Biliary expression of heat shock protein: a non-specific feature of chronic cholestatic liver diseases.

AIM: To analyse the expression of heat shock protein (HSP) 60 in biliary epithelium in auto-immune liver conditions and also in chronic cholestatic and other liver diseases. METHODS: Hepatic expression of HSP-60 in frozen liver biopsy specimens from patients with primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), auto-immune hepatitis (AIH), obstructive jaundice (LDO), alcoholic liver disease (ALD), and from normal controls was studied by immunohistochemistry using the APAAP technique and confocal laser scanning microscopy. RESULTS: Increased expression of HSP-60 was demonstrated in the biliary epithelium of patients with PBC, LDO and, to a lesser extent, with PSC. Focal, weaker, biliary epithelial expression of HSP-60 was observed in AIH, ALD and normal liver tissue. Increased expression was also seen on Kupffer cells in LDO and in hepatocytes in areas of piecemeal necrosis in AIH. CONCLUSION: Enhanced biliary expression of HSP-60 is a common feature of chronic biliary disease irrespective of aetiology and is not specific to auto-immune diseases.     

Hepatic expression of toll-like receptor 4 in primary biliary cirrhosis

Toll-like receptor 4 (TLR4) is a receptor for bacterial lipopolysaccharide, which is suggested to be involved in the pathogenesis of disease of hepatobiliary tracts. To explore a possible role for this receptor in primary biliary cirrhosis (PBC), we investigated the expression of TLR4 in liver tissues from PBC patients. We studied liver biopsy sections from 62 PBC patients and 41 patients with chronic hepatitis C (CHC). Expression of TLR4 in paraffin-embedded sections was analyzed by immunohistochemistry. The bile duct epithelial cells (BECs) of PBC liver tissues markedly expressed TLR4, whereas BECs of CHC liver tissues barely expressed TLR4. The TLR4 expression was also observed in periportal hepatocytes of PBC liver tissues and its expression was extended to interlobular hepatocytes in advanced stage PBC. Although periportal hepatocytes of CHC liver tissues expressed TLR4, its expression levels were not correlated with the fibrosis stage. Our data demonstrated that TLR4 was expressed in BECs and periportal hepatocytes in PBC livers, suggesting the possible involvement of bacterial pathogens and TLR4 in the inflammatory processes of PBC.     

Increased expression of nuclear envelope gp210 antigen in small bile ducts in primary biliary cirrhosis

The sustained antibody response to nuclear envelope gp210 antigen indicates a group of primary biliary cirrhosis (PBC) patients at high risk for the progression to end-stage hepatic failure. To address this issue, we immunohistochemically studied the expression of gp210 antigen in needle liver biopsy specimens from PBC patients using a monoclonal antibody specific for gp210 antigen. The specimens from autoimmune hepatitis (AIH), chronic viral hepatitis B (CHB) and C (CHC) patients served as disease controls. The expression of gp210 antigen was apparently increased on the nuclear envelope of biliary epithelial cells (BECs) of small bile ducts in almost all specimens from PBC. In contrast, the expression of gp210 antigen was negative in BECs of small bile ducts in normal liver, while relatively weak anti-gp210 immunostaining was observed in AIH, CHC and CHB. In addition, the degree of gp210 expression in BECs of small bile ducts was positively correlated to that of portal inflammation, interface hepatitis and lobular inflammation in PBC. These results indicate that the increased expression of gp210 in small bile ducts, which is probably associated with damage to BECs by inflammation, is possibly involved in autoimmune response to gp210 leading to the progression to end-stage hepatic failure in PBC.     

Histological grading and staging in chronic hepatitis: its practical correlation

Although the histological features of various causes of chronic liver disease have been well described, usually the inflammatory activity of the disease is important after the cause has been established. Some patients have co-infection or concomitant liver disease and on occasion it is difficult to decide the treatment. In order to clarify the histological differences, we investigated the inflammatory activity among autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), chronic hepatitis C (CHC) and chronic hepatitis B (CHB) in a standardized way using the modified histological activity index (HAI). According to the modified HAI, inflammatory activity is divided into four categories; categories A/D explains portal/periportal inflammation and categories B/C explains lobular activity. The inflammatory score of AIH tended to be greater in all categories from the early stage of fibrosis, whereas scores of PBC were lower, except for portal inflammation. Chronic hepatitis C patients had portal or periportal inflammation, and their inflammatory scores were linked to the development of fibrosis. Chronic hepatitis B patients tended to have severe lobular injury, but did not have a relationship between the inflammatory score and their stage. To know the distribution of inflammation using the modified HAI scoring system may be helpful and convenient in evaluating patients with chronic inflammatory liver disease.     

cDNA microarray analysis of autoimmune hepatitis, primary biliary cirrhosis and consecutive disease manifestation

While autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) usually have distinct clinical manifestations, some patients present with features of both conditions. Using cDNA microarrays, we analyzed and compared gene expression profiles in 8 patients with AIH, 9 with PBC, 8 with chronic hepatitis C (CHC), 8 with non-alcoholic steatohepatitis (NASH) and 9 with normal livers. We subsequently applied this method to a tissue sample from a 61-year-old woman with overlapping features of both AIH and PBC. A 61-year-old woman was admitted to our hospital for evaluation of elevated serum alkaline phosphatase. A liver biopsy showed accumulation of mononuclear cells around the bile duct cells, a feature characteristic of chronic non-suppurative destructive cholangitis (CNSDC). Three years later, her serum alanine aminotransferase (ALT) level had increased, and a liver biopsy demonstrated evidence of a severe form of hepatitis. A cDNA microarray analysis of both biopsies identified the molecular events associated with her altered histology. The expression profile of this patient, which was originally different from that of the other PBC patients, changed to an AIH pattern. Our results suggest that this patient has characteristics of both AIH and PBC.     

Intrahepatic PD-1/PD-L1 Up-regulation Closely Correlates with Inflammation and Virus Replication in Patients with Chronic HBV Infection

Chronic hepatitis B was characterized by fluctuant immune response to infected hepatocytes resulting in hepatic inflammation and virus persistence. Recently, Programmed Death-1 (PD-1) and its ligand PD-L1 have been demonstrated to play an essential role in balancing antiviral immunity and inflammation in the livers of acute hepatitis B patients, significantly influencing disease outcome. PD-1 up-regulation in peripheral T cells is associated with immune dysfunction in chronic hepatitis B patients. However, the effect of PD-1/PD-L1 on hepatic damage and chronic infective status is still unknown in patients with chronic HBV infection. Here, we report up-regulation of PD-1 and PD-L1 in liver biopsies from 32 chronic HBV patients compared to 4 healthy donors. PD-1/PD-L1 up-regulation was significantly associated with hepatic inflammation and ALT elevation. Moreover, appropriate up-regulation but not overexpression of PD-L1 in the active phase of chronic hepatitis B as well as lower expression of PD-L1 in the inactive phase in liver residential antigen presenting cells (including Kupffer cells and sinusoidal endothelial cells) may contribute to viral inhibition. Our data suggest that the intrahepatic interaction of PD-1 and PD-L1 might play an important role in balancing the immune response to HBV and immune-mediated liver damage in chronic HBV infection.     

Detection of hepatitis A antigen in human liver.

For the first time, hepatitis A viral antigen (HAAg) was shown in liver biopsy tissue from a patient in the acute phase of hepatitis type A by light and electron microscopy, using the peroxidase-antibody technique. Under light microscopy, the staining for HAAg appeared as a fine, granular reaction product, scattered throughout the cytoplasm of hepatocytes and sinusoidal lining cells. Standard thin-section electron microscopy revealed virus-like particles, 24 to 27 nm in diameter, in cytoplasmic vesicles of hepatocytes and Kupffer cells. By immunoperoxidase electron microscopy, HAAg was detected on particles aggregated within cytoplasmic vesicles of hepatocytes, thus demonstrating that the virus-like particles (24 to 27 nm) are hepatitis A virus. The surrounding membrane of the vesicles was also positive for HAAg. The distribution patterns of HAAg in human liver were virtually identical to those described for experimentally infected marmosets. It is notable that most HAAg was detected within vesicles of liver cell cytoplasm, suggesting the possibility of vesicle-oriented morphogenesis of hepatitis A virus.     

Chronic hepatitis B and C in renal graft recipients

Clinical and morphological features of chronic hepatitis B (CHB), C (CHC), and B+C (CHB+C) were studied in 283 renal graft recipients. High total bilirubin serum levels were detected significantly more often in CHB and CHB+C patients vs. CHC patients. High ALT activity was noted in 65% of CHB patients and only in 45% of CHC patients (p = 0.003). Stable low activity of hepatitis prevailed in renal recipients; it was noted in 56.7% of CHB patients, 66.2% of CHC patients, and 62% of CHB+C patients. The character of pathomorphological liver changes in chronic viral hepatitis was studied in 53 renal graft recipients using puncture biopsy. Histopathological activity index (HAI, Knodell R.G. et al., 1981) witnessed a more severe liver lesion in CHB vs. CHC and CHB+C. Thus, inflammatory activity in CHB was found to be minimal or low in 13 patients, and moderate or high in 11 patients, whilst a minimal or low activity in CHC or CHB+C was found in 16 and 10 patients, respectively, and a moderate activity was detected only in two and one, respectively (p = 0.016 and 0.024 compared with CHB). Advanced hepatic fibrosis or cirrhosis was significantly more frequent (p = 0.006) in CHB patients (eight out of 24) than in CHC ones (none out of 18). The rate of advanced sclerotic changes in CHB+C was lower (one out of 10 patients) than that in CHB, and similar to CHC. Thus, clinico-morphological manifestations were more prominent in renal graft recipients with CHB vs. CHC.     

Expression of Perforin and Fas Ligand mRNA in the Liver of Viral Hepatitis

Cytotoxic T lymphocytes (CTLs) play an important role in the pathogenesis of viral hepatitis. We studied the expression of mRNAs of perforin and Fas ligand (Fas-L) in biopsy specimens from chronic hepatitis B (CHB) (15 cases) and hepatitis C (CHC) patients (13 cases). Both perforin and Fas-L mRNAs were detected in all cases of both CHB and CHC. No messages were detected in the control livers from two cases of fatty liver, a case of Gilbert's syndrome, and a case of Dubin–Johnson syndrome. Semiquantitative analysis revealed a positive correlation between the intensity of perforin and Fas-L mRNAs in both CHB and CHC. In CHB, the intensity of both perforin and Fas-L mRNAs showed a positive correlation with the histological activity and serum alanine aminotransferase level, while the correlation was not apparent in CHC. These results suggest that both perforin and Fas/Fas-L systems are involved in the pathogenesis of liver cell injury of CHB and CHC.     

Myeloid Mixed Lineage Kinase 3 Contributes to Chronic Ethanol-Induced Inflammation and Hepatocyte Injury in Mice

Proinflammatory activity of hepatic macrophages plays a key role during progression of alcoholic liver disease (ALD). Since mixed lineage kinase 3 (MLK3)-dependent phosphorylation of JNK is involved in the activation of macrophages, we tested the hypothesis that myeloid MLK3 contributes to chronic ethanol-induced inflammatory responses in liver, leading to hepatocyte injury and cell death. Primary cultures of Kupffer cells, as well in vivo chronic ethanol feeding, were used to interrogate the role of MLK3 in the progression of liver injury. Phosphorylation of MLK3 was increased in primary cultures of Kupffer cells isolated from ethanol-fed rats compared to cells from pair-fed rats. Kupffer cells from ethanol-fed rats were more sensitive to LPS-stimulated cytokine production; this sensitization was normalized by pharmacological inhibition of MLK3. Chronic ethanol feeding to mice increased MLK3 phosphorylation robustly in F4/80⁺ Kupffer cells, as well as in isolated nonparenchymal cells. MLK3^−/− mice were protected from chronic ethanol-induced phosphorylation of MLK3 and JNK, as well as multiple indicators of liver injury, including increased ALT/AST, inflammatory cytokines, and induction of RIP3. However, ethanol-induced steatosis and hepatocyte apoptosis were not affected by MLK3. Finally, chimeric mice lacking MLK3 only in myeloid cells were also protected from chronic ethanol-induced phosphorylation of JNK, expression of inflammatory cytokines, and increased ALT/AST. MLK3 expression in myeloid cells contributes to phosphorylation of JNK, increased cytokine production, and hepatocyte injury in response to chronic ethanol. Our data suggest that myeloid MLK3 could be targeted for developing potential therapeutic strategies to suppress liver injury in ALD patients.Key words: Alcoholic liver disease (ALD), Kupffer cells, Necroptosis, Toll-like receptor 4 (TLR4), Cytokines     

Hepatic progenitor cells in chronic hepatitis C: a phenomenon of older age and advanced liver disease

Hepatic progenitor cells (HPC) appear in a variety of liver diseases. Their occurrence in chronic hepatitis C (CHC) remains unclear, and triggering factors have to be elucidated. The presence of HPC in CHC was examined in relation to histological and virological parameters and patient age. Fifty liver biopsies of HCV-infected patients were examined. The presence of HPC was evaluated by immunohistochemical expression of keratin 7 (K7). Double immunostaining with K7 and cell proliferation marker Ki-67 was undertaken. Ductular reaction at the limiting plate, mean number of isolated progenitor cells (IPC) and isolated ductular structures (IDS) were quantified. The predominant distribution pattern of IPC and IDS and the presence of K7(+) hepatocytes were registered. Relationship between ductular reaction, IPC, IDS, presence of K7(+) hepatocytes, and patient age, hepatitis grade and stage, HCV RNA, and HCV genotype was examined. Prominent ductular reaction and increased numbers of IPC and IDS correlated significantly with older age and severe fibrosis/cirrhosis. The above HPC subtypes were not proliferating. Periportal/periseptal distribution pattern of IPC and IDS and presence of K7(+) hepatocytes were significantly more frequent in advanced hepatitis stages and in patients older than 40 years. Intraparenchymal distribution pattern correlated with younger age, lobular activity, and early fibrosis stage. K7(+) hepatocytes were encountered almost exclusively in the periportal pattern and in the presence of interface hepatitis and were more frequent among HCV genotype-1 patients. HPC activation in CHC is a common but diverse phenomenon closely related to patient age and hepatitis stage.     

Immunoglobulin classes in biopsies of autoimmune liver disease

The immunological isotypes of plasma cell infiltrates in a series of consecutive liver biopsies from patients with chronic active hepatitis (CAH) and primary biliary cirrhosis (PBC) were determined immunohistochemically. The plasma cell infiltrate was more pronounced in PBC than in CAH. IgA and IgG isotypes predominated in CAH, and IgM and IgG in PBC. The expected predominance of kappa light chains was observed in every biopsy in PBC. However, in 8/14 CAH biopsies the plasma cells were predominantly lambda isotype. Lambda predominance was significantly associated with the presence of serum autoantibodies. These findings would suggest that different mechanisms operate in the pathogenesis of these two autoimmune liver diseases.     

Role of sinusoidal cells in the morphogenesis of alcoholic liver cirrhosis]

49 liver biopsies in alcoholic liver damage (fibrosis, chronic persisting hepatitis, cirrhosis) are studied on semithin sections electron microscopically and morphometrically. Morphological varieties of hepatocytes and sinusoidal cells are revealed as well as some rules in their proportions. Domination of stellate reticulo-endotheliocytes with a high secretory and phagocyte activity correlates with high adaptive properties of hepatocytes and transformation of lipocytes into fibroblast-like cells without development of intralobular fibrosis. If nonactive variant of the stellate reticulo-endotheliocytes prevails, the number of damaged hepatocytes and fibroblast-like lipocytes increases, intralobular fibrosis becomes pronounced resulting in hepatocyte death. Role of sinusoidal cells in realization of alcoholic cirrhosis morphogenesis is discussed.     

肝窦及窦周隙病变在慢性乙型肝炎肝微循环障碍中担当关键角色

目的 探讨肝窦及窦周隙病变对肝微循环障碍的影响.方法 对200例慢性乙型肝炎(CHB)肝组织用HE、组化、免疫组化及原位杂交技术,就肝窦及窦周隙病变的形态学、体视学及超微结构改变作了观察.结果 其病变有狭窄、阻塞、扩张、窦周隙淤血、肝窦毛细血管化表现.体视学示肝窦数目减少,总面积、总周长、平均直径增加,与对照组差异有显著性(P<0.01),CHB轻与CHB中、CHB重及CHB/LC之间差异也有显著性(P<0.01).电镜观察示窦内皮有撕裂、窗孔减少、窦内皮细胞增生出芽形成血管腔,贮脂细胞转化为肌成纤维细胞,同时伴相关蛋白表达增加.结论 肝窦及窦周隙病变在肝微循环障碍中担当     

散发性戊型肝炎肝组织病理学及病毒学研究

目的研究散发性戊型肝炎(HE)肝组织病理特征、病毒分布及复制,探讨戊型肝炎病毒(HEV)致肝损伤机制.方法对36例发病急性期(20例)和恢复期(16例)穿刺肝组织,及18例慢性乙型肝炎(CHB)近期重叠HEV感染肝组织,作光、电镜组织学观察,原位杂交HEVRNA检测,Kupffer细胞免疫组化观察.结果HE急性期肝组织病变有其相对特征易见肝细胞羽毛状变性(100.0%)、毛细胆管内淤胆(75.0%)及双核、多核肝细胞(65.0%),肝细胞凋亡小体偏大且不整,Kupffer细胞增生突出等.HEVRNA分布于肝细胞胞质近核周处,急性期肝组织HEVRNA阳性率(100.0%)明显高于恢复期(12.5%,P<0.001),且阳性肝细胞及病毒拷贝数较多,恢复期肝组织趋于复常.CHB重叠HEV感染者肝组织炎症活动度较单纯CHB为重.结论散发性HE相对于其他病毒性肝炎有其病理学特征;HEV感染肝细胞并主要于发病急性期复制活跃;急性散发性HE恢复后转归良好;细胞免疫性肝损伤可能为HEV的主要致肝损伤机制,但不能排除存在病毒直接致肝损伤作用.     

Studies on the relationships of IgA to human liver. IgA deposition in non-alcoholic liver diseases

An investigation was conducted to clarify the relationships of IgA to the human liver. Immunocytochemical studies were performed on biopsy specimens from patients with cirrhosis and chronic hepatitis without any apparent history of alcohol abuse. The results showed that 1) a large amount of IgA is associated with the sinusoidal surface of hepatocytes, endothelial cells and Kupffer cells, 2) this IgA contains J chain and can form a complex with secretory component, and 3) this mainly belongs to the IgA1 subclass, 4) IgA in vesicles within hepatocytes and Kupffer cells is always associated with acid phosphatase activity, and 5) IgA containing vesicles within ductular epithelial cells always lack such enzyme activity. We conclude that 1) the IgA bound to the surface of hepatocytes, sinus endothelial cells and Kupffer cells is polymeric IgA1 uncomplexed with SC, and 2) this IgA occasionally enters these cells, and may be degraded in the lysosomes. 3) Polymeric IgA combines with SC in the ductular epithelium and may be secreted into bile. These findings suggest that J chain-linked polymeric IgA bound to the surface of hepatocytes and Kupffer cells has a certain pathological significance in liver diseases and might be involved in the clearance of excess IgA from the circulation.     

Localisation of intrahepatic interleukin 6 in patients with acute and chronic liver disease.

AIM: To evaluate the role of local interleukin 6 (IL-6) in the pathogenesis of acute and chronic liver disease. METHODS: The cellular site of IL-6 in cryostat sections of liver from 31 patients with liver disease was examined using indirect immunofluorescence with a monoclonal antibody. RESULTS: IL-6 staining in sinusoidal endothelial cells was very noticeable and diffusely distributed in the lobules of specimens of acute viral hepatitis. IL-6 expression in endothelial cells, particularly in necrotic areas of hepatocytes, was increased and was accompanied by enhanced expression in Kupffer cells. In contrast, IL-6 staining in infiltrating mononuclear cells was prominent in portal tracts, and the numbers of cytokine positive cells were greater in specimens of chronic active hepatitis compared with chronic persistent hepatitis. In non-specific reactive hepatitis intrahepatic expression of IL-6 was minimal, while in alcoholic liver fibrosis the cytokine distribution in the lobules was similar to that of acute viral hepatitis. CONCLUSIONS: These results indicate that locally produced IL-6 contributes to the inflammatory process and immunological response in acute and chronic liver disease.     

Distinctive intrahepatic characteristics of paediatric and adult pathogenesis of chronic hepatitis C infection

Mechanisms leading to liver damage in chronic hepatitis C (CHC) are being discussed, but both the immune system and the virus are involved. The aim of this study was to evaluate intrahepatic viral infection, apoptosis and portal and periportal/interface infiltrate in paediatric and adult patients to elucidate the pathogenesis of chronic hepatitis C. HCV-infected, activated caspase-3⁺ and TUNEL⁺ hepatocytes, as well as total, CD4⁺, CD8⁺, Foxp3⁺ and CD20⁺ lymphocytes infiltrating portal and periportal/interface tracts were evaluated in 27 paediatric and 32 adult liver samples by immunohistochemistry or immunofluorescence. The number of infected hepatocytes was higher in paediatric than in adult samples (p 0.0078). In children, they correlated with apoptotic hepatocytes (activated caspase-3⁺r = 0.74, p < 0.0001; TUNEL⁺r = 0.606, p 0.0017). Also, infected (p = 0.026) and apoptotic hepatocytes (p = 0.03) were associated with the severity of fibrosis. In adults, activated caspase-3⁺ cell count was increased in severe hepatitis (p = 0.009). Total, CD4⁺, CD8⁺ and Foxp3⁺ lymphocyte count was higher in adult samples (p < 0.05). Paediatric CD8⁺ cells correlated with infected (r = 0.495, p 0.04) and TUNEL⁺ hepatocytes (r = 0.474, p = 0.047), while adult ones correlated with activated caspase-3⁺ hepatocytes (r = 0.387, p 0.04). In adults, CD8⁺ was associated with hepatitis severity (p < 0.0001) and correlated with inflammatory activity (CD8⁺ r = 0.639, p 0.0003). HCV, apoptosis and immune response proved to be involved in CHC pathogenesis of both paediatric and adult patients. However, liver injury in paediatric CHC would be largely associated with a viral cytopathic effect mediated by apoptosis, while in adults it would be mainly associated with an exacerbated immune response.