Papers by Athanassios Kotsinas
Rationale: SARS-CoV-2 infection of the respiratory system can progress to a life threatening mult... more Rationale: SARS-CoV-2 infection of the respiratory system can progress to a life threatening multi-systemic disease, mediated via an excess of cytokines ("cytokine storm"), but the molecular mechanisms are poorly understood. Objectives: To investigate whether SARS-CoV-2 may induce cellular senescence in lung epithelial cells, leading to secretion of inflammatory cytokines, known as the senescence-associated secretory phenotype (SASP). Methods: Autopsy lung tissue samples from eleven COVID-19 patients and sixty age-matched non-infected controls were analysed by immunohistochemistry for SARS-CoV-2 and markers of cellular senescence (SenTraGor, p16INK4A) and key SASP cytokines (interleukin-1β, interleukin-6). We also investigated whether SARS-CoV-2 infection of an epithelial cell line induces senescence and cytokine secretion. Measurements and Main Results: SARS-CoV-2 was detected by immunocytochemistry and electron microscopy predominantly in alveolar type-2 (AT2) cells, whi...
Archivio italiano di urologia andrologia, Mar 4, 2024
World Journal of Gastroenterology, 2013
The E2F proteins comprise a family of 8 members that function as transcription factors. They are ... more The E2F proteins comprise a family of 8 members that function as transcription factors. They are key targets of the retinoblastoma protein (RB) and were initially divided into groups of activators and repressors. Accumulating data suggest that there is no specific role for each individual E2F member. Instead, each E2F can exert a variety of cellular effects, some of which represent opposing ones. For instance, specific E2Fs can activate transcription and repression, promote or hamper cell proliferation, augment or inhibit apoptosis, all being dependent on the cellular context. This complexity reflects the importance that these transcription factors have on a cell's fate. Thus, delineating the specific role for each E2F member in specific malignancies, although not easy, is a challenging and continuously pursued task, especially in view of potential E2F targeted therapies. Therefore, several reviews are continuously trying to evaluate available data on E2F status in various malignancies. Such reviews have attempted to reach a consensus, often in the simplistic form of oncogenes or tumor suppressor genes for the E2Fs. However they frequently miss spatial and temporal alterations of these factors during tumor development , which should also be considered in conjunction with the status of the regulatory networks that these factors participate in. In the current ''Letter to the Editor'', we comment on the flaws, misinterpretations and omissions in one such review article published recently in the World Journal of Gastroenterology regarding the role of E2Fs in digestive system malignancies.
with biotinylated horse anti-mouse IgG and the ABC-Elite reagent. In all cases 3,3 0diaminobenzid... more with biotinylated horse anti-mouse IgG and the ABC-Elite reagent. In all cases 3,3 0diaminobenzidine (with nickel chloride enhancement for SP-C and CC10 staining) was used as the chromagen and sections were counterstained with methyl green. Analysis of Env RNA expression in lung and airways Total RNA was isolated from lung, from trachea and from epithelial tissue scraped from the inside of the nose, by using a Polytron tissue homogenizer (Brinkmann) and Trizol RNA isolation reagent (Invitrogen). Samples were treated with DNase and with reverse transcriptase in the presence of a 3 0 Env primer; they were then subjected to 30 cycles of PCR amplification with primers flanking the intron in ARJenv (Fig. 1). Products were subjected to electrophoresis in agarose gels and were stained with ethidium bromide.
Genome Biology, Apr 28, 2022
Surgery, Apr 1, 2005
In the present study, we employed an elastase infusion-dependent abdominal aortic aneurysm (AAA) ... more In the present study, we employed an elastase infusion-dependent abdominal aortic aneurysm (AAA) model to examine inducible nitric oxide synthase (iNOS) expression in relation to cellular proliferation and apoptosis in this pathologic condition. Furthermore, we employed N-(3-(aminomethyl)benzyl)acetamidine (1400 W), a previously shown selective iNOS inhibitor, to further explore this relationship. Adult male Wistar rats were randomized into separate groups. Group A served as a control and received an intra-aortic saline infusion, while groups B, C, and D received an intra-aortic elastase infusion according to standard protocols. The animals in group C were administered postoperatively the highly selective iNOS inhibitor, 1400 W, while rats in group D received regularly the same compound preoperatively and postoperatively. The animals were killed at postoperative days 7 and 14. Aorta diameter and nitric oxide (NO), nitrite/nitrate, and MDA levels were measured. iNOS expression was assessed by immunohistochemistry and Western blot analysis, while Ki-67 immunohistochemistry and TUNEL assay were used to evaluate cellular proliferation and apoptosis, respectively. Increased iNOS and NO levels accompanied aneurysm development in groups B, C, and D, but these levels were significantly lower in groups C and D, compared with group B. Interestingly, very low but detectable levels of iNOS were found in the control group, indicating a basal constitutive level. Cell growth parameters were augmented in group B compared with group A. In contrast, groups C and D exhibited a significant decrease of the cellular growth parameters but did not attain normal values. iNOS-derived NO is associated with the cellular growth parameters of the vessel cells, predominantly smooth muscle cells. Selective iNOS blockage ameliorates the cellular remodeling in AAAs.
Journal of Vascular Surgery, Sep 1, 2010
Background: Oxidative stress is an important determinant in atherosclerosis development. Various ... more Background: Oxidative stress is an important determinant in atherosclerosis development. Various markers of oxidative stress, such as oxidation of low-density lipoprotein (LDL), nitrosative stress, lipid peroxidation, and protein oxidation, have been implicated in the initiation and/or progression of atherosclerosis, but their association with plaque erosion and symptomatic carotid disease has not been fully defined. In addition, certain oxidative markers have been shown in various models to promote plaque remodeling through matrix metalloproteinase (MMP) activation. Objective: To perform a global investigation of various oxidative stress markers and assess for potential relationships with destabilization and symptomatic development in human carotid plaques. Methods: Thirty-six patients undergoing endarterectomy were evaluated and compared with 20 control specimens obtained at the time of autopsy. Differences between stable and unstable plaques, symptomatic and asymptomatic patients, and >90% and <90% stenosis were evaluated. Oxidized LDL (ox-LDL), nitrotyrosine (NT), malondialdehyde (MDA), and protein carbonyls (PCs) levels were determined in atheromatic plaques homogenates by corresponding biochemical assays. Immunohistochemical (IHC) analysis was also employed to determine the percentage and topological distribution of cells expressing NT and metalloproteinase-9 (MMP-9) in serial sections from corresponding atheromatic plaques. MMP-9 expression was further verified using Western blot analysis. Results: Ox-LDL was increased in symptomatic patients (P < .05). Also, ox-LDL and NT levels were significantly higher in unstable versus stable carotid plaques (P < .05, respectively). Furthermore, IHC serial section analysis, corroborated by statistical analysis, showed a topological and expressional correlation between NT and MMP-9 (P < .05). MDA and PCs levels, although increased in carotid plaques, did not distinguish stable from unstable carotid plaques as well as symptomatic from asymptomatic patients with various degrees of stenosis. Conclusion: All types of investigated oxidative stress markers were significantly increased in human carotid plaques, but only ox-LDL levels were associated with clinical symptoms, while peroxynitrite products and MMP-9 were specifically related to plaque instability. (J Vasc Surg 2010;52:704-13.) Clinical Relevance: Our results suggest that specific oxidative factors should be taken into consideration as additional future potential markers that could be used to assist in prevention and therapeutic decision.
InTech eBooks, Nov 7, 2011
Springer eBooks, 2010
The rapid technological improvements in molecular medicine and biology during the last decades en... more The rapid technological improvements in molecular medicine and biology during the last decades enriched the armoury of research laboratories and offered new diagnostic tools to the clinician. New insights into the pathogenesis of the diseases and especially cancer, prognostic factors and new therapeutic approaches are the result of the implementation of these techniques. However, their growing complexity demands good knowledge regarding their use and the information they can provide to the clinician. The following chapter analyzes the available techniques for research and diagnostic use, emphasizing mainly on their rationale. Methodologies and available protocols are also provided, as well as their clinical applications.
PubMed, Nov 25, 1998
Time consuming classical diagnostic tests and the increasing incidence of tuberculosis epidemics ... more Time consuming classical diagnostic tests and the increasing incidence of tuberculosis epidemics have rendered the need for new more sensitive diagnostic tools, urgent. This study explored the possibility of a direct and rapid method for the identification and characterization of pathogenic typical and atypical species of mycobacteria from sputum, based on a multiplex PCR assay. Gene bank search on the mycobacterial genome revealed specific sequences that fulfilled the above set criteria. Two pairs of primers were used to amplify a 243 bp fragment of the gene encoding the immunogenic protein MPB 64 and a 133 bp fragment of the gene encoding the 65-KDa mycobacterial antigen. The first pair of primers was selected among others, to detect specifically bacteria of the M. tuberculosis complex, whereas the second, to detect in addition to the latter, those of the M. avium-intracellular complex. Our mutiplex PCR assay, detected and identified correctly, all the mycobacterium tuberculosis and M. avium-intracellulare complex strains provided on pure culture as controls with a sensitivity of 10(-3) colony forming units. Furthermore, by performing our assay on 55 sputum samples from patients with positive culture and Ziehl-Neelsen staining, we identified mycobacterial DNA sequences in all--39 samples with M. tuberculosis complex and 16 with M. avium-intracellulare complex. Out of 300 sputum specimens from patients with clinical evidence of tuberculosis, 149 were positive by our method (95 M. tuberculosis and 54 M. avium-intracellulare complex) whereas 157 samples (95 M. tuberculosis complex, 59 M. avium-intracellulare complex, 1 M. xenopi, and 2 that could not be positively identified) were culture positive and only 95 Ziehl Neelsen positive. These findings suggest that the method described can be applied on sputum, and can identify in one step strains of the M. tuberculosis and M. avium-intracellulare complex and has effectivness comparable to culture methodologies.
PubMed, May 5, 1999
Wild-type (wt) p53 is a tumor-suppressor protein which acts via transcriptional and transcription... more Wild-type (wt) p53 is a tumor-suppressor protein which acts via transcriptional and transcriptional-independent mechanisms. The transcriptional function of p53 is mediated by specific responsive elements (REs). The MDM2, WAF1/Cip1 and Bax genes possess p53REs and their activation by wt p53 induces cell cycle progression, arrest and programmed cell death (apoptosis), respectively. Mutations of the p53 gene are detected in more than 50% of the human malignant tumors. p53 mutants seem to have a more stable conformation and are suggested to exert dominant-negative inhibition of wt p53 in cells containing both wt and mutant (mt) alleles. However, recent studies show that certain mt p53 proteins posses a "gain of function" phenotype. In the present study, we examined the effects of the second most frequent p53 mutant R273H on the p53REs of the MDM2, WAF1/Cip1 and Bax genes in the H1299 non-small cell lung carcinoma cell line. Although mt p53 R273H alone was unable to bind and transactivate the corresponding p53REs, it enhanced the MDM2-p53RE mediated gene transcription of wt p53 (positive-dominant effect) and prevented the wt p53 transactivation of the p53REs of WAF1/Cip1 and Bax genes (negative-dominant effect). Our data suggest that in the appropriate environment, differential transcription of critical p53 target genes by certain p53 mutant proteins may illustrate another mechanism implicated in tumor development.
Frontiers in Genetics, Oct 17, 2017
PubMed, Apr 24, 2012
At the cellular level, free radicals are tightly controlled by an inducible antioxidant program, ... more At the cellular level, free radicals are tightly controlled by an inducible antioxidant program, since at low non-hazardous amounts they contribute to physiological signalling and homeostasis. However, high levels of oxidative stress promote the accumulation of damaged biomolecules, the impairment of cell signalling pathways and the increase of oncogenic hits. As the intracellular and extracellular levels of oxidative stress increase during ageing or in various diseases, so does the amount of damaged biomolecules, since the repair mechanisms are also targets of oxidative damage and thus become gradually ineffective over time. Depending on the severity of the biomolecular damage, the responses of normal human cells to oxidants may range from transient growth arrest to premature senescence, and even to cell death. Although some responses are clearly tumour suppressing (apoptosis), others may be potentially oncogenic as they combine damage accumulation with a retained ability for proliferation (transient growth arrest) or with inflammation (senescence, necrosis). This array of events significantly increases the likelihood of the appearance of tumour-initiating cells, which may then give rise to pre-neoplastic focal lesions and eventually to neoplasia. In the present manuscript, we will focus on the role of free radical-mediated biomolecular damage and inflammation in tumorigenesis.
Handbook of immunohistochemistry and in situ hybridization of human carcinomas, 2002
Current Medicinal Chemistry, Aug 13, 2015
Cardiovascular diseases (CVDs) have become a predominant cause of death worldwide. Although CVDs ... more Cardiovascular diseases (CVDs) have become a predominant cause of death worldwide. Although CVDs encompass a variety of pathological conditions that affect the heart and blood vasculature, they can be classified into two major groups according to their basic pathophysiologic mechanisms, namely atherosclerosis and aneurysm formation. Increasing evidence implicates micro-RNAs (miRNAs or miRs) as vital factors both in the initiation and progression processes of CVDs. Some miRs have a promoting role in CVD development, while others have a protective role. Today, miRs have been shown to be potential biomarkers for several types of CVDs, while strategies targeting miRs are under consideration as potential tools for exploitation in therapeutic approaches for CVD treatments. In this review, we present the available data on the involvement of miRs in CVDs, focusing on their role as regulators of the inflammatory process as an initiating and driving component of CVDs.
Bioengineered, Mar 1, 2012
American Journal of Pathology, Dec 1, 1998
The p16-pRb and p53-MDM2 pathways represent vital cell cycle checkpoints. Recent studies provide ... more The p16-pRb and p53-MDM2 pathways represent vital cell cycle checkpoints. Recent studies provide evidence that these pathways are directly linked via MDM2-pRb interaction and p53 suppression of the RB1 gene. In the present study we investigated the alterations of this G1 phase protein network using immunohistochemical and molecular methods in a series of 68 non-small-cell lung carcinomas (NSCLCs) and correlated the findings with clinicopathological features and prognosis of the patients. Aberrant expression (Ab) of p16 and pRb was observed in 33 (49%) and 27 (40%) of the carcinomas , respectively. Analysis of the region that encodes for p16 by deletion mapping , a polymerase chain reaction (PCR)based methylation assay and PCR single-strand conformation polymorphism (SSCP) analysis revealed that deletions and transcriptional silencing by methylation might represent the main mechanisms of CDKN2/p16 ink4a inactivation in NSCLCs. The results of deletion mapping also suggest that other tumor suppressor genes may reside at the 9p21-22 region, which encodes for CDKN2/MTS1/p16 ink4a , p14 ARF , and MTS2/p15 ink4b. In addition , microsatellite instability was observed with a frequency of 16% in the 9p21-22 chromosome area. Overexpression (P) of p53 and MDM2 proteins was found in 39 (58%) and 47 (70%) of the cases , respectively. A highly significant association was observed between p53 overexpression and p53 mutations (P ؍ 0.006). Statistical analysis of the expression patterns of the biologically relevant molecules (p16/pRb , p53/MDM2 , MDM2/pRb, and p53/pRb) showed coincident overexpression of p53 and MDM2 (P ؍ 0.04) and that abnormal pRb was correlated with elevated levels of MDM2 (P ؍ 0.013) and p53 (P ؍ 0.01), respectively. We suggest that deregulated expression of these molecules may act synergistically. An important finding of the study was that multiple impairments (three and four molecules affected) of the p16/pRb/p53/MDM2 network occurred in a large proportion (43%) of the carcinomas. This finding in addition to the absence of correlation with clinical stage of the tumors suggests that multiple hits of this network may be a relatively early event in the development of a subset of NSCLCs. The relationship between the factors examined in the present study, clinicopathological features, and survival of the patients did not reveal any significant correlations with the exception of smoking, which was associated with microsatellite alterations (loss of heterozygosity and microsatellite instability) at the 9p21-22 locus (P ؍ 0.04) and the immunophenotypes p53(P)/MDM2(P) (P ؍ 0.04) and p16(Ab)/pRb(Ab)/ p53(P)/MDM2(P) (P ؍ 0.03), respectively. We suggest that in a subset of NSCLCs, simultaneous deregulation of the members of this network may represent one way of initiating the oncogenic procedure whereas in other NSCLC subgroups alternative pathways may play this role.
The Journal of Pathology, Sep 5, 2000
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Papers by Athanassios Kotsinas