Increased Bax/Bcl-2 ratio up-regulates caspase-3 and increases apoptosis in the thymus of patients with myasthenia gravis.

BACKGROUND: In this study the possible relation of Bax (an apoptosis promoter) to Bcl-2 (an apoptosis inhibitor) ratio with the apoptosis co-ordination enzyme, caspase-3, in the thymus of patients with myasthenia gravis (MG) was investigated in correlation with long-term clinical prognosis. PATIENTS AND METHODS: The study included 46 patients (17M/29F, mean age 36.60 +/- 16.09 yr) with MG, who underwent thymectomy for treatment. The clinical staging (Osserman classification) included: stage 1-5, IIA-21, IIB-17, III-3. The pathology of the thymus showed: hyperplasia-26, atrophy-8, thymoma B1 and B2 type-9, thymoma B3 type (well differentiated thymic carcinoma)-3. The patients were evaluated 39-166 (mean 91.87 +/- 38.38) months after thymectomy. At the end of the follow-up period, the patients were classified as follows: group A: complete stable remission, group B: pharmacological remission + minimal manifestations + improvement + deterioration. Paraffin sections of thymic tissue were subjected to: a) immunohistochemistry (bax, bcl-2, caspase-3 protein); b) in situ hybridization (bax, bcl-2 mRNA); and c) TUNEL-stain (apoptotic cells). Bax to bcl-2 mRNA and protein ratio was determined for each sample by dividing the % bax (+) cells by the % bcl-2 (+) cells. RESULTS: Follow-up data were available for 39/46 patients: 13/39 patients belonged to group A and 26/39 to group B. The Bax/Bcl-2 mRNA and protein ratios were increased towards advanced disease stages (+370% for mRNA and +391% for protein, from MG stage I to stage III). These ratios were correlated with caspase-3 expression (r = 0.782 and 0.583, p < 0.01) and apoptosis (r = 0.591 and 0.358 p < 0.01 and p < 0.05). All the 13 cases in group A had a Bax/Bcl-2 ratio < 1 (mean +/- SD: 0.58 +/- 0.04 for mRNA and 0.62 +/- 0.03 for protein), whereas all the 26 cases of group B had a ratio > 1 (1.47 +/- 0.07 for mRNA and 1.52 +/- 0.18 for protein). The Kaplan-Meier survival curve showed higher, free of disease, survival in group A (p = 0.0082). Cox regression analysis revealed that the Bax/Bcl-2 ratio was an independent prognostic factor, however the p-value was marginally significant (95% CI: 1.078-44.073, p = 0.041). CONCLUSION: This study has demonstrated that in patients with MG who underwent thymectomy: a) the Bax/Bcl-2 ratio may up-regulate caspase-3 expression and modulate apoptosis associated with progress of the disease; b) the Bax/Bcl-2 ratio < 1 was associated with complete stable remission after thymectomy; and c) Bax/Bcl-2 ratio was an independent predictive marker for therapeutic response after thymectomy.

Immunohistochemical expression of TGF-beta1, p21WAF1, p53, Ki67, and angiogenesis in gastric carcinomas: a clinicopathologic study.

BACKGROUND: Transforming growth factors-beta (TGF-betas) are multifunctional polypeptides with crucial role as regulators of cellular growth and differentiation. It has been reported that TGF-beta1 plays a biphasic action on tumorigenesis thus inducing or inhibiting malignant properties of the epithelial cells. METHODS: TGF-beta1 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-beta1 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-beta1 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-beta1 may be involved in the activation of the cdk inhibitor p21WAF1 in gastric adenocarcinomas, suggesting p53-independent induction of p21 in gastric cells. TGF-beta1 does not seem to contribute to the alteration of the angiogenic status of these tumors.

The effect of ischemia on the regeneration of the cholestatic liver. An experimental study.

BACKGROUND/AIMS: Cholestatic liver is known to be more susceptible to ischemia than normal liver. In this study we assessed the histopathologic features of hepatic ischemic damage and liver regeneration in rats with experimental obstructive jaundice. METHODOLOGY: The study comprised 90 male Wistar rats. These were assigned randomly to 4 groups according to the surgical procedure they underwent: I (n = 10) controls (non-operated), II (n = 10) sham-operated, III (n = 30) occlusion of hepatic artery and portal vein (total liver ischemia), and IV (n = 40) ligation and division of the common bile duct ligation. Rats of group III were sacrificed 15 (IIIa), 30 (IIIb) and 60 min (IIIc) after total liver ischemia was done. Ten days after bile duct ligation, 10 rats of group IV underwent euthanasia, whereas the remaining 30, underwent total liver ischemia and were sacrificed after 15 min (IVb), 30 min (IVc), and 60 min (IVd). Liver wedge biopsies (left anterior lobe) were obtained and histologic examination included hematoxylin and eosin, and immunohistochemical stains for cytokeratin AE1, HEPPAR (hepatocyte paraffin antigen), and antigen Ki67. Immunohistochemical results for Ki67 were expressed following morphometric analysis. RESULTS: Liver sections from category IVa showed large duct obstruction features, and those from group III, ischemic chages including centrilobular hepatocellular swelling and necrosis, hepatocanalicular cholestasis, and mild portal mononuclear/mixed inflammation. Sections from groups IVB, IVc, IVd displayed together changes of large duct obstruction and ischemia, and in categories IVc (bile duct ligation +30 min total liver ischemia), and IVd (bile duct ligation +60 min total liver ischemia) necrosis of the large bile ducts was present. The total liver parenchymal area affected (% necrosis) was higher in categories IVd, and IVc compared to categories IVb (P < 0.05), and IIIc, IIIb, IIIa (P < 0.01). All 60 total liver ischemia-liver biopsies, developed features of liver regeneration that originated from zone 2, extended to zone 1 and occasionally to zone 3. Immunohistochemical stains revealed cells positive to AE1 and cells positive to HEPPAR. Proliferation rate (% Ki67+ cells) was higher in category IIIa compared to categories IIIb, IIIc, IVb, IVc, and IVd (P < 0.05). CONCLUSIONS: Our study shows that liver ischemia induces more severe hepatocyte damage in livers with obstructive cholangiopathy compared to normal ones. Liver regenerative process is mediated mainly by proliferation of non-necrotic cells that express hepatocellular or ductular epithelial features. Proliferation rate of hepatocytes is lower when liver ischemia and obstructive jaundice coexist.

Expression of apoptosis-related proteins bcl-2 and bax along with transforming growth factor (TGF-beta1) 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-beta1 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-beta1) 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-beta1 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-beta1 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-beta1 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-beta1 expression with that of apoptosis-related proteins might represent an implication of this growth factor with apoptotic process in the human diseased kidney.

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-beta(1) (TGF-beta(1)) 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-beta(1) 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-beta(1) 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-beta(1) 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-beta(1) 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-beta(1) expression are associated with the development of interstitial fibrosis in patients with glomerular disease.

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 TGFbeta1, TGFbeta2 and TGFbeta3 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 TGFbeta1, TGFbeta2 and TGFbeta3 was applied. Morphological and immunohistochemical results were correlated with clinicopathologic parameters. RESULTS: TGFbeta1 protein was expressed in 88 out of 124 (71%) carcinomas, whereas TGFbeta2 and TGFbeta3 proteins were detected in all tumors examined. Normal colonic mucosal epithelial cells expressed TGFbeta2 (significantly less as compared to neoplastic cells; p < 0.01) and TGFbeta3 (p > 0.05 compared to neoplastic cells), but not TGFbeta1. Statistical analysis revealed a higher expression of TGFbeta1 in low-grade carcinomas (p = 0.009) and a higher presence of TGFbeta2 in advanced tumors (p = 0.008). TGFbeta1 expression was related with increased disease-free and overall survival (p < 0.05 each). The presence of TGFbeta2 was correlated with worse prognosis (p < 0.05). Cox analysis revealed that besides tumor grade and stage, TGFbeta1 expression constituted an independent prognostic factor. CONCLUSION: This study shows that in adenocarcinomas of the colon, there is a differential expression of TGFbeta1, TGFbeta2 and TGF3. TGFbeta1 may be implicated in the pathogenesis of these tumors, since it is expressed only in neoplastic but not in normal cells. TGFbeta1 is related with an increased disease-free and overall survival and constitutes an independent prognostic factor. In advanced stages, TGFbeta2 seems to be involved in tumor progression and is related with worse prognosis.