The role of exosomes in hepatitis,liver cirrhosis and hepatocellular carcinoma

Exosomes are small vesicles that were initially thought to be a mechanism for discarding unneeded membrane proteins from reticulocytes. Their mediation of intercellular communication appears to be associated with several biological functions. Current studies have shown that most mammalian cells undergo the process of exosome formation and utilize exosome‐mediated cell communication. Exosomes contain various microRNAs, mRNAs and proteins. They have been reported to mediate multiple functions, such as antigen presentation, immune escape and tumour progression. This concise review highlights the findings regarding the roles of exosomes in liver diseases, particularly hepatitis B, hepatitis C, liver cirrhosis and hepatocellular carcinoma. However, further elucidation of the contributions of exosomes to intercellular information transmission is needed. The potential medical applications of exosomes in liver diseases seem practical and will depend on the ingenuity of future investigators and their insights into exosome‐mediated biological processes.     

Cytokeratin 8 and 18 expression in imprint smears of chronic viral hepatitis, autoimmune hepatitis and hepatocellular carcinoma. A preliminary study

OBJECTIVE: To investigate whether the expression of cytokeratin (CK) 8 and 18 is altered in chronic active viral hepatitis, autoimmune hepatitis and hepatocellular carcinoma. STUDY DESIGN: Cytologic imprint smears were obtained from 53 liver core biopsy specimens and were studied immunocytochemically for the expression of CK8 and 18. RESULTS: CK8-positive expression was observed in 45.5% of chronic active hepatitis B (CH-B), 20% of chronic active hepatitis C (CH-C), 90% of autoimmune hepatitis (AIH) and 83.3% of hepatocellular carcinoma (HCC) cases. CK18-positive expression was observed in 36.4% of CH-B, 26.7% of CH-C, 70% of AIH and 83.3% of HCC cases. A statistically significant association was found between CK8- and CK18-positive expression and the diagnosis of AIH and HCC. In contrast, CH-C and CH-B were associated with negative CK8 and CK18 expression. In addition, a negative [CK8(-)/CK18(-)] or imbalanced [CK8(-)/CK18(+), CK8(+)/CK18(-)] expression pattern was found in 100.0% and 81.18% of CH-C and CH-B cases, respectively, while the relative percentages of AIH and HCC cases were significantly lower (30.0% and 16.7%, respectively) (p < 0.0001). CONCLUSION: Our results indicate that CK8 and 18 expression is maintained in AIH and HCC and altered in CH-B and CH-C. The pathogenetic mechanism of this alteration remains to be clarified.     

Altered glycosylation,expression of serum haptoglobin and alpha-1-antitrypsin in chronic hepatitis C,hepatitis C induced liver cirrhosis and hepatocellular carcinoma patients

Liver cirrhosis with hepatitis C viral infection (HCV-LC) causes high risk to develop hepatocellular carcinoma (HCC). Besides diagnosis of liver cirrhosis by biochemical test, imaging techniques, assessment of structural liver damage by biopsy shows several disadvantages. Our aim was to monitor the changes in the expression level of serum proteins and their glycosylation pattern among chronic hepatitis C (HCV-CH), HCV-LC and HCC patients with respect to controls. 2D gel electrophoresis of HCV-CH, HCV-LC and HCC patients’ sera showed several protein spots, which were identified by LC-MS. The change in the expression of two prominent protein spots, haptoglobin (Hp) and alpha 1-antitrypsin (AAT) was evaluated by western blot and ELISA. The changes in glycosylation pattern of these serum proteins were assayed using different lectins. Increased level of Hp and AAT was observed in HCV-LC and HCC patients’ group whereas those were found to be present less in HCV-CH patient groups with respect to control as determined by ELISA using monoclonal antibodies. Decreased level of sialylation in both Hp and AAT was observed in HCV-LC and HCV-CH patients’ group whereas increased level of sialylation was observed in HCC patient groups by ELISA using Sambucus nigra agglutinin. On the other hand increased level of fucosylation in two serum glycoproteins was observed in HCV-LC and HCC patients’ group using Lens culinarris agglutinin. High glycan branching was found in HCV-LC and HCC patient groups in Hp but not in HCV-CH as determined by Datura stramonium agglutinin. However, there was no such change observed in glycan branching in AAT of HCV-CH and HCV-LC patients’ groups, to the contrary high glycan branching was observed in HCC patients’ group. Increased level of exposed galactose in both serum proteins was observed in both HCC patients’ group as determined by Ricinus communis agglutinin. The present glycoproteomics study could predict the progression of HCV-CH, HCV-LC and HCC without the need of liver biopsy.     

Serial analysis of gene expression in chronic hepatitis C and hepatocellular carcinoma

Hepatitis C virus (HCV) causes chronic hepatitis C (CH-C) and is epidemiologically linked with the occurrence of hepatocellular carcinoma (HCC). To elucidate the comprehensive gene expression profiles of CH-C and HCC, serial analysis of gene expression (SAGE) libraries were made from CH-C and HCC tissues of a patient, and compared with a reported SAGE library of a normal liver (NL). Scatter plots of the distribution of tags from the HCC library exhibited the existence of many differentially expressed genes compared with those from the CH-C and NL libraries. Up-regulation of IFN-gamma inducible genes and oxidative stress-inducible genes were identified in both the CH-C and HCC libraries, and some unpublished new genes were specifically up- or down-regulated in the HCC library. This genome-wide scanning study discloses the molecular portraits of CH-C and HCC, and provides novel candidate genes that should help clarify the mechanism of hepatocarcinogenesis in the chronically HCV-infected liver.     

Role of fibrogenic markers in chronic hepatitis C and associated hepatocellular carcinoma

Detection and follow up of fibrogenesis in chronic hepatitis C (CHC) is mandatory for early treatment and risk stratification. The current study included 120 patients with CHC, of whom 30 had liver cirrhosis (LC) and 30 had hepatocellular carcinoma (HCC). 15 wedge liver biopsies, taken during laparoscopic cholecystectomy, were included as normal controls. Cases were subjected to laboratory investigations, serologic markers for viral hepatitis and assessment of circulating levels of hyaluronic acid (HA) and platelet-derived growth factor (PDGF). Immunohistochemical expression of connective tissue growth factor (CTGF), PDGF and transforming growth factor-β1 (TGF-β1) was also carried out. A significant increase (p < 0.01) in serum HA was noticed in CHC, LC and HCC compared to controls. Although, a significant decrease in serum PDGF was detected in CHC and LC compared to controls, HCC values were comparable. A significant up-regulation of CTGF was detected in CHC, LC and HCC (p < 0.01) in contrast to its limited mild expression in normal livers. Intense PDGF positive staining was noticed in CHC, LC and HCC compared to scattered faint expression in controls. The significant expression and marked intensity of PDGF staining matched the progress to tumorigenesis. A positive TGF-β1 immunostaining was also noticed in CHC, LC and HCC. An intense and extensive cytoplasmic expression of TGF-β1 was encountered in patients with LC revealing that CTGF, PDGF and TGF-β1 act synergistically in LC. Data revealed that HA and CTGF may be implicated as important diagnostic parameters for assessment of hepatic fibrosis and PDGF for monitoring malignant transformation in CHC.     

Artichoke leave extract for chronic hepatitis C – A pilot study

BackgroundArtichoke leave extracts (ALE) have hepatoprotektive properties and are used by patients with chronic liver disease. Effects in patients with chronic hepatitis C are unclear.Methods17 patients with chronic hepatitis C and persistently elevated aminotransferase levels were treated for 12 weeks with 3200 mg standardized ALE per day. Primary outcome parameter was the rate of alanine aminotransferase (ALT) normalisation after 12 weeks. Secondary parameters were the course of ALT, aspartate aminotransferase and γ glutamyltransferase levels, quantitative HCV RNA, subjective symptoms frequently associated with chronic hepatitis C (fatigue, discomfort upper abdomen, joint problems) and safety.ResultsNone of the patients had normalized ALT levels after 12 weeks of treatment. There was no significant change of aminotransferase levels or viral load compared to baseline levels. Fatigue and joint problems significantly improved after 4 weeks of treatment. However, after 12 weeks, there was no significant difference to baseline. Tolerability of ALE was rated as good to excellent. Severe side effects did not occur.ConclusionALE seem not to be effective to improve aminotransferase levels in patients with chronic hepatitis C     

Viral hepatitis and hepatocellular carcinoma

Background

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The incidence of HCC varies considerably with the geographic area because of differences in the major causative factors. Chronic hepatitis B and C, mostly in the cirrhotic stage, are responsible for the great majority of cases of HCC worldwide. The geographic areas at the highest risk are South-East Asia and sub-Saharan Africa, here hepatitis B is highly endemic and is the main cause of HCC. In areas with an intermediate rate of HCC such as Southern Europe and Japan, hepatitis C is the predominant cause, whereas in low rate areas such as Northern Europe and the USA, HCC is often related to other factors as alcoholic liver disease. There is a rising incidence in HCC in developed countries during the last two decades, due to the increasing rate of hepatitis C infection and improvement of the clinical management of cirrhosis.

Methods

This article reviews the literature on hepatitis and hepatocellular carcinoma. The Medline search was carried out using these key words and articles were selected on epidemiology, risk factors, screening, and prevention of hepatocellular carcinoma.

Results

Screening of patients with advanced chronic hepatitis B and C with hepatic ultrasound and determination of serum alfa-fetoprotein may improve the detection of HCC, but further studies are needed whether screening improves clinical outcome. Hepatitis B and C viruses (HBV/HCV) can be implicated in the development of HCC in an indirect way, through induction of chronic inflammation, or directly by means of viral proteins or, in the case of HBV, by creation of mutations by integration into the genome of the hepatocyte.

Conclusion

The most effective tool to prevent HCC is avoidance of the risk factors such as viral infection. For HBV, a very effective vaccine is available. Preliminary data from Taiwan indicate a protective effect of universal vaccination on the development of HCC. Vaccination against HBV should therefore be a health priority. In patients with chronic hepatitis B or C, interferon-alfa treatment in a noncirrhotic stage is protective for HCC development in responders, probably by prevention of cirrhosis development. When cirrhosis is already present, the protective effect is less clear. For cirrhosis due to hepatitis B, a protective effect was demonstrated in Oriental, but not in European patients. For cirrhosis due to hepatitis C, interferon-alfa treatment showed to be protective in some studies, especially in Japan with a high incidence of HCC in untreated patients. Virological, but also merely biochemical response, seems to be associated with a lower risk of development of HCC. As most studies are not randomized controlled trials, no definitive conclusions on the long-term effects of interferon-alfa in HBV or HCV cirrhosis can be established. Especially in hepatitis C, prospective studies should be performed using the more potent reference treatments for cirrhotics, namely the combination of peginterferon and ribavirin.     

Oxidative stress and hepatic Nox proteins in chronic hepatitis C and hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common liver cancer and a leading cause of cancer-related mortality in the world. Hepatitis C virus (HCV) is a major etiologic agent of HCC. A majority of HCV infections lead to chronic infection that can progress to cirrhosis and, eventually, HCC and liver failure. A common pathogenic feature present in HCV infection, and other conditions leading to HCC, is oxidative stress. HCV directly increases superoxide and H₂O₂ formation in hepatocytes by elevating Nox protein expression and sensitizing mitochondria to reactive oxygen species generation while decreasing glutathione. Nitric oxide synthesis and hepatic iron are also elevated. Furthermore, activation of phagocytic NADPH oxidase (Nox) 2 of host immune cells is likely to exacerbate oxidative stress in HCV-infected patients. Key mechanisms of HCC include genome instability, epigenetic regulation, inflammation with chronic tissue injury and sustained cell proliferation, and modulation of cell growth and death. Oxidative stress, or Nox proteins, plays various roles in these mechanisms. Nox proteins also function in hepatic fibrosis, which commonly precedes HCC, and Nox4 elevation by HCV is mediated by transforming growth factor β. This review summarizes mechanisms of oncogenesis by HCV, highlighting the roles of oxidative stress and hepatic Nox enzymes in HCC.     

CD137-mediated pathogenesis from chronic hepatitis to hepatocellular carcinoma in hepatitis B virus-transgenic mice

Chronic hepatitis B virus (HBV) infection is characterized by sustained liver inflammation with an influx of lymphocytes, which contributes to the development of cirrhosis and hepatocellular carcinoma. The mechanisms underlying this immune-mediated hepatic pathogenesis remain ill defined. We report in this article that repetitive infusion of anti-CD137 agonist mAb in HBV-transgenic mice closely mimics this process by sequentially inducing hepatitis, fibrosis, cirrhosis, and, ultimately, liver cancer. CD137 mAb initially triggers hepatic inflammatory infiltration due to activation of nonspecific CD8(+) T cells with memory phenotype. CD8(+) T cell-derived IFN-γ plays a central role in the progression of chronic liver diseases by actively recruiting hepatic macrophages to produce fibrosis-promoting cytokines and chemokines, including TNF-α, IL-6, and MCP-1. Importantly, the natural ligand of CD137 was upregulated significantly in circulating CD14(+) monocytes in patients with chronic hepatitis B infection and closely correlated with development of liver cirrhosis. Thus, sustained CD137 stimulation may be a contributing factor for liver immunopathology in chronic HBV infection. Our studies reveal a common molecular pathway that is used to defend against viral infection but also causes chronic hepatic diseases.     

Birch bark extract as therapy for chronic hepatitis C - A pilot study

The hepatoprotective effect of birch bark extract (BBE) in patients with chronic hepatitis C (CHC) was studied. Forty-two patients with serologically confirmed chronic hepatitis C were treated for 12 weeks with 160 mg standardized BBE per day. The primary outcome parameter measured was the rate of alanine aminotransferase (ALT) normalization after 12 weeks. Secondary parameters included the course of ALT, aspartate aminotransferase (AST) levels, quantitative HCV RNA levels, subjective symptoms associated with CHC (fatigue, abdominal discomfort, depression, and dyspepsia), safety and compliance. The qualitative-quantitative analysis of BBE was made using high performance liquid chromatography to confirm the presence of 75% betulin and 3.5% betulinic acid. Significant differences in the mean ALT and HCV RNA levels were observed after 12 weeks of treatment. The level of ALT was decreased in 54.0% and normalized (p = 0.046). HCV RNA was reduced in 43.2% (p = 0.016). After 12 weeks of treatment, reports of fatigue and abdominal discomfort were reduced by 6-fold (p = 0.028) and 3-fold (p = 0.05), respectively. Dyspepsia was no longer reported (p = 0.042) and the effect was significantly different from baseline. Because this study lacks a control group clinical relevance of the data can only be estimated in future by following controlled clinical trials.     

ABO blood group and the risk of hepatocellular carcinoma: a case-control study in patients with chronic hepatitis B

Background

Studies have observed an association between the ABO blood group and risk of certain malignancies. However, no studies of the association with hepatocellular carcinoma (HCC) risk are available. We conducted this hospital-based case-control study to examine the association with HCC in patients with chronic hepatitis B (CHB).

Methods

From January 2004 to December 2008, a total of 6275 consecutive eligible patients with chronic hepatitis B virus (HBV) infection were recruited. 1105 of them were patients with HBV-related HCC and 5,170 patients were CHB without HCC. Multivariate logistic regression models were used to investigate the association between the ABO blood group and HCC risk.

Results

Compared with subjects with blood type O, the adjusted odds ratio (AOR) for the association of those with blood type A and HCC risk was 1.39 [95% confidence interval (CI), 1.05–1.83] after adjusting for age, sex, type 2 diabetes, cirrhosis, hepatitis B e antigen, and HBV DNA. The associations were only statistically significant [AOR (95%CI) = 1.56(1.14–2.13)] for men, for being hepatitis B e antigen positive [AOR (95%CI) = 4.92(2.83–8.57)], for those with cirrhosis [AOR (95%CI), 1.57(1.12–2.20)], and for those with HBV DNA≤10⁵copies/mL [AOR (95%CI), 1.58(1.04–2.42)]. Stratified analysis by sex indicated that compared with those with blood type O, those with blood type B also had a significantly high risk of HCC among men, whereas, those with blood type AB or B had a low risk of HCC among women.

Conclusions

The ABO blood type was associated with the risk of HCC in Chinese patients with CHB. The association was gender-related.     

Multiple integration site of hepatitis B virus DNA in hepatocellular carcinoma and chronic active hepatitis tissues from children.

Attention was directed to hepatitis B virus (HBV) integration in tissues obtained from an hepatocellular carcinoma (HCC) of an 11-year-old boy and from the liver of his 6-year-old brother, who had chronic active hepatitis. Multiple HBV DNA integration sites were demonstrated in both tissues. Cell population(s) in the HCC and liver from the patient with chronic active hepatitis were assumed to be heterogeneous with regard to HBV integration. The integrated forms in the two tissues showed similar genetic organization without gross rearrangement. The location of one of the virus-chromosomal junctions was restricted to the 5'-end region of the minus-strand DNA of HBV. The experimental results support our previous model for the mechanism of HBV integration, in which minus-strand replicative intermediates integrate into chromosomal DNA. The integrated HBV DNAs were conserved in the same region of the viral genome, spanning from the C gene through the S gene to the X gene, which contains intrinsic promoter-enhancer sequences.     

Gremlin in the pathogenesis of hepatocellular carcinoma complicating chronic hepatitis C: an immunohistochemical and PCR study of human liver biopsies

ABSTRACT: BACKGROUND: The possible role of secretory products of fibrous tissue in the development of hepatocellular carcinoma (HCC) complicating chronic hepatitis C was investigated. Our hypothesis was that gremlin, secreted by fibroblasts, inhibited bone morphogenic protein (BMP), which mediates stem cell maturation into adult functioning hepatocytes, and thus, arrest stem cell maturation and promoted their proliferation in an immature state possibly culminating into development of HCCs. RESULTS: Protein expression of cytokeratin 19 (CK19) and fibroblast growth factor 2 (FGF-2), and mRNA expression of gremlin and BMP-7 were studied in 35 cases of chronic hepatitis, cirrhosis and HCC complicating chronic hepatitis C. CK19 expression was higher in cases of cirrhosis (0.004), which correlated with the grade (r=0.64, p=0.009) and stage (r=0.71, p=0.001). All HCCs were negative for CK19. Stem cell niche activation (as indicated as a ductular reaction) was highest in cases of cirrhosis (p=0.001) and correlated with CK19 expression (r=0.42, p=0.012), the grade(r=0.56, p=0.024) and stage (0.66, p=0.006). FGF-2 expression was highest in HCCs and correlated with the grade (r=0.6, p=0.013), stage (0.72, p=0.002), CK19 expression (r=0.71, p=002) and ductular reaction (0.68, p=0.004) in hepatitis cases. Higher numbers of cirrhosis cases and HCCs (p=0.009) showed gremlin expression, which correlated with the stage (r=0.7, p=0.002). Gremlin expression correlated with that of CK19 (r=0.699, p=0.003) and FGF2 (r=0.75, p=0.001) in hepatitis cases. CONCLUSIONS: Fibrosis promotes carcinogenesis by fibroblast-secreted gremlin that blocks BMP function and promotes stem cell activation and proliferation as well as possibly HCC development.