CD40 expression in HCV-associated chronic liver diseases.

CD40 is expressed primarily on B cells and plays an important role in antigen presentation, B cell proliferation, and T cell activation. It has been reported that the CD40 signal modulates apoptosis and has an anti-viral effect in certain cells. Therefore, we investigated the expression and the function of CD40 in HCV-associated chronic liver disease. The expression of CD40 on liver tissues was determined through immunohistochemistry on 50 liver specimens obtained from HCV-positive patients. The effect of CD40 signaling on apoptosis of HepG2 cells was assessed using the MTT assay. The effect of CD40 stimulation on NF-kappaB activation was determined in NF-kappaB reporter gene-transfected HepG2 cells with the Luciferase assay. CD40 positive hepatocytes were observed in both periportal and lobular areas, accompanied by inflammation. In both areas, CD40 staining intensity became significantly stronger, correlating with the histological grading. Similarly, it became stronger with the progression of the histological staging in F1, F2 and F3 cases; however, the expression level decreased in F4 cases. CD40 ligation induced apoptosis in HepG2 cells in the presence of 500 ng/ml of actinomycin D, while CD40 ligation alone could not. Anti-CD40 monoclonal antibody caused NF-kappaB activation in HepG2 cells in a dose-dependent manner. These results suggest that hepatocyte over-expression of CD40 might play an important role in regulating hepatocyte survival and death in HCV-associated chronic liver diseases.

Cellular apoptosis susceptibility protein and proliferation in human hepatocellular carcinoma.

The cellular apoptosis susceptibility protein (CAS) is the human homologue of the product of the essential yeast chromosome segregation gene, CSE1, and has important roles in tumor necrosis factor (TNF)-induced apoptosis and cell proliferation. In this study, we used immunoblotting and immunohistochemistry to look at CAS expression in human hepatocellular carcinoma (HCC) cells. We also studied the correlation between CAS expression and cell proliferation. To do this, we studied the expression of proliferating cell nuclear antigen (PCNA) by immunostaining and at apoptosis by in situ nick end-labeling (TUNEL), followed by calculation of the PCNA labeling index (PCNA LI) and TUNEL labeling index (TUNEL LI). CAS was constitutively expressed in human HCC cell lines and was primarily confined to the cytoplasm of the cells. PCNA LI and TUNEL LI were significantly higher in HCC than in non-tumor tissue (p<0.01); however, the ratio of TUNEL LI/PCNA LI in HCC was significantly lower than that of non-tumor tissue. Immunohistochemistry revealed that the staining intensity score of CAS in HCC was significantly higher than that of non-tumor tissue (p<0.05). These results indicate that there is an augmentation of pro-liferative activity and apoptosis in HCC tissue, as compared to non-tumor tissue. There was a significant positive correlation between CAS and PCNA LI (p<0.05). In addition, we observed an inverse relationship between CAS expression and TUNEL LI, although the correlation did not reach statistical significance. These results suggest that CAS is expressed at higher levels in human HCC tissue than in non-tumor tissue. CAS may be associated with cell proliferation rather than apoptosis, and further, CAS might play an important role in the development of human HCCs.

Expression of CD34-positive sinusoidal endothelial cells in patients with HBV-associated chronic liver diseases.

It has been demonstrated that CD34-positive cells isolated from human peripheral blood differentiate into endothelial cells and contribute to neoangiogenesis in adults. We investigated the role of CD34-positive endothelial cells in liver samples from patients with hepatitis B virus (HBV)-associated chronic liver diseases. Tissue sections were obtained by liver biopsy from 25 patients with HBV-associated chronic liver diseases and were examined by immunohistochemistry using anti-CD34, anti-von Willebrand factor (vWF), and anti-vascular endothelial growth factor (VEGF) antibodies. CD34-positive, but vWF-negative endothelial cells were observed, particularly in the sinusoids and vascular endothelial cells. We counted these cells and expressed the results as a CD34-labeling index (LI). The CD34 LI did not correlate with VEGF expression and the CD34 LI of patients who progressed to hepatocellular carcinoma (HCC) tended to increase compared to those that did not progress to HCC. CD34 LI was an independent risk factor for development of HCC (relative risk, 35.689; P = 0.033). We conclude that CD34-positive endothelial cells in patients with HBV-associated chronic liver diseases might play a role in hepatocarcinogenesis.

Overexpression of X-linked inhibitor of apoptosis in human hepatocellular carcinoma.

The X-linked inhibitor of apoptosis (XIAP) is a member of a novel family of inhibitors of apoptosis. Since suppression of apoptosis is fundamentally important for carcinogenesis and tumor growth, we investigated the expression and function of XIAP in human hepatocellular carcinomas (HCCs). XIAP was expressed constitutively in HCC cell lines. Fourteen out of 20 (70%) HCC tissues demonstrated moderate or strong cytoplasmic staining for XIAP, whereas non-tumor parts showed negative or weak staining for XIAP by immunohistochemistry. In addition, XIAP expression was inversely correlated with apoptosis, but not with proliferation in HCC tissues. These results indicated that XIAP is a principal inhibitor of apoptosis overexpressed in human HCCs and that XIAP may be a potential target for gene therapy of human HCCs.

Cellular FLICE/caspase-8-inhibitory protein as a principal regulator of cell death and survival in human hepatocellular carcinoma.

Human hepatocellular carcinomas (HCCs) show resistance to apoptosis mediated by several death receptors. Because cellular FLICE/caspase-8-inhibitory protein (cFLIP) is a recently identified intracellular inhibitor of caspase-8 activation that potently inhibits death signaling mediated by all known death receptors, including Fas, TNF-receptor (TNF-R), and TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs), we investigated the expression and function of cFLIP in human HCCs. We found that cFLIP is constitutively expressed in all human HCC cell lines and is expressed more in human HCC tissues than in nontumor liver tissues. Metabolic inhibitors, actinomycin D (ActD) or cycloheximide (CHX), dramatically rendered HCC cells sensitive to Fas-mediated apoptosis. Neither caspase-8 nor caspase-3 was activated by agonistic anti-Fas antibody alone, but both caspases were activated by Fas stimulation in the presence of ActD or CHX, indicating the importance of caspase-8 inhibitors that are sensitive to metabolic inhibitors. Actually, cFLIP expression was decreased in ActD or CHX treatment. cFLIP down-regulation induced by cFLIP antisense oligodeoxynucleotides sensitized HLE cells to Fas, TNF-R, and TRAIL-R-mediated apoptosis. Furthermore, cFLIP over-expression activated nuclear factor (NF)-kappaB and cFLIP down-regulation attenuated NF-kappaB activation induced by TNF-alpha or TRAIL. Pretreatment with pan-caspase-inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD-fmk), restored NF-kappaB activity attenuated by cFLIP down-regulation. cFLIP expression was increased by TNF-alpha, TRAIL, or vascular endothelial growth factor but decreased by wortmannin, indicating that cFLIP expression is regulated by both the NF-kappaB and phosphatidylinostiol-3 kinase (PI-3)/Akt pathways. These results suggest that cFLIP plays an important role in cell survival not simply by inhibiting death-receptor-mediated apoptosis but also by regulating NF-kappaB activation in human HCCs.

Over-expression of Smac promotes TRAIL-induced cell death in human hepatocellular carcinoma.

The second mitochondria-derived activator of caspase, Smac, is an apoptosis-related protein. Smac releases inhibition of the IAP family from caspase-3 to induce apoptosis. Smac is expressed in some malignant tumor cells and is released from mitochondria into the cytosol after death receptor stimulation to promote apoptosis of tumor cells. In this study, we found down-regulated Smac protein expression in hepatocellular carcinoma (HCC) tissues, compared to that in non-tumor hepatic tissues. Simultaneously, caspase-3 expression also decreased in HCC tissues. HCC cell lines did not undergo apoptosis after TRAIL stimulation, although Smac was expressed in these HCC cells. Ectopic Smac alone did not induce cell death, but could sensitize HCC cells to TRAIL stimulation. With over-expression of Smac in HCC cells, TRAIL induced by 10% HCC cell death. The role of Smac in apoptosis signaling pathway in HCC cells warrants further study.

Hepatocellular carcinoma development in sustained viral responders to interferon therapy in patients with chronic hepatitis C.

The incidence of hepatocellular carcinoma (HCC) tends to decrease in sustained responders (SR) to interferon (IFN) therapy for chronic hepatitis C rather than in non-responders (NR). However, some SR develop HCC and their details and prognosis are not well-known, so we investigated such cases. Among 462 patients who underwent IFN therapy and were available for follow-up, 142 patients (30.7%) were SR and six of these (4.2%) developed HCCs. The interval between interferon therapy and diagnosis of HCC was 32-99 months (average 59.2 months). Five of the six cases were single HCCs sized 20-125 mm. The remainder was multiple HCCs. After initial treatment, five patients (83.3%) relapsed and three patients (60%) died due to the HCC. The interval between initial treatment and recurrence was 2-14 months (average 5.8 months). Among the three fatal cases, the interval between initial treatment and their death was 11-66 months (average 29.3 months). Though the prognosis of the one patient who did not relapse after initial treatment was good, the other five patients relapsed and three of them died due to the HCC. These results suggest that the prognosis of sustained responders who develop HCC after IFN therapy is not necessarily good, so close follow-up remains necessary, despite their response to IFN therapy.

Long-term follow-up of chronic hepatitis C in Japan.

BACKGROUND/AIMS: Chronic hepatitis C which exhibits a varied natural course, is becoming a major problem worldwide. METHODOLOGY: In this study, we investigated 161 patients with chronic hepatitis C by repeated liver biopsies. From initial biopsies, we diagnosed 56 patients with chronic persistent hepatitis, 74 with chronic active hepatitis 2A, and 31 with chronic active hepatitis 2B. RESULTS: During the follow-up period, a progression from chronic hepatitis to liver cirrhosis was recognized among all stages, however the rate of progression to liver cirrhosis was less in chronic persistent hepatitis than in chronic active hepatitis 2A and chronic active hepatitis 2B. Hepatocellular carcinoma was detected in chronic active hepatitis 2A and chronic active hepatitis 2B at the initial stage, however, no tumors developed in chronic persistent hepatitis at the initial stage. Most hepatocellular carcinomas were concomitant with liver cirrhosis. CONCLUSIONS: We suggest a close follow-up of patients with chronic hepatitis C, especially those patients with chronic active hepatitis 2A or 2B and exhibiting successive active inflammation of liver.

Focal spared area in fatty liver mimicking a tumor.

A focal fatty liver change may be associated with several conditions related to diffuse hepatic steatosis, such as a diffuse fatty liver change. Using ultrasonography, the focal fatty liver change appears more frequently as hyperechoic and less frequently as hypoechoic areas in the liver. We report a rare case of a focal fatty liver change in which an area was spared in fatty liver. The patient was a 42-year-old man. Abdominal ultrasonography showed focal hypoechogenicity with an irregular margin in S8 within a bright liver. Abdominal computed tomography and enhanced computed tomography showed a high-density mass in S8 of the right lobe. A microscopic examination of the specimens from the liver biopsy from the hypoechoic region revealed normal hepatic parenchymal cells, while tissue samples from the surrounding liver had a high fat deposition.

Pneumothorax after diagnostic laparoscopy.

Here we want to call laparoscopist's attention to pneumothorax after diagnostic laparoscopy. Diagnostic laparoscopy has less complications, compared with laparoscopic surgery. In our experience, only one case (0.04%) developed pneumothorax during routine diagnostic laparoscopic procedure. This complication is presented in a 50-year-old female. She complained of dyspnea just after the decrease of intraabdominal pressure and deflation of intraperitoneal gas. The chest roentgenogram showed a right pneumothorax, and a right chest tube was inserted with immediate relief of tension. Pneumothorax during diagnostic laparoscopy is relatively rare but a major complication; Medline literature research showed six reported cases of pneumothorax after diagnostic laparoscopy from 1983 to 1998 including our case. The etiology was idiopathy in 5 and diaphragmatic injury in 1. Hypotension, elevation of inspiratory pressure, dyspnea, a decrease in systemic oxygen saturation, and loss of breath sounds suggest tension pneumothorax. The clinical condition of the patients improved rapidly because of the easy diffusion of the gas used in laparoscopy. Diagnostic laparoscopists should be aware of this complication and treat ventilatory problems.

High expression of p21WAF1/CIP1 is correlated with human hepatocellular carcinoma in patients with hepatitis C virus-associated chronic liver diseases.

p21(WAF1/CIP1) (p21) protein is a universal inhibitor of cyclin-dependent kinases and is regulated transcriptionally by p53, which is activated by DNA stress. Hepatocytes in chronic hepatitis receive several DNA stresses by lymphocytes and Kupffer cells. Therefore, we analyzed p21 expression of hepatocytes in hepatitis C virus (HCV)-associated chronic liver diseases and investigated the possible involvement of p21 in hepatocarcinogenesis. We examined p21 expression in 35 cases of HCV-associated chronic hepatitis and 25 cases of HCV-associated liver cirrhosis by immunohistochemical analysis. The p21 labeling index (LI) was calculated as the ratio of positive cells to total cells. p21-positive hepatocytes were more numerous in areas of intense inflammation and spotty necrosis and areas close to fibrosis, and were increased according to the degrees of grading and staging. The p21 LI with liver cirrhosis was significantly higher than that with chronic hepatitis (14.4 +/- 5.9 versus 11.1 +/- 4.2, P = 0.014). The cumulative incidence of hepatocellular carcinoma (HCC) was significantly higher in the p21 LI >or=14% group than in the p21 LI <14% group (P = 0.0079). Multivariate analysis demonstrated that p21 expression can be recognized as an independent significant factor for HCC development (relative risk 5.00, P = 0.039). p21 LI decreased significantly after interferon therapy. These results suggested that p21 is up-regulated by the stress of inflammation and fibrosis in HCV-associated chronic liver diseases and that high p21 expression might be related to hepatocarcinogenesis in cirrhotic patients.

Peroxisome proliferator-activated receptor gamma augments tumor necrosis factor family-induced apoptosis in hepatocellular carcinoma.

Proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor, which mainly associates with adipogenesis, but also appears to facilitate cell differentiation or apoptosis in certain malignant cells. This apoptosis induction by PPARgamma is increased by co-stimulation with tumor necrosis factor (TNF)-alpha-related apoptosis-inducing ligand (TRAIL), a member of the TNF family. In this study, we investigated the effect of PPARgamma on Fas-mediated apoptosis in hepatocellular carcinoma (HCC) cell lines. PPARgamma was expressed on all seven HCC cell lines and located in their nuclei. 15-Deoxy-Delta-12,14-prostaglandin J2 (15d- PGJ2), a PPARgamma ligand, inhibited cellular proliferation in HepG2, SK-Hep1 or HLE cells, unlike pioglitazone, another PPARgamma ligand, which did not have a significant influence on proliferation of these cells. However, 15d-PGJ2 facilitated Fas-mediated HCC apoptosis that could not be induced by Fas alone. These results suggest that PPARgamma can augment TNF-family-induced apoptosis.