Progenitor cell activation in chronic viralhepatitis.

BACKGROUND/AIM: Oval cell proliferation is known to occur in experimental models of hepatic regeneration and carcinogenesis. Recent studies have suggested that activation of progenitor cells, representing the human counterpart of oval cells, may play a role in hepatic diseases. Therefore, we evaluated putative progenitor cells in chronic viral hepatitis. METHODS: Forty-one needle liver biopsy specimens from patients with chronic hepatitis B and 43 specimens from patients with chronic hepatitis C were examined histologically. The grade (histological activity index (HAI)) and stage (degree of fibrosis) were determined on routinely stained sections. The number of progenitor cells was assessed semiquantitatively on cytokeratin 7- (CK 7-) stained sections. RESULTS: In both aetiological categories of chronic viral hepatitis, progenitor cell numbers were found to increase in parallel to the HAI, as well as to the stage of disease. Features suggestive of hepatocytic differentiation of progenitor cells were also noted on immunohistochemical stains for CK 7 and 'hepatocyte-specific' antigen. CONCLUSIONS: In chronic hepatitis B and chronic hepatitis C, progenitor cell activation is correlated with the grade and stage of disease. Proliferating progenitor cells may play a role in hepatic regeneration occurring in this setting.     

Upregulation of lymphotoxin beta expression in liver progenitor (oval) cells in chronic hepatitis C

BACKGROUND: Bipotent liver progenitor (oval) cells with the ability to differentiate into hepatocytes and biliary epithelium have recently been identified in human subjects with hepatitis C. Animal studies suggest that members of the tumour necrosis factor family, including lymphotoxin beta (LT-beta), regulate oval cell proliferation in liver disease, but its role in human liver disease is unclear. AIMS: This study seeks to establish a role for LT-beta in hepatitis C related liver injury and to provide evidence that its increased expression is related to the presence of oval cells. METHODS: Liver biopsy specimens were obtained from patients with chronic hepatitis C virus (HCV) infection (n=20). Control liver samples (n=5) were obtained from liver resection or transplant surgery. LT-beta expression in liver biopsy specimens was studied using quantitative real time polymerase chain reaction and immunohistochemistry. RESULTS: LT-beta mRNA levels were similar in control and HCV liver in the absence of fibrosis. In subjects with portal fibrosis, LT-beta mRNA levels were elevated 2.2-fold over control liver levels (p=0.04). In subjects with bridging fibrosis, LT-beta mRNA levels increased 4.4-fold over control liver levels (p=0.02). LT-beta mRNA levels in subjects with established cirrhosis were increased 3.3-fold compared with controls and 2.6-fold compared with mild liver damage (p=0.02). Immunohistochemical analysis established that LT-beta was expressed by oval cells, inflammatory cells, and small portal hepatocytes. CONCLUSIONS: In chronic HCV infection, LT-beta expression is observed in multiple hepatic cell types, including oval cells. LT-beta expression is significantly increased when fibrosis or cirrhosis is present, suggesting a role for LT-beta in the pathogenesis of chronic hepatitis C and a possible role in oval cell mediated liver regeneration.     

Cholangiolocellular carcinoma: an innocent-looking malignant liver tumor mimicking ductular reaction

The authors present an interesting case of a 60-year-old man who underwent right hepatectomy for a diagnosis of hepatocellular carcinoma (HCC) on a background of noncirrhotic chronic hepatitis C. Pathologic examination confirmed the presence of HCC near the porta hepatis, which invaded the right portal vein branch. In addition, a well-demarcated 13.5 × 7.8 × 4.0 cm yellow and firm area upstream of the HCC was noted. This yellow area corresponded to a tumoral ductular proliferation, which cytologically was extremely bland, but invaded portal tracts and the adjacent liver parenchyma. This tumoral proliferation mimicked ductular reaction, except that it had more anastomosing structures and was associated with abundant hyalinized fibrotic stroma. Cytologically, the tumor cells had round to oval nuclei with fine chromatin, indistinct nucleoli, and scant cytoplasm. They exhibited immunohistochemical features of hepatic progenitor cells, i.e., expressing CK7, CK19, and N-CAM; and their malignancy was supported by the p53 and Ki67 immunoreactivity. The authors concluded that the patient had cholangiolocellular carcinoma with an aggressive hepatocellular carcinoma component. Cholangiolocellular carcinoma has been reported to be associated with chronic hepatitis C viral infection and to derive from hepatic progenitor cells, which explains why hepatocellular carcinoma and/or cholangiocarcinoma component may be present.     

Attenuated liver progenitor (oval) cell and fibrogenic responses to the choline deficient, ethionine supplemented diet in the BALB/c inbred strain of mice

BACKGROUND/AIMS: Liver regeneration following chronic injury is associated with inflammation, the proliferation of liver progenitor (oval) cells and fibrosis. Previous studies identified interferon-gamma as a key mediator of oval cell proliferation. Interferon-gamma is known to regulate Th1 cell activities during immune challenge. Therefore, we hypothesised that progenitor cell-mediated regeneration is associated with a Th1 immune response. METHODS: C57Bl/6 (normal Th1 response) and BALB/c mice (deficient in Th1 signalling) were placed on a carcinogenic diet to induce liver injury, progenitor cell proliferation and fibrosis. RESULTS: Serum transaminases and mortality were elevated in BALB/c mice fed the diet. Proliferation of liver progenitor cells was significantly attenuated in BALB/c animals. The pattern of cytokine expression and inflammation differed between strains. Liver fibrosis and hepatic stellate cell activation were significantly inhibited in BALB/c mice compared to C57Bl/6. In addition, interferon-gamma knockout mice also showed reduced fibrosis compared to wild type. These findings are in contrast to published results, in which interferon-gamma is shown to be anti-fibrogenic. CONCLUSIONS: Our data demonstrate that the hepatic progenitor cell response to a CDE diet is inhibited in mice lacking Th1 immune signalling and further show that this inhibition is associated with reduced liver fibrosis.     

Anticarcinogenic impact of interferon therapy on the progression of hepatocellular carcinoma in patients with chronic viral infection

Hepatocellular carcinoma (HCC) is mainly caused by a persistent infection due to the hepatitis B or hepatitis C virus. The number of HCC cases is increasing in Asian and African countries, as well as in European and American countries. Interferon (IFN) therapy, used for type B chronic liver diseases, inhibits hepatic carcinogenesis in patients with compensated cirrhosis. However, there is insufficient evidence that IFN therapy inhibits hepatic carcinogenesis in patients with chronic hepatitis B. There are few cases of HCC due to chronic hepatitis B, and long-term follow-up periods verifying the inhibitory effect of IFN on hepatic carcinogenesis have not been obtained. To improve the prognosis of type B chronic liver diseases, it is important that hepatitis treatment follows guidelines in which a patient's age and the extent of hepatic fibrosis are taken into account. As for chronic hepatitis C, since a sustained virological response (SVR) in IFN therapy inhibits hepatic carcinogenesis and improves prognosis, treatment that aims for an SVR while taking into consideration host-sided and virus-sided factors is recommended for patients with type C chronic liver diseases. In areas with low incidence of HCC (e.g. USA), a large number of cases and a long-term follow-up period are needed before it can be accepted that IFN therapy inhibits hepatic carcinogenesis. After locally curative treatment of HCC, IFN therapy suppresses recurrence and improves survival rates.     

Inhibition of activated blood platelets by interferon alpha 2b in chronic hepatitis C

BACKGROUND/AIMS: Interferon alpha used in treatment of chronic hepatitis C significantly influences the blood platelets. The role of platelets in initiating and developing pathological processes in hepatic diseases is still barely known. We studied the effects of interferon alpha 2b (IFN alpha2b) on blood platelets in chronic hepatitis C. METHODOLOGY: The studies were conducted in 16 patients who underwent IFN alpha2b treatment 3 times a week at 6MU. The examination was carried out before and on the 14th day of the treatment of IFN alpha2b. Morphological parameters of blood platelets were determined by hematological methods and flow cytometry. Expression of receptors on blood platelet surfaces (CD41, CD42a, CD62P) and thrombopoietin, platelet-derived growth factor, soluble form sP-selectin, IL-6, and tumor necrosis factor alpha were also determined. RESULTS: The use of IFN alpha2b in patients with chronic hepatitis C significantly effects blood platelets morphology by causing the decrease in their number, the change in population size, and the increase in large platelet count. Interferon decreases P-selectin expression on platelets, sP-selectin and platelet-derived growth factor concentration in plasma. During interferon therapy we noted increase concentration of thrombopoietin, tumor necrosis factor alpha, IL-6 in chronic hepatitis C. CONCLUSIONS: IFN alpha2b stabilizes activated platelets and probably decreases their participation in inflammatory and fibrotic processes in the liver.     

Differential regulation of rodent hepatocyte and oval cell proliferation by interferon gamma

Hepatocytes and intrahepatic progenitor cells (oval cells) have similar responses to most growth factors but rarely proliferate together. Oval cells constitute a reserve compartment that is activated when hepatocyte proliferation is inhibited. Interferon gamma (IFN-gamma) increases in liver injury that involves oval cell responses, but it is not upregulated during liver regeneration after partial hepatectomy. Based on these observations, we used well-characterized lines of hepatocytes (AML-12 cells) and oval cells (LE-6 cells) to investigate the potential mechanisms that regulate differential growth responses in hepatocytes and oval cells. We show that IFN-gamma blocks hepatocyte proliferation in vivo, and that in combination with either tumor necrosis factor (TNF) or lipopolysaccharide (LPS), it causes cell cycle arrest in hepatocytes but stimulates oval cell proliferation in cultured cells. The hepatocyte cell cycle arrest is reversible, is p53-independent, and is not associated with apoptosis. Treatment of AML-12 hepatocytes with IFN-gamma/LPS or IFN-gamma/TNF, but not with individual cytokines, induced NO synthase and generated NO, while similarly treated oval cells produced little if any NO. Generation of NO by an NO donor reproduced the inhibitory effect of the cytokine combinations on AML-12 cell replication, while NO inhibitors abolish the replication deficiency. In conclusion, we propose that IFN-gamma, in conjunction with TNF or LPS, can both inhibit hepatocyte proliferation through the generation of NO and stimulate oval cell replication. The response of hepatocytes and oval cells to cytokine combinations may contribute to the differential proliferation of these cells in hepatic growth processes.     

Sympathetic nervous system inhibition increases hepatic progenitors and reduces liver injury

Recovery from liver damage might be enhanced by encouraging repopulation of the liver by endogenous hepatic progenitor cells. Oval cells are resident hepatic stem cells that promote liver regeneration and repair. Little is known about the mediators that regulate the accumulation of these cells in the liver. Parasympathetic nervous system inhibition reduces the number of oval cells in injured livers. The effect of sympathetic nervous system (SNS) inhibition on oval cell number is not known. Adrenergic inhibition mobilizes hematopoietic precursors into the circulation and has also been shown to promote liver regeneration. Thus, we hypothesized that SNS inhibition would promote hepatic accumulation of oval cells and reduce liver damage in mice fed antioxidant-depleted diets to induce liver injury. Our results confirm this hypothesis. Compared with control mice that were fed only the antioxidant-depleted diets, mice fed the same diets with prazosin (PRZ, an alpha-1 adrenoceptor antagonist) or 6-hydroxydopamine (6-OHDA, an agent that induces chemical sympathectomy) had significantly increased numbers of oval cells. Increased oval cell accumulation was accompanied by less hepatic necrosis and steatosis, lower serum aminotransferases, and greater liver and whole body weights. Neither PRZ nor 6-OHDA affected the expression of cytokines, growth factors, or growth factor receptors that are known to regulate progenitor cells. In conclusion, stress-related sympathetic activity modulates progenitor cell accumulation in damaged livers and SNS blockade with alpha-adrenoceptor antagonists enhances hepatic progenitor cell accumulation.     

Immunohistochemical study of hepatic oval cells in human chronic viral hepatitis

AIM: To detect immunohistochemically the presence of oval cells in chronic viral hepatitis with antibody against c-kit. METHODS: We detected oval cells in paraffin embedded liver sections of 3 normal controls and 26 liver samples from patients with chronic viral hepatitis, using immunohistochemistry with antibodies against c-kit, piclass glutathione S-transferase (pi-GST) and cytokeratins 19 (CK19). RESULTS: Oval cells were not observed in normal livers. In chronic viral hepatitis, hepatic oval cells were located predominantly in the periportal region and fibrosis septa,characterized by an ovoid nucleus, small size,and scant cytoplasm. Antibody against stem cell factor receptor, c-kit, had higher sensitivity and specificity than pi-GST and CK19. About 50%-70% of c-kit positive oval cells were stained positively for either pi-GST or CK19. CONCLUSION: Oval cells are frequently detected in human livers with chronic viral hepatitis, suggesting that oval cell proliferation is associated with the liver regeneration in this condition.     

Epithelioid granulomas in chronic hepatitis C: A transient pathological feature

Multiple hepatic granulomas in chronic hepatitis C patients treated with alpha interferon were recently observed. To assess the presence of hepatic granulomas in chronic hepatitis C, and to determine whether their presence is related to interferon therapy or primarily related to chronic hepatitis C viral (HCV) infection, 446 liver biopsy specimens from 239 Japanese patients with chronic hepatitis C were reviewed. Well-formed non-caseating epithelioid granulomas were found in five (1.1%) of 446 liver biopsy specimens from five (2.0%) of 239 patients. All five patients had been followed up for 1 to 3 years, having between one and six liver biopsy specimens taken at intervals of 6 months to 1 year. Four of these five patients received alpha interferon therapy during the follow-up period. Hepatic granulomas were found in one of the pretherapy liver biopsy specimens in four patients and in one of the post-therapy specimens in one patient. Extensive investigation of the aetiology of hepatic granulomas yielded no conclusive findings. The presence of hepatic granulomas could not be demonstrated in follow-up liver biopsy specimens taken from the four patients who had undergone alpha interferon therapy. These findings suggest that hepatic granulomas may appear as an expression of non-specific reaction in HCV-related chronic hepatitis, and are not related to alpha interferon therapy.     

Interferon regulatory factor-3 activation, hepatic interferon-stimulated gene expression, and immune cell infiltration in hepatitis C virus patients

Interferon regulatory factor-3 (IRF-3) activation directs alpha/beta interferon production and interferon-stimulated gene (ISG) expression, which limits virus infection. Here, we examined the distribution of hepatitis C virus (HCV) nonstructural 3 protein, the status of IRF-3 activation, and expression of IRF-3 target genes and ISGs during asynchronous HCV infection in vitro and in liver biopsies from patients with chronic HCV infection, using confocal microscopy and functional genomics approaches. In general, asynchronous infection with HCV stimulated a low-frequency and transient IRF-3 activation within responsive cells in vitro that was associated with cell-to-cell virus spread. Similarly, a subset of HCV patients exhibited the nuclear, active form of IRF-3 in hepatocytes and an associated increase in IRF-3 target gene expression in hepatic tissue. Moreover, ISG expression profiles formed disease-specific clusters for HCV and control nonalcoholic fatty liver disease patients, with increased ISG expression among the HCV patients. We identified the presence of T cell and plasmacytoid dendritic cell infiltrates within all biopsy specimens, suggesting they could be a source of hepatic interferon in the setting of hepatitis C and chronic inflammatory condition. CONCLUSION: These results indicate that HCV can transiently trigger IRF-3 activation during virus spread and that in chronic HCV, IRF-3 activation within infected hepatocytes occurs but is limited.     

The origin and liver repopulating capacity of murine oval cells

The appearance of bipotential oval cells in chronic liver injury suggests the existence of hepatocyte progenitor/stem cells. To study the origin and properties of this cell population, oval cell proliferation was induced in adult mouse liver by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and a method for their isolation was developed. Transplantation into fumarylacetoacetate hydrolase (Fah) deficient mice was used to determine their capacity for liver repopulation. In competitive repopulation experiments, hepatic oval cells were at least as efficient as mature hepatocytes in repopulating the liver. In mice with chimeric livers, the oval cells were not derived from hepatocytes but from liver nonparenchymal cells. This finding supports a model in which intrahepatic progenitors differentiate into hepatocytes irreversibly. To determine whether oval cells originated from stem cells residing in the bone marrow, bone marrow transplanted wild-type mice were treated with DDC for 8 months and oval cells were then serially transferred into Fah mutants. The liver repopulating cells in these secondary transplant recipients lacked the genetic markers of the original bone marrow donor. We conclude that hepatic oval cells do not originate in bone marrow but in the liver itself, and that they have valuable properties for therapeutic liver repopulation.     

Ductular proliferation in liver tissues with severe chronic hepatitis B： An immunohistochemical study

AIM: To clarify the pathogenesis of ductular proliferation and its possible association with oval cell activation and hepatocyte regeneration. METHODS: Immunohistochemical staining and image analysis of the ductular structures in the liver tissues from 11 patients with severe chronic hepatitis B and 2 healthy individuals were performed. The liver specimens were sectioned serially, and then cytokeratin 8 (CK8), CK19, OV6, proliferating cell nuclear antigens (PCNA), glutathione-S-transferase (GST),α-fetal protein (AFP) and albumin were stained immunohistochemically. RESULTS: Typical and atypical types of ductular proliferation were observed in the portal tracts of the liver tissues in all 11 patients. The proliferating ductular cells were positive for CK8, CK19, OV6 and PCNA staining. Some atypical ductular cells displayed the morphological and immunohistochemical characteristics of hepatic oval cells. Some small hepatocyte-like cells were between hepatic oval cells and mature hepatocytes morphometri-cally and immunohistochemically. CONCLUSION: The proliferating ductules in the liver of patients with severe chronic liver disease may have different origins. Some atypical ductular cells are actually activated hepatic oval cells. Atypical ductular proliferation is related to hepatocyte regeneration and small hepatocyte-like cells may be intermediate transient cells between hepatic oval cells and mature hepatocytes.     

Hepatic Fas protein expression might be a predictive factor for hepatocellular carcinoma development in patients with chronic hepatitis C undergoing interferon therapy

BACKGROUND: Several studies have revealed that interferon treatment may reduce the incidence of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C virus (HCV). However, even after eradication of HCV, patients remain at risk for developing HCC. STUDY: Of 153 consecutive HCV patients who were treated with interferon and followed up for 5 years, 17 (11.1%) developed HCC. To elucidate predictive factors of HCC development, multivariate analysis was done for the 153 patients, and Fas protein expression in the biopsied specimens of liver before interferon treatment was examined in 17 patients who developed HCC and 17 patients who did not. RESULTS: Among the independent factors (sex, age, HCV genotype, HCV-RNA level, effect of interferon therapy, serum alanine aminotransferase before interferon therapy, and histologic stage and grade) tested by Cox proportional-hazards analysis, histologic stage (hepatic fibrosis) before interferon was significantly associated with HCC development (p = 0.01). In addition, the intensity of Fas protein expression was significantly greater in the liver specimens of the patients who developed HCC than in those who did not (p = 0.015). CONCLUSION: Histologic stage (hepatic fibrosis) and Fas protein expression before interferon treatment might be indicative of the need for intensive follow-up in patients with chronic hepatitis C undergoing interferon therapy.     

Immunohistochemical evidence for hepatic progenitor cells in liver diseases

BACKGROUND/AIM: Proliferative bile ductular reactions occur in a variety of liver diseases in humans. It is a matter of debate whether such reactions result from progenitor cell proliferation with biliary and hepatocytic differentiation, versus biliary metaplasia of damaged hepatocytes. We investigated bile ductular reactions in liver diseases, paying particular attention to the presence of cells with intermediate (hepatocytic/biliary) features (oval-like cells). METHODS: Five specimens each were selected of submassive hepatic necrosis and cirrhosis due to hepatitis B, hepatitis C, autoimmune hepatitis, alcohol injury, primary biliary cirrhosis and primary sclerosing cholangitis. Immunohistochemical stains were performed for biliary markers (cytokeratins [CKs] 7 and 19), as well as hepatocytic markers (HepParl and alpha-fetoprotein[AFP]) in sequential sections. The degree of staining of each cell type (biliary, hepatocytic, intermediate) was graded semiquantitatively. RESULTS: Hepatocytes always stained diffusely for HepParl, occasionally for CK7, and rarely for CK19. Biliary cells were always diffusely positive for CK7 and CK19, and rarely for HepParl. Intermediate cells were identified in all cases and showed widespread staining for both HepParl and CK7, and less commonly for CK19. AFP was not expressed in any cell type. The morphologic and immunohistochemical features of bile ductular reactions were similar in the different diseases. CONCLUSIONS: Proliferating hepatic parenchymal cells with intermediate (hepatocytic/biliary) morphologic features and combined immunophenotype can be identified in a variety of acute and chronic liver diseases. The similarity of bile ductular reactions among chronic hepatitic, alcoholic and biliary diseases suggests that they result from proliferation of oval-like progenitor cells.     

慢性重型肝炎患者肝组织内胆管增生的免疫组织化学研究

在慢性重型肝炎患者的肝组织切片中,除肝细胞大面积坏死、纤维组织增生、炎症细胞浸润及不同程度的肝细胞再生等病理特征外,还常有明显的胆管增生现象,但这种胆管增生的本质及意义尚不清楚。新近提出肝组织内胆管增生与肝干细胞     

Dose- and time-dependent oval cell reaction in acetaminophen-induced murine liver injury

We examined the response of murine oval cells, that is, the putative liver progenitor cells, to acetaminophen. Female C57BL/6J mice were injected intraperitoneally with varying doses of N-acetyl-paraaminophen (APAP) (250, 500, 750, and 1,000 mg/kg of weight) and sacrificed at 3, 6, 9, 24, and 48 hours. In preliminary studies, we showed that anticytokeratin antibodies detected A6-positive cells with a sensitivity and specificity of greater than 99%. The oval cell reaction was quantified, on immunostaining for biliary-type cytokeratins, as both number and density of oval cells per portal tract, analyzed by size of portal tract. Acetaminophen injury was followed by periportal oval cell accumulation displaying a moderate degree of morphological homogeneity. Oval cell response was biphasic, not temporally correlating with the single wave of injury seen histologically. Increases in oval cells were largely confined to the smallest portal tracts, in keeping with their primary derivation from the canals of Hering, and increased in a dose-dependent fashion. The timing of the two peaks of the oval cell reaction also changed with increasing dose, the first becoming earlier and the second later. In conclusion, our studies indicate a marked oval cell activation during the height of hepatic injury. Oval cells appear to be resistant to acetaminophen injury. The close fidelity of mechanism and histology of acetaminophen injury between mouse and human livers makes it a useful model for investigating liver regeneration and the participation of stem/progenitor cells in that process.     

Characterization and enrichment of hepatic progenitor cells in adult rat liver

AIM: To detect the markers of oval cells in adult rat liver and to enrich them for further analysis of characterization in vitro. METHODS: Rat model for hepatic oval cell proliferation was established with 2-acetylaminofluorene and two third partial hepatectomy (2-AAF/PH). Paraffin embedded rat liver sections from model (11 d after hepatectomy) and control groups were stained with HE and OV6, cytokeratin19 (CK19), albumin, alpha fetoprotein (AFP), connexin43, and c-kit antibodies by immunohistochemistry. Oval cell proliferation was measured with BrdU incorporation test. C-kit positive oval cells were enriched by using magnetic activated cell sorting (MACS).The sorted oval cells were cultured in a low density to observe colony formation and to examine their characterization in vitro by immunocytochemistry and RT-PCR. RESULTS: A 2-AAF/PH model was successfully established to activate the oval cell compartment in rat liver. BrdU incorporation test of oval cell was positive. The hepatic oval cells coexpressed oval cell specific marker OV6, hepatocyte-marker albumin and cholangiocyte-marker CK19. They also expressed AFP and connexin 43. C-kit, one hematopoietic stem cell receptor, was expressed in hepatic oval cells at high levels. By using c-kit antibody in conjunction with MACS, we developed a rapid oval cell isolation protocol. The sorted cells formed colony when cultured in vitro. Cells in the colony expressed albumin or CK19 or coexpressed both and BrdU incorporation test was positive. RT-PCR on colony showed expression of albumin and CK19 gene. CONCLUSION: Hepatic oval cells in the 2-AAF/PH model had the properties of hepatic stem/progenitor cells. Using MACS, we established a method to isolate oval cells. The sorted hepatic oval cells can form colony in vitro which expresses different combinations of phenotypic markers and genes from both hepatocytes and cholangiocyte lineage.