Dionysis S. Bonikos

Potential Role of Bcl-2 and Bax mRNA and Protein Expression in Chronic Hepatitis Type B and C: A Clinicopathologic Study

Bcl-2 oncoprotein regulates programmed cell death by providing a survival advantage to rapidly proliferating cells, and bax protein promotes apoptosis by enchanting cell susceptibility to apoptotic stimuli. In this study, we assessed the expression of bcl-2 and bax in liver biopsies from patients with chronic hepatitis (CH) Type B (HBV) and C (HCV). The study comprised 65 liver biopsies from 65 patients with HBV (n = 37) and HCV (n = 28) and 10 normal liver biopsies as controls. The HAI score ranged from 3/18–13/18, and the fibrosis Stage, from 1–6 (7 HBV/10 HCV). Pathologic examination included the following: (1) immunohistochemical stains in paraffin sections for bcl-2 and bax protein expression, (2) Western blot analysis (bcl-2 and bax protein levels evaluation), (3) ISH (detection of bcl-2 and bax mRNA), and (4) ISH (TUNEL-ABI [apoptotic body index]). In CH cases, both bcl-2 and bax protein and mRNA were detected in portal and intralobular lymphocytes and in cholangiolar epithelial cells in interface areas and fibrous bands. Bax protein and mRNA was expressed within hepatocytes and epithelial cells of interlobular ducts in portal tracts. Bcl-2 mRNA was present in periportal hepatocytes only in cases with Stage 5–6 fibrosis. Western blot analysis showed a decreased bcl-2 and an increased bax expression toward advanced fibrotic stages. In CH cases, ABI was reverse correlated with the percentage of bcl-2 expression and was correlated directly with the percentage of bax expression (P < .001). The results of this study suggest that in cases of chronic HBV or HCV infection, bax may be involved in the hepatocyte cycle regulation during infection, whereas its expression in intraportal bile duct epithelium implies that this protein enhances susceptibility of these particular cells to apoptosis. The increased bax expression and ABI in fibrosis Stages 1–5, imply that they are responsible for hepatocytes depletion through apoptosis, during progress of liver fibrosis and fibrous tissue accumulation, until cirrhosis is established. Bcl-2 mRNA expression in periportal hepatocytes only in Stages 5 and 6 suggests that this oncogene is involved in the late stages of progressive liver fibrosis and failure and furthermore that periportal hepatocytes are resistant to apoptosis. Bcl-2 expression, in cholangioles of interface area, suggests that this oncoprotein may be involved in growth regulation of these epithelial cells. Further research is warranted to specify the exact role of apoptosis and apoptotic genes involved in liver fibrosis process in cases of chronic HBV and HCV infection. This may lead to new strategies in the management of human liver disease to prevent the progression to chronic liver failure.

The potential role of TGFbeta1, TGFbeta2 and TGFbeta3 protein expression in colorectal carcinomas. Correlation with classic histopathologic factors and patient survival.

Purpose:This study investigates the expression of tumor growth factors TGFβ1, TGFβ2 and TGFβ3 in tissue material from patients with colorectal carcinoma and evaluates their correlation with known prognostic markers and patient survival.Patients and Methods:The study included 124 patients with colorectal carcinoma. According to the TNM classification of malignant tumors, 26 tumors were identified as being stage I, 30 stage II, 48 stage III, and 20 stage IV, whereas 106 tumors were low-grade and 18 high-grade malignancies. On paraffin sections, the streptavidin-biotin technique using antibodies against TGFβ1, TGFβ2 and TGFβ3 was applied. Morphological and immunohistochemical results were correlated with clinicopathologic parameters.Results:TGFβ1 protein was expressed in 88 out of 124 (71%) carcinomas, whereas TGFβ2 and TGFβ3 proteins were detected in all tumors examined. Normal colonic mucosal epithelial cells expressed TGFβ2 (significantly less as compared to neoplastic cells; p < 0.01) and TGFβ3 (p > 0.05 compared to neoplastic cells), but not TGFβ1. Statistical analysis revealed a higher expression of TGFβ1 in low-grade carcinomas (p = 0.009) and a higher presence of TGFβ2 in advanced tumors (p = 0.008). TGFβ1 expression was related with increased disease-free and overall survival (p < 0.05 each). The presence of TGFβ2 was correlated with worse prognosis (p < 0.05). Cox analysis revealed that besides tumor grade and stage, TGFβ1 expression constituted an independent prognostic factor.Conclusion:This study shows that in adenocarcinomas of the colon, there is a differential expression of TGFβ1, TGFβ2 and TGF3. TGFβ1 may be implicated in the pathogenesis of these tumors, since it is expressed only in neoplastic but not in normal cells. TGFβ1 is related with an increased disease-free and overall survival and constitutes an independent prognostic factor. In advanced stages, TGFβ2 seems to be involved in tumor progression and is related with worse prognosis.Ziel:Diese Studie untersuchte die Expression der Tumorwachstumsfaktoren TGFβ1, TGFβ2 and TGFβ3 in Gewebeproben von Patienten mit kolorektalen Karzinomen und prüfte ihre Korrelation mit bekannten prognostischen Markern und mit dem Überleben der Patienten.Patienten und Methodik:Die Studie umfasste 124 Patienten mit kolorektalen Karzinomen. Nach der TNM-Klassifikation wurden 26 Tumoren als Stadium I, 30 als Stadium II, 48 als Stadium III und 20 als Stadium IV eingeordnet, während 106 Tumoren Low-Grade- und 18 High-Grade-Malignome waren. Paraffinschnittpräparate wurden nach der Streptavidin-Biotin-Methode mit Antikörpern gegen TGFβ1, TGFβ2 und TGFβ3 behandelt. Die morphologischen und immunhistochemischen Befunde wurden mit klinisch-pathologischen Parametern korreliert.Ergebnisse:TGFβ1 wurde in 88 von 124 (71%) Karzinomen exprimiert, während TGFβ2 und TGFβ3 in allen untersuchten Tumoren gefunden wurden. Normale Epithelzellen der Dickdarmschleimhaut exprimierten TGFβ2 (signifikant weniger verglichen mit neoplastischen Zellen; p < 0,01) und TGFβ3 (p > 0,05 verglichen mit neoplastischen Zellen), aber kein TGFβ1. Die statistische Analyse ergab stärkere TGFβ1-Expression in Low-Grade-Karzinomen (p = 0,009) und eine verstärkte Präsenz von TGF2 in fortgeschrittenen Tumoren (p = 0,008). Die TGFβ1-Expression korrelierte mit verlängertem krankheitsfreien und Gesamtüberleben (jeweils p < 0,05). Das Vorliegen von TGFβ2 korrelierte mit schlechterer Prognose (p < 0,05). Die Cox-Analyse ergab, dass neben Tumorgrad und -stadium die TGFβ1-Expression einen unabhängigen prognostischen Faktor darstellte.Schlussfolgerung:Diese Studie zeigt für Adenokarzinome des Kolons und Rektums Unterschiede in der Expression von TGFβ1, TGFβ2 und TGFβ3. TGFβ1 könnte in der Pathogenese dieser Tumoren eine Rolle spielen, da es nur in neoplastischen, nicht aber in normalen Zellen exprimiert wird. TGFβ1 geht mit verlängertem krankheitsfreien und Gesamtüberleben einher und ist ein unabhängiger prognostischer Faktor. In fortgeschrittenen Stadien scheint TGFβ2 für die Tumorprogression relevant zu sein und ist mit einer schlechteren Prognose verbunden.

The potential role of bcl-2, bax, and Ki67 expression in thymus of patients with myasthenia gravis, and their correlation with clinicopathologic parameters

OBJECTIVE The aim of this study was to evaluate bcl-2, bax (apoptotic-oncoproteins), and Ki67 (cell proliferation-marker) expression in thymus of patients with myasthenia gravis (MG) and to determine the potential correlation with clinicopathologic parameters. METHODS The study was done on 38 patients (16 males, 22 females; mean age 38+/-10 years) with MG who underwent modified maximal thymectomy (MMT). Clinical staging (Osserman classification) included stage I in three, IIA in 19, IIB in 13 and III in three. Microscopic examination of thymus showed thymic hyperplasia in 19, atrophy in eight, thymoma in nine and thymic carcinoma in two. On paraffin sections, the streptavidin-biotin technique, using antibodies to bcl-2, bax, and Ki67, was employed, and in situ hybridization with digoxigenin-labeled probes to bcl-2 and bax was performed. In addition, the apoptotic body index (ABI) was assessed via the TUNEL method. Staining results were correlated with clinicopathologic parameters. RESULTS Bcl-2 expression was higher in hyperplasia and thymoma cases, compared to thymic carcinomas (P<0.001). Higher expression in carcinomas, compared to hyperplasia and thymomas, was observed for bax (P<0.001), Ki67 (P<0.001) and ABI (P<0.001). Statistical analysis demonstrated: (A) positive correlation of bax+ cells with MG stage (P<0.001), ABI and %Ki67+ cells with MG stage (P<0.001, respectively), %Ki67+ and %bax+ cells with ABI (P<0.05); and (B) reverse correlation between %bcl-2+ cells and MG stage (P<0.05). CONCLUSIONS In patients with MG who underwent MMT, bcl-2, bax, and Ki67 expression correlates positively or reversibly with the microscopic findings of thymus. Increased apoptosis and proliferation accompany advanced disease stage and possible worse prognosis.

The Potential Role of TGFβ1, TGFβ2 and TGFβ3 Protein Expression in Colorectal Carcinomas

Purpose:This study investigates the expression of tumor growth factors TGFβ1, TGFβ2 and TGFβ3 in tissue material from patients with colorectal carcinoma and evaluates their correlation with known prognostic markers and patient survival.Patients and Methods:The study included 124 patients with colorectal carcinoma. According to the TNM classification of malignant tumors, 26 tumors were identified as being stage I, 30 stage II, 48 stage III, and 20 stage IV, whereas 106 tumors were low-grade and 18 high-grade malignancies. On paraffin sections, the streptavidin-biotin technique using antibodies against TGFβ1, TGFβ2 and TGFβ3 was applied. Morphological and immunohistochemical results were correlated with clinicopathologic parameters.Results:TGFβ1 protein was expressed in 88 out of 124 (71%) carcinomas, whereas TGFβ2 and TGFβ3 proteins were detected in all tumors examined. Normal colonic mucosal epithelial cells expressed TGFβ2 (significantly less as compared to neoplastic cells; p < 0.01) and TGFβ3 (p > 0.05 compared to neoplastic cells), but not TGFβ1. Statistical analysis revealed a higher expression of TGFβ1 in low-grade carcinomas (p = 0.009) and a higher presence of TGFβ2 in advanced tumors (p = 0.008). TGFβ1 expression was related with increased disease-free and overall survival (p < 0.05 each). The presence of TGFβ2 was correlated with worse prognosis (p < 0.05). Cox analysis revealed that besides tumor grade and stage, TGFβ1 expression constituted an independent prognostic factor.Conclusion:This study shows that in adenocarcinomas of the colon, there is a differential expression of TGFβ1, TGFβ2 and TGF3. TGFβ1 may be implicated in the pathogenesis of these tumors, since it is expressed only in neoplastic but not in normal cells. TGFβ1 is related with an increased disease-free and overall survival and constitutes an independent prognostic factor. In advanced stages, TGFβ2 seems to be involved in tumor progression and is related with worse prognosis.Ziel:Diese Studie untersuchte die Expression der Tumorwachstumsfaktoren TGFβ1, TGFβ2 and TGFβ3 in Gewebeproben von Patienten mit kolorektalen Karzinomen und prüfte ihre Korrelation mit bekannten prognostischen Markern und mit dem Überleben der Patienten.Patienten und Methodik:Die Studie umfasste 124 Patienten mit kolorektalen Karzinomen. Nach der TNM-Klassifikation wurden 26 Tumoren als Stadium I, 30 als Stadium II, 48 als Stadium III und 20 als Stadium IV eingeordnet, während 106 Tumoren Low-Grade- und 18 High-Grade-Malignome waren. Paraffinschnittpräparate wurden nach der Streptavidin-Biotin-Methode mit Antikörpern gegen TGFβ1, TGFβ2 und TGFβ3 behandelt. Die morphologischen und immunhistochemischen Befunde wurden mit klinisch-pathologischen Parametern korreliert.Ergebnisse:TGFβ1 wurde in 88 von 124 (71%) Karzinomen exprimiert, während TGFβ2 und TGFβ3 in allen untersuchten Tumoren gefunden wurden. Normale Epithelzellen der Dickdarmschleimhaut exprimierten TGFβ2 (signifikant weniger verglichen mit neoplastischen Zellen; p < 0,01) und TGFβ3 (p > 0,05 verglichen mit neoplastischen Zellen), aber kein TGFβ1. Die statistische Analyse ergab stärkere TGFβ1-Expression in Low-Grade-Karzinomen (p = 0,009) und eine verstärkte Präsenz von TGF2 in fortgeschrittenen Tumoren (p = 0,008). Die TGFβ1-Expression korrelierte mit verlängertem krankheitsfreien und Gesamtüberleben (jeweils p < 0,05). Das Vorliegen von TGFβ2 korrelierte mit schlechterer Prognose (p < 0,05). Die Cox-Analyse ergab, dass neben Tumorgrad und -stadium die TGFβ1-Expression einen unabhängigen prognostischen Faktor darstellte.Schlussfolgerung:Diese Studie zeigt für Adenokarzinome des Kolons und Rektums Unterschiede in der Expression von TGFβ1, TGFβ2 und TGFβ3. TGFβ1 könnte in der Pathogenese dieser Tumoren eine Rolle spielen, da es nur in neoplastischen, nicht aber in normalen Zellen exprimiert wird. TGFβ1 geht mit verlängertem krankheitsfreien und Gesamtüberleben einher und ist ein unabhängiger prognostischer Faktor. In fortgeschrittenen Stadien scheint TGFβ2 für die Tumorprogression relevant zu sein und ist mit einer schlechteren Prognose verbunden.

Apoptosis and Myofibroblast Expression in Human Glomerular Disease: A Possible Link with Transforming Growth Factor-Beta-1

Background/Aims: The pathophysiological pathways involved in the pathogenesis and evolution of renal fibrosis, have not been fully elucidated. Transforming growth factor-beta1 (TGF-β1) is involved in the development of renal scarring. Apoptosis is responsible for intrinsic cell deletion observed in end-stage kidney disease. Myofibroblasts are involved in the development of renal fibrosis. This study investigates whether there is a potential relationship between apoptosis, myofibroblast infiltration and TGF-β1 expression in the kidney of patients with glomerulonephritis (GN). Methods: Forty patients with various types of GN were included in the study. Myofibroblasts and TGF-β1 positive cells were detected in kidney biopsies by immunohistochemistry, while apoptotic cells were detected by the in situ end labelling of fragmented DNA. Results: Myofibroblasts were identified in the glomeruli of some patients with severe mesangioproliferative GN and glomerulosclerosis but a more intensive myofibroblast expression was found in the renal interstitium. TGF-β1 was expressed in the cytoplasm of tubular epithelial cells, in the renal interstitium and in the glomeruli of patients with GN. Apoptotic cells were mainly detected in the tubules and interstitium and were present in areas with intense myofibroblast infiltration. Positive correlations were observed between the intensity of myofibroblast expression in the interstitium and apoptosis in the tubulointerstitial area (r = 0.521, p < 0.01) as well as TGF-β1 expression (r = 0.462, p < 0.05) and degree of renal impairment (r = 0.430, p < 0.05). Conclusions: These observations suggest that myofibroblast infiltration and apoptosis along with TGF-β1 expression are associated with the development of interstitial fibrosis in patients with glomerular disease.

Immunohistochemical expression of TGF-β1, p21WAF1, p53, Ki67, and angiogenesis in gastric carcinomas

AbstractBackground: Transforming growth factors-beta (TGF-βs) are multifunctional polypeptides with crucial role as regulators of cellular growth and differentiation. It has been reported that TGF-β1 plays a biphasic action on tumorigenesis thus inducing or inhibiting malignant properties of the epithelial cells. Methods: TGF-β1 expression was analyzed in 56 patients with gastric carcinoma by immunohistochemical methods and compared with the expression of p21, p53, and Ki67, as well as with angiogenesis. The correlation of these markers with clinicopathological parameters was also evaluated. Results: TGF-β1 expression was detected in 71% of tumors and was more frequent in adenocarcinomas of the intestinal type (p<0.001). Positivity of p21WAF1, and p53 was observed in 32% and 51% of the tumors, respectively. A high Ki67 proliferating index was detected in 53.5% of the tumors. TGF-β1 expression was significantly correlated with p21 expression (p<0.001) and was inversely correlated with microvessel density. p21 expression was also higher in tumors with low proliferating index (p<0.01). There was no apparent correlation between the expression of these markers and tumor stage, depth of invasion, or lymphnode metastases. Conclusion: The findings show that TGF-β1 may be involved in the activation of the cdk inhibitor p21WAF1 in gastric adenocarcinomas, suggesting p53-independent induction of p21 in gastric cells. TGF-β1 does not seem to contribute to the alteration of the angiogenic status of these tumors.

Expression of Apoptosis‐Related Proteins Bcl‐2 and Bax Along with Transforming Growth Factor (TGF‐β1) in the Kidney of Patients with Glomerulonephritides

Background. Apoptosis, a gene‐directed form of cell death, has been involved in the resolution of renal injury but also in the development of scarring. Bcl‐2 and bax are proteins related to apoptotic process that either provides a survival advantage to rapidly proliferating cells (bcl‐2) or promote cell death by apoptosis (bax). Various cytokines and growth factors are involved in this process. This study investigates the expression of bcl‐2 and bax and the presence of apoptotic bodies in relation to the TGF‐β1 expression at the time of diagnosis in the renal biopsies of patients with glomerulonephritis (GN). Methods. Fifty patients with various types of GN and ten controls were included in the study. Bcl‐2, bax and Transforming Growth Factor (TGF‐β1) positive cells were detected in kidney biopsies by immunohistochemistry, while apoptotic cells were detected by in situ end labeling of fragmented DNA (ISEL). Morphometric analysis was used for quantitation of immunostaining. Results. The intensity of bcl‐2, bax and TGF‐β1 immunostaining in the renal tissue of patients with GN was significantly more to the observed in the control biopsies. Bcl‐2 and bax were expressed within the epithelial cells of proximal, distal and collecting tubules and in the renal interstitium. Bax and bcl‐2 proteins were also identified within the glomeruli in a few patients but their distribution was not related to the type of GN. TGF‐β1 was expressed in the cytoplasm of tubular epithelial cells and to a lesser extent in the renal interstitium and glomeruli. A positive correlation of TGF‐β1 with the extent of bax immunostaining (r = 0.498, p < 0.05) and an inverse correlation with that of bcl‐2 (r = − 0.490, p < 0.05) were identified. Apoptotic bodies were identified only in the renal tissue of patients with GN and were mainly localized among tubular epithelial and interstitial cells. Conclusion. The intensity of bcl‐2 and bax proteins expression and the presence of apoptotic bodies in the renal tissue of patients with GN suggest that apoptotic process is ongoing during the evolution of renal disease. The correlation of TGF‐β1 expression with that of apoptosis‐related proteins might represent an implication of this growth factor with apoptotic process in the human diseased kidney.

Trends of Mortality Rates During the Last Thirty Years in Greece

General mortality rates and specific mortality rates by major causes of death (coronary disease, cancer, tuberculosis, traffic accidents, suicide, vascular disorders of central neural system) in Greece have been studied throughout the 30-year period 1967–1996 and time trends have being calculated by nonlinear models of trend analysis. The results confirm the general pattern of decrease of mortality in Greece during the particular period of time as a result of the combination of the impact of two controversial trends: on one hand the decrease of infant mortality and mortality from infectious disorders and on the other the relatively slower and more gradual increase in mortality rates by disorders related to the western lifestyle culture, such as cardiovascular disorders and cancer. During the period under study in Greece the impact of the second trend seems to inflict less severe implication in the formulation of the pattern of general mortality especially as women are concerned. This statistically significant and gradually increasing difference in mortality between men and women in Greece has been confirmed in general mortality time trends as well as in the evaluation of the relatively better pattern of mortality trends of women from particular “western lifestyle” disorders.

Coordinated Increased Expression of Cyclooxygenase2 and Nuclear Factor κB Is a Steady Feature of Urinary Bladder Carcinogenesis

Objectives. The inescapable relationship between chronic inflammation and carcinogenesis has long been established. Our objective was to investigate COX-2 and NF-κB immunohistochemical expression in a large series of normal epithelium and bladder carcinomas. Methods. Immunohistochemical methodology was performed on formalin-fixed, paraffin-embedded sections from urinary bladder carcinomas of 140 patients (94 males and 46 females with bladder carcinomas). Results. COX-2 expression is increased in the cytoplasm of bladder cells, during loss of cell differentiation (r s = 0.61, P-value < .001) and in muscle invasive carcinomas (P-value < .001). A strong positive association between tumor grade and nuclear expression of NFκB has been established. A positive correlation between COX-2 and nuclear NFκB immunoreactivity was observed. Conclusions. The possible coordinated upregulation of NFκB and COX-2, during bladder carcinogenesis, indicates that agents inhibitors of these two molecules may represent a possible new treatment strategy, by virtue of their role in bladder carcinogenesis.