Comparative hepatology最新文献

Background: Biliary atresia (BA) is an idiopathic inflammatory obliterative cholangiopathy of neonates, leading to progressive biliary cirrhosis. Hepatoportoenterostomy (Kasai procedure) can cure jaundice in 30% to 80% of patients. Postoperative clearance of jaundice is one of the most important factors influencing long-term outcomes of BA patients. Multidrug resistance protein 2 (MRP2) is one of the canalicular export pumps located in hepatocytes; it exports organic anions and their conjugates (e.g., bilirubin) into bile canaliculus. Although MRP2 is an essential transporter for the excretion of bilirubin, its role in the clinical course of BA patients is unclear. The present study investigated the relationship between hepatic MRP2 expression and clinical course in BA patients, with particular emphasis in curing jaundice after hepatoportoenterostomy.

Results: No significant differences in hepatic MRP2 expression level were observed between BA and controls groups. There was no correlation between MRP2 expression and age at time of surgery in BA and control groups. In BA patients, MRP2 expression level in the jaundice and jaundice-free group did not differ significantly (2.0 × 10-4 vs 3.1 × 10-4, p = 0.094). Although the serum level of total bilirubin just before surgery did not correlate with MRP2 expression level (rs = 0.031, p = 0.914), the serum level of total bilirubin measured at 2 weeks (rs = -0.569, p = 0.034) and 4 weeks after surgery (rs = -0.620, p = 0.018) were significantly correlated with MRP2 expression level. Furthermore, MRP2 expression level was inversely correlated with ratio of change in serum total bilirubin level over 4 weeks (rs = -0.676, p = 0.008), which represents the serum bilirubin level measured at 4 weeks after surgery divided by value just before surgery. There was no correlation between expression level of MRP2 and nuclear receptors, such as retinoid × receptor α, farnesoid × receptor, pregnane × receptor, or constitutive androstane receptor.

Conclusions: Hepatic MRP2 expression level was associated with postoperative clearance of jaundice in BA patients, at least within 1 month after hepatoportoenterostomy. This finding suggests that not only morphological appearance of the liver tissue but also the biological status of hepatocytes is important for BA pathophysiology.

Situs inversus totalis is is a congenital anomaly associated with various visceral abnormalities, but there is no data about the relationship between secondary biliary cirrhosis and that condition. We here present a case of a 58 year-old female with situs inversus totalis who was admitted to our clinic with extrahepatic cholestasis. After excluding all potential causes of biliary cirrhosis, secondary biliary cirrhosis was diagnosed based on the patient's history, imaging techniques, clinical and laboratory findings, besides histolopathological findings. After treatment with tauroursodeoxycholic acid, all biochemical parameters, including total/direct bilirubin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and gama glutamyl transferase, returned to normal ranges at the second month of the treatment. We think that this is the first case in literature that may indicate the development of secondary biliary cirrhosis in a patient with situs inversus totalis. In conclusion, situs inversus should be considered as a rare cause of biliary cirrhosis in patients with situs inversus totalis which is presented with extrahepatic cholestasis.

Background: To understand the complex and largely not well-understood apoptotic pathway and immune system evasion mechanisms in hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC) and HCV associated chronic hepatitis (CH), we studied the expression patterns of a number of pro-apoptotic and anti-apoptotic genes (Fas, FasL, Bcl-2, Bcl-xL and Bak) in HepG2 cell line harboring HCV- genotype-4 replication. For confirmation, we also assessed the expression levels of the same group of genes in clinical samples obtained from 35 HCC and 34 CH patients.

Methods: Viral replication was assessed in the tissue culture medium by RT-PCR, quantitative Real-Time PCR (qRT-PCR); detection of HCV core protein by western blot and inhibition of HCV replication with siRNA. The expression level of Fas, FasL, Bcl-2, Bcl-xL and Bak was assessed by immunohistochemistry and RT-PCR whereas caspases 3, 8 and 9 were assessed by colorimetric assay kits up to 135 days post infection.

Results: There was a consistent increase in apoptotic activity for the first 4 weeks post-CV infection followed by a consistent decrease up to the end of the experiment. The concordance between the changes in the expression levels of Fas, FasL, Bcl-2, Bcl-xL and Bak in vitro and in situ was statistically significant (p < 0.05). Fas was highly expressed at early stages of infection in cell lines and in normal control liver tissues followed by a dramatic reduction post-HCV infection and an increase in the expression level of FasL post HCV infection. The effect of HCV infection on other apoptotic proteins started very early post-infection, suggesting that hepatitis C modulating apoptosis by modulating intracellular pro-apoptotic signals.

Conclusions: Chronic HCV infection differently modulates the apoptotic machinery during the course of infection, where the virus induces apoptosis early in the course of infection, and as the disease progresses apoptosis is modulated. This study could open a new opportunity for understanding the various signaling of apoptosis and in the developing a targeted therapy to inhibit viral persistence and HCC development.

Background: Ischemic pre- and postconditioning protects the liver against ischemia/reperfusion injuries. The aim of the present study was to examine how ischemic pre- and postconditioning affects gene expression of hypoxia inducible factor 1α (HIF-1α), vascular endothelial growth factor A (VEGF-A) and transforming growth factor β (TGF-β) in liver tissue.

Methods: 28 rats were randomized into five groups: control; ischemia/reperfusion; ischemic preconditioning (IPC); ischemic postconditioning (IPO); combined IPC and IPO. IPC consisted of 10 min of ischemia and 10 min of reperfusion. IPO consisted of three cycles of 30 sec. reperfusion and 30 sec. of ischemia.

Results: HIF-1α mRNA expression was significantly increased after liver ischemia compared to controls (p = 0.010). HIF-1α mRNA expression was significantly lower in groups subjected to IPC or combined IPC and IPO when compared to the ischemia/reperfusion group (p = 0.002). VEGF-A mRNA expression increased in the ischemia/reperfusion or combined IPC and IPO groups when compared to the control group (p < 0.05).

Conclusion: Ischemic conditioning seems to prevent HIF-1α mRNA induction in the rat liver after ischemia and reperfusion. This suggests that the protective effects of ischemic conditioning do not involve the HIF-1 system. On the other hand, the magnitude of the HIF-1α response might be a marker for the degree of I/R injuries after liver ischemia. Further studies are needed to clarify this issue.

Background: Kupffer cells are well known macrophages of the liver, however, the developmental characteristics of Kupffer cells in mice are not well understood. To clarify this matter, the characteristics of Kupffer macrophages in normal developing mouse liver were studied using light microscopy and immunocytochemistry.

Methods: Sections of liver tissue from early postnatal mice were prepared using immunocytochemical techniques. The Kupffer cells were identified by their immunoreactivity to the F4/80 antibody, whereas endothelial cells were labelled with the CD-34 antibody. In addition, Kupffer cells and endothelial cells were labelled by systemically injected fluorescently labelled latex microspheres. Tissue slices were examined by fluorescence microscopy.

Results: Intravenous or intraperitonal injections of microspheres yielded similar patterns of liver cell labelling. The F4/80 positive Kupffer cells were labelled with both large (0.2 μm) and small (0.02 μm) diameter microspheres, while endothelial cells were labelled only with the smaller diameter microspheres. Microsphere labelling of Kupffer cells appeared stable for at least 6 weeks. Cells immunoreactive for F4/80 were identified as early as postnatal day 0, and these cells also displayed uptake of microspheres. Numbers of F4/80 Kupffer cells, relative to numbers of albumin positive hepatocytes, did not show a significant trend over the first 2 postnatal weeks.

Conclusions: Kupffer cells of the developing mouse liver appear quite similar to those of other mammalian species, confirming that the mouse presents a useful animal model for studies of liver macrophage developmental structure and function.

Background: Repeated apnoea events cause intermittent hypoxia (IH), which alters the function of various systems and produces free radicals and oxidative stress.

Methods: We investigated hepatic oxidative stress in adult mice subjected to intermittent hypoxia, simulating sleep apnoea. Three groups were submitted to 21 days of IH (IH-21), 35 days of IH (IH-35), or 35 days of sham IH. We assessed the oxidative damage to lipids by TBARS and to DNA by comet assay; hepatic tissue inflammation was assessed in HE-stained slides. Antioxidants were gauged by catalase, superoxide dismutase, glutathione peroxidase activity and by total glutathione.

Results: After IH-21, no significant change was observed in hepatic oxidative stress. After IH-35, significant oxidative stress, lipid peroxidation, DNA damage and reduction of endogenous antioxidants were detected.

Conclusions: In an animal model of sleep apnoea, intermittent hypoxia causes liver damage due to oxidative stress after 35 days, but not after 21 days.

Background: Under compromised biliary regeneration, transdifferentiation of hepatocytes into biliary epithelial cells (BEC) has been previously observed in rats, upon exposure to BEC-specific toxicant methylene dianiline (DAPM) followed by bile duct ligation (BDL), and in patients with chronic biliary liver disease. However, mechanisms promoting such transdifferentiation are not fully understood. In the present study, acquisition of biliary specific transcription factors by hepatocytes leading to reprogramming of BEC-specific cellular profile was investigated as a potential mechanism of transdifferentiation in two different models of compromised biliary regeneration in rats.

Results: In addition to previously examined DAPM + BDL model, an experimental model resembling chronic biliary damage was established by repeated administration of DAPM. Hepatocyte to BEC transdifferentiation was tracked using dipetidyl dipeptidase IV (DDPIV) chimeric rats that normally carry DPPIV only in hepatocytes. Following DAPM treatment, ~20% BEC population turned DPPIV-positive, indicating that they are derived from DPPIV-positive hepatocytes. New ductules emerging after DAPM + BDL and repeated DAPM exposure expressed hepatocyte-associated transcription factor hepatocyte nuclear factor (HNF) 4α and biliary specific transcription factor HNF1β. In addition, periportal hepatocytes expressed biliary marker CK19 suggesting periportal hepatocytes as a potential source of transdifferentiating cells. Although TGFβ1 was induced, there was no considerable reduction in periportal HNF6 expression, as observed during embryonic biliary development.

Conclusions: Taken together, these findings indicate that gradual loss of HNF4α and acquisition of HNF1β by hepatocytes, as well as increase in TGFβ1 expression in periportal region, appear to be the underlying mechanisms of hepatocyte-to-BEC transdifferentiation.

Background: Proliferation of oval cells, the bipotent precursor cells of the liver, requires impeded proliferation and loss of hepatocytes as well as a specific micro-environment, provided by adjacent sinusoidal cells of liver. Despite their immense importance for triggering the oval cell response, cells of hepatic sinusoids are rarely investigated. To elucidate the response of sinusoidal liver cells we have employed a choline-deficient, ethionine-supplemented (CDE) diet, a common method for inducing an oval cell response in rodent liver. We have utilised selected expression markers commonly used in the past for phenotypic discrimination of oval cells and sinusoidal cells: cytokeratin, E-cadherin and M2-pyruvate kinase for oval cells; and glial fibrillary acidic protein (GFAP) was used for hepatic stellate cells (HSCs).

Results: CDE diet leads to an activation of all cells of the hepatic sinusoid in the mouse liver. Beside oval cells, also HSCs and Kupffer cells proliferate. The entire fraction of proliferating cells in mouse liver as well as endothelial cells and cholangiocytes express M2-pyruvate kinase. Concomitantly, GFAP, long considered a unique marker of quiescent HSCs was upregulated in activated HSCs and expressed also in cholangiocytes and oval cells.

Conclusions: Our results point to an important role of all types of sinusoidal cells in regeneration from CDE induced liver damage and call for utmost caution in using traditional marker for identifying specific cell types. Thus, M2-pyruvate kinase should no longer be used for estimating the oval cell response in mouse liver. CDE diet leads to activation of GFAP positive HSCs in the pericentral zone of liver lobulus. In the periportal zone the detection of GFAP in biliary cells and oval cells, calls other cell types as progenitors of hepatocytes into question under CDE diet conditions.

Background: Hepatitis C virus (HCV) is a major cause of chronic hepatitis and a health problem affecting over 170 million people around the world. We previously studied transgenic mice that express HCV Core, Envelope 1 and Envelope 2 proteins predominantly in the liver, resulting in steatosis, liver and lymphoid tumors, and hepatocellular carcinoma. Herein, the immune-mediated cell response to hepatitis C antigens was evaluated by adoptive transfers of carboxyfluorescein succinimidyl ester (CFSE) labelled splenocytes from HCV immunized mice into HCV transgenic mice.

Results: In comparison to non-transgenic mice, there was a significant decrease in the percentage of CFSE-labeled CD4+ and CD8+ T cells in transgenic mouse peripheral blood receiving adoptive transfers from immunized donors. Moreover, the percentage of CFSE-labeled CD4+ and CD8+ T cells were significantly higher in the spleen of transgenic and non-transgenic mice when they received splenocytes from non-immunized than from immunized mice. On the other hand, the percentages of CD4+ and CD8+ T cells in the non-transgenic recipient mouse lymph nodes were significantly higher than the transgenic mice when they received the adoptive transfer from immunized donors. Interestingly, livers of transgenic mice that received transfers from immunized mice had a significantly higher percentage of CFSE labeled T cells than livers of non-transgenic mice receiving non-immunized transfers.

Conclusions: These results suggest that the T cells from HCV immunized mice recognize the HCV proteins in the liver of the transgenic mouse model and homed to the HCV antigen expression sites. We propose using this model system to study active T cell responses in HCV infection.

Background: ABCB4 functions as a phosphatidylcholine translocater, flipping phosphatidylcholine across hepatocyte canalicular membranes into biliary canaliculi. In people, ABCB4 gene mutations are associated with several disease syndromes including intrahepatic cholestasis of pregnancy, progressive familial intrahepatic cholestasis (type 3), primary biliary cirrhosis, and cholelithiasis. Hepatobiliary disease, specifically gallbladder mucocele formation, has been recognized with increased frequency in dogs during the past decade. Because Shetland Sheepdogs are considered to be predisposed to gallbladder mucoceles, we initially investigated ABCB4 as a candidate gene for gallbladder mucocele formation in that breed, but included affected dogs of other breeds as well.

Results: An insertion (G) mutation in exon 12 of canine ABCB4 (ABCB4 1583_1584G) was found to be significantly associated with hepatobiliary disease in Shetland Sheepdogs specifically (P < 0.0001) as well as other breeds (P < 0.0006). ABCB4 1583_1584G results in a frame shift generating four stop codons that prematurely terminate ABCB4 protein synthesis within exon 12, abolishing over half of the protein including critical ATP and a putative substrate binding site.

Conclusions: The finding of a significant association of ABCB4 1583_1584G with gallbladder mucoceles in dogs suggests that this phospholipid flippase may play a role in the pathophysiology of this disorder. Affected dogs may provide a useful model for identifying novel treatment strategies for ABCB4-associated hepatobiliary disease in people.