Comparative hepatology最新文献

Background: Temporal restriction of food availability entrains circadian behavioral and physiological rhythms in mammals by resetting peripheral oscillators. This entrainment underlies the activity of a timing system, different from the suprachiasmatic nuclei (SCN), known as the food entrainable oscillator (FEO). So far, the precise anatomical location of the FEO is unknown. The expression of this oscillator is associated with an enhanced arousal prior to the food presentation that is called food anticipatory activity (FAA). We have focused on the study of the role played by the liver as a probable component of the FEO. The aim of this work was to identify metabolic and structural adaptations in the liver during the expression of the FEO, as revealed by histochemical assessment of hepatic glycogen and triacylglycerol contents, morphometry, and ultrastructure in rats under restricted feeding schedules (RFS).

Results: RFS promoted a decrease in the liver/body weight ratio prior to food access, a reduction of hepatic water content, an increase in cross-sectional area of the hepatocytes, a moderate reduction in glycogen content, and a striking decrease in triacylglyceride levels. Although these adaptation effects were also observed when the animal displayed FAA, they were reversed upon feeding. Mitochondria observed by electron microscopy showed a notorious opacity in the hepatocytes from rats during FAA (11:00 h). Twenty four hour fasting rats did not show any of the modifications observed in the animals expressing the FEO.

Conclusions: Our results demonstrate that FEO expression is associated with modified liver handling of glycogen and triacylglycerides accompanied by morphometric and ultrastructural adaptations in the hepatocytes. Because the cellular changes detected in the liver cannot be attributed to a simple alternation between feeding and fasting conditions, they also strengthen the notion that RFS promotes a rheostatic adjustment in liver physiology during FEO expression.

Background: The expression of Keratin 19 (K19) was reported in a subset of hepatocellular carcinomas (HCCs). K19 positive HCCs are associated with an increased malignancy compared to K19 negative HCCs. No suitable mouse models exist for this subtype of HCC, nor is the incidence of K19 expression in hepatocellular neoplasia in model animals known. Therefore, we compared the occurrence and tumour behaviour of K19 positive hepatocellular neoplasias in dog and man.

Results: The expression of hepatocellular differentiation (HepPar-1), biliary/progenitor cell (K7, K19), and malignancy (glypican-3) markers was semi-quantitatively assessed by immunohistochemistry. The histological grade of tumour differentiation was determined according to a modified classification of Edmondson and Steiner; the staging included intrahepatic, lymph node or distant metastases. Four of the 34 canine hepatocellular neoplasias showed K19 positivity (12%), of which two co-expressed K7. K19 positive tumours did not express HepPar-1, despite the histological evidence of a hepatocellular origin. Like in human HCC, all K19 positive hepatocellular neoplasias were glypican-3 positive and histologically poorly differentiated and revealed intra- or extrahepatic metastases whereas K19 negative hepatocellular neoplasias did not.

Conclusions: K19 positive hepatocellular neoplasias are highly comparable to man and occur in 12% of canine hepatocellular tumours and are associated with a poorly differentiated histology and aggressive tumour behaviour.

Background: Perihepatitis is rare but consistently occurring extragenital manifestation of untreated Chlamydia trachomatis infection. Despite of possible liver involvement in generalized C. trachomatis infection, the ability of the pathogen to propagate in the hepatic cells and its impact on liver functions is not thoroughly investigated. The effect of mevastatin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase, on C. trachomatis growth in human hepatoma cell line HepG2 has been studied. Bacterial growth was assessed by immunostaining with FITC-labeled monoclonal antibody against chlamydial lipopolysaccharide and by RT-PCR for two chlamydial genetic markers (16S rRNA and euo).

Results: Chlamydial inclusion bodies were seen in approximately 50% of hepatocytes at 48 hours in the post infection period. Lysates obtained from infected hepatocytes were positive in the infective progeny test at 48 and especially in 72 hours after infection initiation. It has been shown that chlamydial infection in hepatocytes also leads to the decline of LDL-receptor mRNA which reflects infection multiplicity rate. Additions of mevastatin (1, 20 and 40 microM) 1 hour before inoculation restored and upregulated LDL-receptor mRNA level in a dose-dependent manner. Mevastatin treatment had no effect on internalization of chlamydial particles. However it reduced drastically the number of chlamydial 16S rRNA and euo transcripts as well as overall infection rate in HepG-2 cells. Complete eradication of infection has been seen by immunofluorescent staining at 40 microM mevastatin concentration, when expression level of chlamydial 16S rRNA and euo was undetectable. Lower concentration of mevastatin (20 microM) promoted euo expression level and the appearance of atypically small chlamydial inclusions, while there was a noticeable reduction in the number of infected cells and 16S rRNA transcripts.

Conclusions: C. trachomatis can efficiently propagate in hepatocytes affecting transcription rate of some liver-specific genes. Ongoing cholesterol synthesis is essential for chlamydial growth in hepatocytes. Inhibitors of cholesterol biosynthesis can supplement conventional strategy in the management of C. trachomatis infection.

Background: Hemodynamic changes in the liver remnant following partial hepatectomy (PHx) have been suggested to be a primary stimulus in triggering liver regeneration. We hypothesized that it is the increased sinusoidal flow per se and hence the shear-stress stimulus on the endothelial surface within the liver remnant which is the main stimulus to regeneration. In order to test this hypothesis we wanted to increase the sinusoidal flow without performing a concomitant liver resection. Accordingly, we constructed an aorto-portal shunt to the left portal vein branch creating a standardized four-fold increase in flow to segments II, III and IV. The impact of this manipulation was studied in both an acute model (6 animals, 9 hours) using a global porcine cDNA microarray chip and in a chronic model observing weight and histological changes (7 animals, 3 weeks).

Results: Gene expression profiling from the shunted segments does not suggest that increased sinusoidal flow per se results in activation of genes promoting mitosis. Hyperperfusion over three weeks results in the whole liver gaining a supranormal weight of 3.9% of the total body weight (versus the normal 2.5%). Contrary to our hypothesis, the weight gain was observed on the non-shunted side without an increase in sinusoidal flow.

Conclusions: An isolated increase in sinusoidal flow does not have the same genetic, microscopic or macroscopic impact on the liver as that seen in the liver remnant after partial hepatectomy, indicating that increased sinusoidal flow may not be a sufficient stimulus in itself for the initiation of liver regeneration.

Background: Liver disease progression from chronic hepatitis C virus (HCV) infection to hepatocellular carcinoma (HCC) is associated with an imbalance between T-helper 1 and T-helper 2 cytokines. Evaluation of cytokines as possible candidate biomarkers for prediction of HCC was performed using soluble Fas(sFas), soluble tumor necrosis factor receptor-II (sTNFR-II), interleukin-2 receptor (IL-2R) and interleukin-8 (IL-8).

Results: The following patients were recruited: 79 with HCV infection, 30 with HCC, 32 with chronic liver disease associated with elevated liver enzyme levels (with or without cirrhosis) in addition to 17 with chronic HCV with persistent normal alanine aminotransferase levels (PNALT). Nine normal persons negative either for HCV or for hepatitis B virus were included as a control group. All persons were tested for sFas, sTNFR-II, IL-2R and IL-8 in their serum by quantitative ELISA. HCC patients had higher levels of liver enzymes but lower log-HCV titer when compared to the other groups. HCC patients had also significantly higher levels of sFas, sTNFR-II and IL-2R and significantly lower levels of IL-8 when compared to the other groups. Exclusion of HCC among patients having PNALT could be predicted with 90 % sensitivity and 70.6 % specificity when sTNFR-II is [greater than or equal to] 389 pg/ml or IL-8 is < 290 pg/ml.

Conclusions: Serum TNFR-II, IL-2Ralpha and IL-8, may be used as combined markers in HCV-infected cases for patients at high risk of developing HCC; further studies, however, are mandatory to check these findings before their application at the population level.

Background: In adult liver, the mesenchymal cells, portal fibroblasts and vascular smooth muscle cells can transdifferentiate into myofibroblasts, and are involved in portal fibrosis. Differential expression of markers, such as alpha-smooth muscle actin (ASMA), h-caldesmon and cellular retinol-binding protein-1 allows their phenotypic discrimination. The aim of our study was to explore the phenotypic evolution of the mesenchymal cells during fetal development in normal liver and in liver with portal fibrosis secondary to ductal plate malformation in a series of Meckel-Gruber syndrome, autosomal recessive polycystic kidney disease and Ivemark's syndrome.

Results: At the early steps of the portal tract maturation, portal mesenchymal cells expressed only ASMA. During the maturation process, these cells were found condensed around the biliary and vascular structures. At the end of maturation process, only cells around vessels expressed ASMA and cells of the artery tunica media also expressed h-caldesmon. In contrast, ASMA positive cells persisted around the abnormal biliary ducts in fibrous livers.

Conclusion: As in adult liver, there is a phenotypic heterogeneity of the mesenchymal cells during fetal liver development. During portal tract maturation, myofibroblastic cells disappear in normal development but persist in fibrosis following ductal plate malformation.

Background: It has been postulated that ethanol affects hepatic sinusoidal and perisinusoidal cells. In the current experimental study, we investigated the early effect of a single intravenous dose of ethanol on the diameter of liver sinusoidal endothelial fenestrae in New Zealand White rabbits. The diameter of fenestrae in these rabbits is similar to the diameter found in humans with healthy livers. The effect of ethanol on the size of fenestrae was studied using transmission electron microscopy, because plastic embedding provides true measures for the diameter of fenestrae.

Results: After intravenous administration of a single dose of 0.75 g/kg, ethanol concentration peaked at 1.1 +/- 0.10 g/l at ten minutes after injection. Compared to control rabbits (103 +/- 1.1 nm; n = 8), the average diameter of fenestrae in ethanol-injected rabbits determined at 10 minutes after injection was significantly (p < 0.01) smaller (96 +/- 2.2 nm; n = 5). Detailed analysis of distribution histograms of the diameters of fenestrae showed that the effect of ethanol was highly homogeneous.

Conclusion: A decrease of the diameter of fenestrae 10 minutes after ethanol administration is likely the earliest morphological alteration induced by ethanol in the liver and underscores the potential role of liver sinusoidal endothelial cells in alcoholic liver injury.

Background: To minimize the necessary number of biopsies for molecular and histological research we evaluated different sampling techniques, fixation methods, and storage procedures for canine liver tissue. For addressing the aim, three biopsy techniques (wedge biopsy, Menghini, True-cut), four storage methods for retrieval of RNA (snap freezing, RNAlater, Boonfix, RLT-buffer), two RNA isolation procedures (Trizol and RNAeasy), and three different fixation protocols for histological studies (10% buffered formalin, RNAlater, Boonfix) were compared. Histological evaluation was based on hematoxylin-eosin (HE) and reticulin (fibrogenesis) staining, and rubeanic acid and rhodanine stains for copper. Immunohistochemical evaluation was performed for cytokeratin-7 (K-7), multidrug resistance binding protein-2 (MRP-2) and Hepar-1.

Results: RNA quality was best guaranteed by the combination of a Menghini biopsy with NaCl, followed by RNAlater preservation and RNAeasy mini kit extraction. These results were confirmed by quantitative RT-PCR testing. Reliable histological assessment for copper proved only possible in formalin fixed liver tissue. Short formalin fixation (1-4 hrs) improved immunohistochemical reactivity and preservation of good morphology in small liver biopsies.

Conclusion: At least two biopsies (RNAlater and formalin) are needed. Since human and canine liver diseases are highly comparable, it is conceivable that the protocols described here can be easily translated into the human biomedical field.

Background: Golden-mantled ground squirrels (S. lateralis) are anorexic during the winter and survive by exploiting hibernation to reduce energetic demands. The liver normally plays a critical role in fueling and regulating metabolism and one might expect significant changes in hepatobiliary function with hibernation. We analyzed bile collected from animals in summer, animals in winter that were either torpid, active between bouts of torpor, or which failed to enter hibernation in order to characterize the effects of hibernation on hepatobiliary function per se.

Results: Surprisingly, hibernator bile did not differ from summer squirrel bile in key characteristics including [bile acids], [cholesterol], [free fatty acids], [lecithin], and osmolality. One major distinction between summer and winter squirrels was that winter squirrels experience >5 fold increases in [bilirubin]. Such an increase may have significant physiological consequences that could aid in survivorship of torpor. Animals that failed to hibernate, despite being anorexic, were very similar to summer squirrels in all measured parameters except they had lower bile acid and lecithin concentrations.

Conclusion: The data indicate that despite extended anorexia, differences in metabolic fuel privation, and bouts of reduced body temperatures, hibernators normally do not experience broad changes in hepatobiliary function.

Background: Pregnane X receptor (PXR) agonists inhibit liver fibrosis. However, the rodent PXR activator pregnenolone 16alpha carbonitrile (PCN) blocks, in vitro, hepatic stellate cell-to-myofibroblast trans-differentiation and proliferation in cells from mice with a disrupted PXR gene, suggesting there is an additional anti-fibrogenic drug target for PCN. The role of the low affinity glucocorticoid binding site (LAGS) - which may be identical or associated with the progesterone receptor membrane component 1 (PGRMC1) - in mediating this anti-fibrogenic effect has been examined, since binding of dexamethasone to the LAGS in liver microsomal membranes has previously been shown to be inhibited by PCN.

Results: Quiescent rat and human hepatic stellate cells (HSC) were isolated from livers and cultured to generate liver myofibroblasts. HSC and myofibroblasts expressed PGRMC1 as determined by RT-PCR and Western blotting. Quiescent rat HSC also expressed the truncated HC5 variant of rPGRMC1. Rat PGRMC1 was cloned and expression in COS-7 cells gave rise to specific binding of radiolabelled dexamethasone in cell extracts that was inhibited by PCN, suggesting that PGRMC1 may be identical to LAGS or activates LAGS binding activity. Liver microsomes were used to screen a range of structurally related compounds for their ability to inhibit radiolabelled dexamethasone binding to rat LAGS. These compounds were also screened for their ability to activate rat and human PXR and to inhibit rat HSC-to-myofibroblast trans-differentiation/proliferation. A compound (4 androstene-3-one 17beta-carboxylic acid methyl ester) was identified which bound rat LAGS with high affinity and inhibited both rat and human HSC trans-differentiation/proliferation to fibrogenic myofibroblasts without showing evidence of rat or human PXR agonism. However, despite potent anti-fibrogenic effects in vitro, this compound did not modulate liver fibrosis severity in a rat model of liver fibrosis. Immunohistochemical analysis showed that rat liver myofibroblasts in vivo did not express rPGRMC1.

Conclusion: LAGS ligands inhibit HSC trans-differentiation and proliferation in vitro but show little efficacy in inhibiting liver fibrosis, in vivo. The reason(s) for this disparity is/are likely associated with an altered myofibroblast phenotype, in vitro, with expression of rPGMRC1 in vitro but not in vivo. These data emphasize the limitations of in vitro-derived myofibroblasts for predicting their activity in vivo, in studies of fibrogenesis. The data also demonstrate that the anti-fibrogenic effects of PCN in vivo are likely mediated entirely via the PXR.