atherosclerotic plaque. The presence of Ama has important biological implications because the malonic acid moiety potentially imparts calcium binding properties to protein. Ama was obtained by anaerobic alkaline hydrolysis and identified by chromatographic behavior, quantitative acid-mediated decarboxylation to glycine, and unambiguous gas chromatographic/mass spectral detection. The chromatographic, chemical, and mass spectral properties of naturally occurring Ama were identical to those of the synthetic compound. Amino acid analysis and GC/mass spectrometry also revealed the presence of beta-carboxyaspartic acid and gamma-carboxyglutamic acid in the base hydrolysate of human atherosclerotic plaque. The ratio of Ama to beta-carboxyaspartic acid to gamma-carboxyglutamic acid was 20:1:10, and the quantity of Ama per 1,000 glycine residues was 0.2. Ama is a relatively unstable, minor amino acid in co...
ISSATISFACTION with currently marketed devices for the treatment of hydrocephalus has provided th... more ISSATISFACTION with currently marketed devices for the treatment of hydrocephalus has provided the impetus for the development of a new shunting system. Problems attendant to shunt placement are many, 2,3,s,1~ consisting primarily of: 1) erratic performance or blocking of the device in vivo, especially in the presence of particulate contamination or unusually elevated protein levels of the cerebrospinal fluid; 2) a significant incidence of infectious complications, conceivably related to nonautoclavable devices with metallic components or plastic devices with complex recessing; 5,9 and 3) the appreciable expense of the shunting device itself. The latter factor limits the availability of the operative procedure to a restricted population of patients within a favored social setting. Our objective has been to develop an inexpensive device of simple yet rugged construction, devoid of metallic parts, capable of passing fluid of high viscosity or particulate contamination without obstruction. This report deals with a description of a new shunting system, "'the Denver shunt"* designed to meet these specifications, and our initial surgical experiences with this device in 57 patients at the University of Colorado Medical Center over an 18-month period. Description and Flow Characteristics The bodies of the Denver shunt are molded and vulcanized in one piece from Silastic.t The proximal valve of the shunt is a simple slit contained within the molded part and analogous to a venous valve. The distal
Certain tumor cells have a remarkable viability despite the absence of glucose or oxygen, even in... more Certain tumor cells have a remarkable viability despite the absence of glucose or oxygen, even in the presence of cyanide (23). The ascites tumor cell, an extreme example, can remain viable for 24 hr without glucose or oxygen (18, 22). This unusual property has important implications for the in vivo existence of tumors and deserves intensive investigation. This is a quantitative study of changes in the metabolites of glycolysis in a series of intracranial tumors during pro longed anaerobic incubation. No opportunity was given during the incubation for gain or loss of metabolites from the environment. By measuring the levels of adenosine triphosphate (ATP), P-creatine, and certain substrates of the glycolytic system at intervals, an attempt has been made (a) to determine the rate of energy expenditure, (b) to identify the source or sources of high energy phosphate bond generation, and (c) to measure the length of time that significant levels of ATP are maintained under the status of complete ischemia. The investigation has defined more problems than it has resolved, but it does indicate that brain tumors as a group have some very unusual metabolic properties which merit full-scale investigation. MATERIALS AND METHODS SOURCE OF MATERIAL Thirteen human tumors and 3 experimentally induced tumors were studied. The tumors were arbitrarily divided 1 Supported by grant 2B-5221 from the American Cancer So ciety. 2 Research fellow under a training grant in Neuropharmacology of the National Institute of Neurological Diseases and Blindness of the USPHS.
Cell cultures were prepared from nine human brain tumors. Fibrin plate assays showed plasminogen-... more Cell cultures were prepared from nine human brain tumors. Fibrin plate assays showed plasminogen-dependent fibrinolytic activity in lysates and in material released by these neoplastic cells but not in those from normal adult human white matter. Antibodies against human urokinase caused catalytic inhibition of the urokinase and of the plasminogen activator from WI-38 cells, simian virus 40-transformed WI-38 cells, human prostatic cells, and human ovarian carcinoma cells. However, the anti-urokinase immunoglobulin G did not inhibit the plasminogen activator activity of any of the human brain tumor preparations. These studies indicate that the plasminogen activator produced by human brain tumor cells is antigenically different from the plasminogen activator of other human normal and neoplastic cells.
The function of the amyloid precursor protein (APP) in brain health remains unclear. This study e... more The function of the amyloid precursor protein (APP) in brain health remains unclear. This study elucidated a novel cytoprotective signaling pathway initiated by the APP transcriptionally active intracellular domain (AICD) in response to 27-hydroxycholesterol (27OHC), an oxidized cholesterol metabolite associated with neurodegeneration. The cellular response to 27OHC was hormetic, such that low, but not high, doses promoted AICD transactivation of microtubule associated serine/threonine kinase family member 4 (MAST4). MAST4 in turn phosphorylated and inhibited FOXO1-dependent transcriptional repression of rhotekin 2 (RTKN2), an oxysterol stress responder, to optimize cell survival. A palmitate-rich diet, which increases serum 27OHC, or APP ablation, abrogated this response in vivo. Further, this pathway was downregulated in human Alzheimer's Disease (AD) brains but not in frontotemporal dementia brains. These results unveil MAST4 as functional kinase of FOXO1 in a 27OHC AICD-driv...
The role of iron metabolism in Alzheimer's disease (AD) is well documented. Regulation of the pro... more The role of iron metabolism in Alzheimer's disease (AD) is well documented. Regulation of the proteins that maintain cellular iron metabolism is mediated by two cytoplasmic RNA-binding proteins, the Iron Regulatory Proteins (IRP1 and IRP2), that function through post-transcriptional interactions with RNA stem loop structures called iron-responsive elements. As the primary mediator of iron homeostasis in neuronal cells, IRP2 is a strong candidate for polymorphisms that could impact AD pathogenesis. Thus, we performed a pilot study to assess polymorphisms in the gene encoding IRP2 (IREB2) on clinically well-characterized, post-mortem samples (50 AD and 50 controls). DNA sequence analysis of the IREB2 gene region revealed 14 polymorphisms. Two (rs2656070 and rs13180) showed statistically significant skewing of allelic and genotypic distributions between AD patients and controls. In silico analyses revealed that rs2656070 lies within a probable promoter and disrupts the binding sites of at least two known transcription factors. Though silent and likely not functionally relevant, rs13180 is in complete LD with rs2656070 (D' > 0.999), creating an IREB2-haplotype that is significantly associated with AD. Confirmation of this association in a larger cohort of cases and controls would further support the role of iron regulation in the pathogenesis of this catastrophic and increasingly common neurodegenerative disorder.
Chitosan has great potential as a pharmaceutical excipient. In this study, chitosan flake was mic... more Chitosan has great potential as a pharmaceutical excipient. In this study, chitosan flake was micronized using cryo-ball and cryo-jet milling and subsequently sterilized with nitrogen plasma. Micronized chitosan was characterized by laser diffraction, scanning electron microscopy (SEM), conductometric titration, viscometry, loss on drying, FTIR, and limulus amebocyte lysate (LAL) assays. Cryo-jet milling produced mean particle size of 16.05μm, 44% smaller than cryo-ball milling. Cryomilled chitosan demonstrated increased hygroscopicity, but reduced molecular weight and degree of deacetylation (DD). SEM imaging showed highly irregular shapes. FTIR showed changes consistent with reduced DD and an unexplained shift at 1100cm(-1). Plasma treated chitosan was sterile with <2.5EU/g after low-pressure plasma and <1.3EU/g after atmospheric pressure plasma treatment. Plasma treatment decreased the reduced viscosity of chitosan flake and powder, with a greater effect on powder. In concl...
Base of Skull Surgery Exquisite surgical demands are imposed by manipulations within the anatomic... more Base of Skull Surgery Exquisite surgical demands are imposed by manipulations within the anatomic confines of the skull base. Vital structures must be precisely and confidently dissected, then rejoined under conditions of limited exposure. These circumstances can make conventional needle and suture approximation cumbersome and dangerous, particularly for attenuated dura, venous channels, and arteries. Thus, skull base reconstructions merit trials of alternative, nonsuture methods for tissue approximation.
Because of the growing impact of late onset cognitive loss, considerable effort has been directed... more Because of the growing impact of late onset cognitive loss, considerable effort has been directed toward the development of improved diagnostic techniques for Alzheimer's disease (AD) that may pave the way for earlier (and more effective) therapeutic efforts. Serum-based biomarkers are the least expensive and invasive modality for screening and routine monitoring. We systematically reviewed the literature to assemble a list of serum biomarkers relevant to AD. In parallel, we conducted a proteomic LC-MS/MS analysis of serum collected from neurologically normal subjects and subjects with mild cognitive impairment (MCI) and early AD (n = 6 in all). Complement C3 and alpha-2-macroglobulin were identified from both the literature review and our proteomic screen for further validation. For these two candidates, ELISA was performed on serum collected from a small independent cohort of subjects for longitudinal analysis. Serum was serially collected from neurologically normal subjects (...
Accumulation of amyloid-β (Aβ) peptide and the hyperphosphorylation of tau protein are major hall... more Accumulation of amyloid-β (Aβ) peptide and the hyperphosphorylation of tau protein are major hallmarks of Alzheimer's disease (AD). The causes of AD are not well known but a number of environmental and dietary factors are suggested to increase the risk of developing AD. Additionally, altered metabolism of iron may have a role in the pathogenesis of AD. We have previously demonstrated that cholesterol-enriched diet causes AD-like pathology with iron deposition in rabbit brain. However, the extent to which chelation of iron protects against this pathology has not been determined. In this study, we administered the iron chelator deferiprone in drinking water to rabbits fed with a 2% cholesterol diet for 12 weeks. We found that deferiprone (both at 10 and 50 mg/kg/day) significantly decreased levels of Aβ40 and Aβ42 as well as BACE1, the enzyme that initiates cleavage of amyloid-β protein precursor to yield Aβ. Deferiprone also reduced the cholesterol diet-induced increase in phosph...
Alzheimer's disease (AD) brain is marked by severe neuronal death which has been partly attri... more Alzheimer's disease (AD) brain is marked by severe neuronal death which has been partly attributed to increased oxidative stress. The pathophysiology accounting for this free radical injury is not well-delineated at this point, but one hypothesis is that a derangement in transition metal metabolism contributes to the process. We tested the hypothesis that peripheral derangement of transition metal metabolism is present early in the dementing process. We analyzed non-heme iron and copper levels in serum from subjects with normal cognition, mild cognitive impairment, and early stage senile dementia and followed these subjects over 5 years. An increase in the ratio of serum copper to non-heme iron levels predicted which subjects with mild cognitive impairment would progress to dementia versus those that would remain cognitively stable. This increase did not correlate with changes in expression of iron regulatory protein 2 or selected downstream targets in peripheral lymphocytes. A ...
Cerebral amyloid angiopathy (CAA) is a vascular lesion associated with Alzheimer's disease (A... more Cerebral amyloid angiopathy (CAA) is a vascular lesion associated with Alzheimer's disease (AD) present in up to 95% of AD patients and produces MRI-detectable microbleeds in many of these patients. It is possible that CAA-related microbleeding is a source of pathological iron in the AD brain. Because the homeostasis of copper, iron, and zinc are so intimately linked, we determined whether CAA contributes to changes in the brain levels of these metals. We obtained brain tissue from AD patients with severe CAA to compare to AD patients without evidence of vascular amyloid-β. Patients with severe CAA had significantly higher non-heme iron levels. Histologically, iron was deposited in the walls of large CAA-affected vessels. Zinc levels were significantly elevated in grey matter in both the CAA and non-CAA AD tissue, but no vascular staining was noted in CAA cases. Copper levels were decreased in both CAA and non-CAA AD tissues and copper was found to be prominently deposited on th...
One of the remaining challenges in Alzheimer's disease (AD) research is the establishment of ... more One of the remaining challenges in Alzheimer's disease (AD) research is the establishment of biomarkers for early disease detection. As part of a prospective study spanning a period of five years, we have collected serial serum samples from cognitively normal, mild cognitively impaired (MCI), and mild AD participants, including same patient samples before and after cognitive decline. Using mass spectrometry we identified several promising leads for biomarker development, such as prosaposin, phospholipase D1, biliverdin reductase B, and S100 calcium binding protein A7. Selected candidate markers were verified using reverse phase protein microarray assays. Of 15 protein/protein abundance ratios that were significantly altered in sera from subjects with mild AD compared to Normal or MCI subjects, 14 were composed of ratios containing heme oxygenase-1, biliverdin reductase A, or biliverdin reductase B. Moreover, an increase in the protein abundance ratio of matrix metallopeptidase 9...
A new iron sensitive MR sequence (susceptibility weighted imaging-SWI) enabling the simultaneous ... more A new iron sensitive MR sequence (susceptibility weighted imaging-SWI) enabling the simultaneous quantitation of regional brain iron levels and brain microbleeds (BMB) has been acquired serially to study dementia. Cohorts of mildly cognitively impaired (MCI) elderly (n=73) and cognitively normal participants (n=33) have been serially evaluated for up to 50 months. SWI phase values (putative iron levels) in 14 brain regions were measured and the number of brain microbleeds (BMB) were counted for each SWI study. SWI phase values showed a left putaminal mean increase of iron (decrease of phase values) over the study duration in 27 participants who progressed to dementia compared to Normals (p=0.035) and stable MCI (p=0.01). BMB were detected in 9 of 26 (38%) of MCI participants who progressed to dementia and are a significant risk factor for cognitive failure in MCI participants (risk ratio = 2.06 (95% confidence interval 1.37-3.12)). SWI is useful to measure regional iron changes and presence of BMB, both of which may be important MR based biomarkers for neurodegenerative diseases.
Dysfunctional homeostasis of transition metals is believed to play a role in the pathogenesis of ... more Dysfunctional homeostasis of transition metals is believed to play a role in the pathogenesis of Alzheimer's disease (AD). Although questioned by some, brain copper, zinc, and particularly iron overload are widely accepted features of AD which have led to the hypothesis that oxidative stress generated from aberrant homeostasis of these transition metals might be a pathogenic mechanism behind AD. This meta-analysis compiled and critically assessed available quantitative data on brain iron, zinc and copper levels in AD patients compared to aged controls. The results were very heterogeneous. A series of heavily cited articles from one laboratory reported a large increase in iron in AD neocortex compared to age-matched controls (p<0.0001) while seven laboratories failed to reproduce these findings reporting no significant difference between the groups (p=0.76). A more than threefold citation bias was found to favor outlier studies reporting increases in iron and this bias was particularly prominent among narrative review articles. Additionally, while zinc was not significantly changed in the neocortex (p=0.29), copper was significantly depleted in AD (p=0.0003). In light of these findings, it will be important to re-evaluate the hypothesis that transition metal overload accounts for oxidative injury noted in AD.
High-resolution susceptibility weighted MR imaging at high field strength provides excellent depi... more High-resolution susceptibility weighted MR imaging at high field strength provides excellent depiction of venous structures, blood products, and iron deposits, making it a promising complementary imaging modality for cerebral cavernous malformations (CCMs). Although already introduced in 1997 and being constantly improved, susceptibility weighted imaging is not yet routine in clinical neuroimaging protocols for CCMs. In this article, the authors review the recent literature dealing with clinical and scientific susceptibility weighted imaging of CCMs to summarize its prospects and drawbacks and provide their first experience with its use in ultra–high field (7-T) MR imaging.
Accumulating evidence implicates a role for altered iron and copper metabolism in the pathogenesi... more Accumulating evidence implicates a role for altered iron and copper metabolism in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). However, imbalances in the levels of the various forms of iron at different stages of AD have not been examined. In this pilot study we extracted and measured the levels of loosely bound, non-heme and total iron and copper in the frontal cortex and hippocampus of patients with mild-moderate AD (n = 3), severe AD (n = 8) and dementia with Lewy bodies (DLB, n = 6), using graphite furnace atomic absorption spectrometry (GFAAS). Additionally, the expression of iron regulatory protein 2 (IRP2) was examined in relation to the pathological hallmarks of AD and DLB, amyloid plaques, neurofibrillary tangles (NFT), and Lewy bodies, by immunohistochemistry. We found significantly decreased loosely bound iron in the hippocampal white matter of mild-moderate and severe AD patients and a trend towards increased non-heme iron in the hippocampal gray matter of severe AD patients. Furthermore, decreased levels of total copper were seen in severe AD and DLB frontal cortex compared to controls, suggesting an imbalance in brain metal levels in both AD and DLB. The decrease in loosely bound iron in mildmoderate AD patients may be associated with myelin breakdown seen in the beginning stages of AD and implicates that iron dysregulation is an early event in AD pathogenesis.
Alterations in the peripheral immune system are associated with dementia and the neuropathology o... more Alterations in the peripheral immune system are associated with dementia and the neuropathology of Alzheimer's disease, but have yet to be studied early in the disease process. To test the hypothesis that the balance of immune cell phenotypes is disrupted in the early progression of memory deterioration, patients with mild cognitive impairment (MCI) and healthy elderly controls were examined for the distribution of subpopulations of leukocytes (lymphocytes, granulocytes, and monocytes) and lymphocyte subtypes (helper/inducer and suppressor/cytotoxic T lymphocytes and B lymphocytes) in blood. MCI subjects had a significantly higher percentage of total lymphocytes and a lower percentage of granulocytes compared to elderly controls. Furthermore, the expression of cell surface amyloid precursor protein (APP) and intracellular amyloid-β peptide (Aβ) in lymphocytes and monocytes were determined. We found lymphocyte APP expression to be significantly increased in MCI subjects compared to controls. Our data indicate that changes in immunological parameters may be detected early in MCI, and an alteration of the immune response may precede clinical AD.
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Papers by Wolff Kirsch