Carsten Gartung

Bile acid concentrations in human and rat liver tissue and in hepatocyte nuclei

BACKGROUND & AIMS Bile acids exert cellular and molecular effects in the liver, but little is known about tissue concentrations. The aim of this study was to characterize bile acid composition in human and rat liver tissue and hepatocyte nuclei and examine the effects of experimental cholestasis and bile acid administration. METHODS Bile acids were measured by gas chromatography-mass spectrometry. RESULTS Liver tissue concentrations of sham-operated rats were 130.8 +/- 21.3 nmol/g, representing 2%-4% of the bile acid pool; cholic and delta 22-beta-muricholic acids were the major bile acids identified. Concentrations increased 7-8-fold with bile duct ligation; deoxycholate and hyodeoxycholate disappeared. Lithocholate concentrations were higher in ligated rats (6.4 +/- 0.4 vs. 3.9 +/- 0.5 nmol/g for sham-operated). Total bile acid concentrations in human liver tissue were 61.6 +/- 29.7 nmol/g and comprised mainly chenodeoxycholic and cholic acids. Concentrations were higher during ursodeoxycholate or tauroursodeoxycholate administration (157.2 +/- 45.6 and 161.6 +/- 43.4 nmol/g, respectively), and liver tissue was enriched 30% in ursodeoxycholate at the expense of hydrophobic bile acids. Bile acids were identified in rat hepatic nuclei (50-110 pmol/4 x 10(7) nuclei), accounting for < 0.1% of liver tissue levels. CONCLUSIONS Human and rat liver tissue bile acid concentrations are low, increase with bile acid administration or bile duct ligation, and account for only a small fraction of the bile acid pool.

Association of polymorphisms of the transforming growth factor-β1 gene with the rate of progression of HCV-induced liver fibrosis

BACKGROUND The objective of the present study was to elucidate possible relationships between four polymorphisms of the TGF-beta1 gene (-800G>A; -509C>T; Leu10Pro; Arg25Pro) and stage, histological activity grade and progression rate of liver fibrosis, classified according to the METAVIR-score. METHODS Three study groups, i.e. 48 patients with hepatic fibrosis (26 with known duration of hepatitis C virus infection), 47 patients with non-fibrotic diseases and 50 healthy blood donors, were analyzed for TGF-beta1 polymorphisms using ARMS-PCR and sequence analysis. The concentrations of total TGF-beta1 in plasma and of hyaluronan, P-III-NP and activities of transminases in serum were measured. RESULTS The presence of proline at codons 10 and/or 25 was associated with a faster progression of fibrosis than other polymorphisms. Patients with the genotype (25)ArgPro developed fibrosis significantly faster (0.23 units/year) than those having (25)ArgArg (0.08 units/year). Similarly, the fibrosis progression rate of patients with genotypes (10)LeuPro and (10)ProPro was almost three times as fast as of those having genotype (10)LeuLeu. Stage and histological activity grade of fibrosis in (25)ArgPro in comparison to (25)ArgArg were higher. Also (10)LeuPro showed a higher average stage of fibrosis than (10)LeuLeu. The TGF-beta1 plasma concentrations of patients with hepatic fibrosis were not significantly different between carriers of (25)ArgArg and (25)ArgPro genotypes. The frequency of the genotype (25)ArgPro in liver fibrotic patients was about three times that of the control group whereas the frequency distribution of the genotype (25)ArgArg tended to lower frequency in the fibrosis group. TGF-beta1-promoter polymorphisms did not show any correlation with stage, grade or progression of liver fibrosis. CONCLUSION Our results indicate that the heterozygous ArgPro of codon 25 predicts significantly faster fibrotic progression of chronic hepatitis C than the homozygous (25)ArgArg genotype. The homozygous LeuLeu genotype of codon 10 showed a slow progression of fibrosis.

Differential expression of basolateral and canalicular organic anion transporters during regeneration of rat liver

BACKGROUND & AIMS Liver regeneration in response to various forms of injury or surgical resection is a complex process resulting in restoration of the original liver mass and maintenance of liver-specific functions such as bile formation. However, liver regeneration is frequently associated with cholestasis, whose molecular pathogenesis remains unknown. METHODS To study the molecular mechanisms leading to cholestasis, expression of all major hepatic organic anion transporters contributing to bile formation was determined for up to 2 weeks in rats after 70% partial hepatectomy. RESULTS Inversely related to serum bile acid levels, basolateral transporters including the sodium-taurocholate cotransporter (Ntcp) and the organic anion transporting polypeptides Oatp1 and Oatp2 were markedly down-regulated at both protein and steady-state mRNA levels by 50%-60% of controls (P < 0.05) during early replicative stages of regeneration (12 hours to 2 days) with a slightly delayed time course for Oatp2. Expression of all basolateral transporters returned to control values between 4 and 4 days after partial hepatectomy. In contrast, protein and mRNA expression of both the canalicular ATP-dependent bile salt export pump (Bsep) and the multiorganic anion transporter Mrp2 remained unchanged or were slightly increased during liver regeneration, but also returned to control values 7-14 days after partial hepatectomy. CONCLUSIONS The data suggest a differential regulation of basolateral and canalicular organic anion transporters in the regenerating liver. Unaltered expression of Bsep and Mrp2 provides a potential molecular mechanism for regenerating liver cells to maintain or even increase bile secretion expressed per weight of remaining liver. However, down-regulation of basolateral organic anion transporters might protect replicating liver cells by diminishing uptake of potentially hepatotoxic bile salts, because the remaining liver initially cannot cope with the original bile acid pool size.

Hepatobiliary organic anion transporters are differentially regulated in acute toxic liver injury induced by carbon tetrachloride

BACKGROUND/AIMS Hepatobiliary transporters are down-regulated in cholestasis, but their expression in acute, non-cholestatic, cytokine-mediated liver injury is unknown. Thus we studied the molecular mechanisms, by which sodium taurocholate cotransporting polypeptide (Ntcp), organic anion transporting polypeptide 1 (Oatp1), Oatp2, Oatp4, multidrug-resistance protein 2 (Mrp2) and bile salt export pump (Bsep) are regulated in liver injury induced by carbon tetrachloride (CCl(4)). METHODS mRNA and protein levels were determined in rats 24 and 72h after CCl(4) injection. Transporter gene transcription and binding activities of Ntcp and Mrp2 transactivators were assessed by nuclear runoff and electrophoretic mobility shift assays. RESULTS mRNA levels significantly declined to 41+/-44% for Ntcp, 65+/-41% for Oatp1 and 64+/-28% for Oatp2, but remained unchanged for Oatp4, canalicular Mrp2 and Bsep. Protein levels declined only for Oatp4 (-50+/-17%) and Ntcp (-23+/-13%) at 24h. Reduced mRNA levels (Ntcp, Oatp1, Oatp2) were associated with decreased transcriptional activities. Binding activity of Ntcp transactivators (hepatocyte nuclear factor 1 alpha (HNF1alpha) and CAAT enhancer binding protein alpha (C/EBPalpha) were reduced by 24h, whereas retinoid X receptor alpha (RXRalpha):retinoid acid receptor alpha (RARalpha) as transactivator of both Ntcp and Mrp2 remained unaltered. Recovery of acute hepatitis and changes in gene expression occurred after 72h. CONCLUSIONS Acute liver injury results in down-regulation of basolateral organic anion transporters similar to liver regeneration after partial hepatectomy, but in contrast to endotoxin-induced cholestasis. Maintained binding activity of RXRalpha:RARalpha may explain differences in Mrp2 expression.

Regulation of basolateral organic anion transporters in ethinylestradiol-induced cholestasis in the rat

BACKGROUND/AIMS Estrogen-mediated cholestasis is an important clinical entity, but its molecular pathophysiology is still not fully understood. Impaired sodium-dependent uptake of bile acids has been associated with diminished expression of a basolateral Na(+)/bile acid cotransporter (Ntcp), whereas sodium-independent uptake is maintained despite a down-regulation of the organic anion transporter Oatp1. Thus, expression of the two other rat Oatps (Oatps2 and -4) was determined in estrogen-induced cholestasis. In addition, known transactivators of Oatp2 and Ntcp were studied to further characterize transcriptional regulation of these transporter genes. METHODS Hepatic protein and mRNA expression of various Oatps (1, 2, 4) in comparison to Ntcp were analyzed after 0.5, 1, 3 and 5 days of ethinylestradiol (EE) treatment (5 mg/kg) in rats. Binding activities of Oatp2 and Ntcp transactivators were assessed by electrophoretic mobility shift assays. RESULTS All basolateral Oatps (1, 2 and 4) were specifically down-regulated at the protein level by 30-40% of controls, but less pronounced than Ntcp (minus 70-80%). In contrast to unaltered Oatp4 mRNA levels, Oatp1 and Oatp2 mRNAs were reduced to various extents (minus 40-90% of controls). Binding activity of known transactivators of Ntcp and Oatp2 such as hepatocyte nuclear factor 1 (HNF1), CAAT enhancer binding protein alpha (C/EBPalpha) and pregnane X receptor (PXR) were also diminished during the time of cholestasis. CONCLUSIONS Estrogen-induced cholestasis results in a down-regulation of all basolateral organic anion transporters. The moderate decline in expression of Oatp1, -2 and -4 may explain the unchanged sodium-independent transport of bile acids due to overlapping substrate specificity. Reduction in transporter gene expression seems to be mediated by a diminished nuclear binding activity of transactivators such as HNF1, C/EBP and PXR by estrogens.

Dynamics of esophageal bolus transport in healthy subjects studied using multiple intraluminal impedancometry

The dynamics of a bolus transport through the esophagus are largely unexplored. To study this physiological process, we applied multiple intraluminal impedancometry in 10 healthy subjects. Three different protocols were used: 1) liquid bolus administered with subject supine, 2) liquid bolus with subject upright, or 3) semisolid bolus with subject supine. Transit of different parts of a bolus (bolus head, body, and tail) was analyzed at different anatomic segments, namely the pharynx and the proximal, middle, and distal thirds of the esophagus. A characteristic pattern of bolus transport was seen in all subjects. Impedance changes related to air were observed preceding the bolus head. The bolus head propelled significantly faster than did the bolus body and tail. Pharyngeal bolus transit was significantly faster than esophageal bolus transit. Within the esophagus, bolus propulsion velocity gradually decreased. Bolus transport was significantly accelerated in the upright position and delayed with increase of bolus viscosity. In conclusion, the dynamics of a bolus transport from the pharynx into the stomach are complex. It varies within both different anatomic segments and different parts of the bolus and depends on bolus characteristics and test conditions. The spatial and temporal resolution of a bolus transport can be obtained by the impedance technique.The dynamics of a bolus transport through the esophagus are largely unexplored. To study this physiological process, we applied multiple intraluminal impedancometry in 10 healthy subjects. Three different protocols were used: 1) liquid bolus administered with subject supine, 2) liquid bolus with subject upright, or 3) semisolid bolus with subject supine. Transit of different parts of a bolus (bolus head, body, and tail) was analyzed at different anatomic segments, namely the pharynx and the proximal, middle, and distal thirds of the esophagus. A characteristic pattern of bolus transport was seen in all subjects. Impedance changes related to air were observed preceding the bolus head. The bolus head propelled significantly faster than did the bolus body and tail. Pharyngeal bolus transit was significantly faster than esophageal bolus transit. Within the esophagus, bolus propulsion velocity gradually decreased. Bolus transport was significantly accelerated in the upright position and delayed with increase of bolus viscosity. In conclusion, the dynamics of a bolus transport from the pharynx into the stomach are complex. It varies within both different anatomic segments and different parts of the bolus and depends on bolus characteristics and test conditions. The spatial and temporal resolution of a bolus transport can be obtained by the impedance technique.

Common heterozygous hemochromatosis gene mutations are risk factors for inflammation and fibrosis in chronic hepatitis C

Abstract: 
Background: Chronic hepatitis C is frequently associated with increased hepatic iron stores. It remains controversial whether heterozygous mutations of hemochromatosis genes affect fibrosis progression. Therefore our aim was to assess associations between HFE mutations and hepatic inflammation and stage of fibrosis in German hepatitis C patients.

The presence of non-organ-specific autoantibodies is associated with a negative response to combination therapy with interferon and ribavirin for chronic hepatitis C

BackgroundNon-organ-specific autoantibodies are found in a considerable number of anti-HCV positive patients. Previous studies investigated the clinical relevance of these antibodies in patients treated with interferon monotherapy, but not combination therapies.MethodsAnti-nuclear, anti-smooth muscle, anti-mitochondrial, anti-neutrophil-cytoplasmatic and anti-liver/kidney microsomal antibodies were determined in 78 consecutive anti-HCV positive patients by indirect immunofluorescence. The presence of these antibodies was related to demographic variables and to the outcome of antiviral combination therapy with interferon-α and ribavirin in 65 patients.ResultsIn our study, positivity for autoantibodies was associated with higher alanine aminotransferase levels and higher mean values for HCV-RNA (p < 0.01). Furthermore, negativity for non-organ-specific autoantibodies was associated with a favourable treatment outcome of combination therapy with at least one negative RT-PCR for HCV-RNA during treatment (OR 4.65, 95% CI 1.31 to 16.48, p = 0.02). ANA and SMA staining patterns and titers were not correlated to treatment response. With multiple logistic regression analysis, positivity for autoantibodies and HCV genotype were independently associated with outcome of antiviral combination therapy (p = 0.02).ConclusionsThe absence of non-organ-specific autoantibodies might indicate a significantly higher chance for viral clearance in response to combination therapy for chronic hepatitis C infection. Therefore, despite of an overall higher treatment response, the addition of the immunomodulatory drug ribavirin could accentuate immunological differences that affect treatment outcome and might have been less obvious in earlier studies analysing interferon monotherapy.

Internalization of blocking antibodies against mannose-6-phosphate specific receptors

Antibodies against mannose‐6‐phosphate specific receptors inhibit the receptor‐dependent endocytosis of exogenous lysosomal enzymes as well as the sorting of endogenous lysosomal enzymes. This inhibition was correlated with an apparent loss of the receptors. We report here that treatment of cells with the antibody results in the formation of receptor‐antibody complexes that are not extracted by the procedure used for the solubilization of receptors prior to immunoprecipitation and detection of the receptor. The apparent loss of receptors is observed with both native antibody and the F(ab)2 fragments, but not with Fab fragments. In contrast the transport of lysosomal enzymes is inhibited by all three forms of the antibody. The inhibition is ascribed to masking by the antibody of the enzyme‐binding site in the receptor. The inhibition of the sorting of endogenous lysosomal enzymes by antibodies added to the medium indicates that the mannose‐6‐phosphate specific receptors at the sorting site are in dynamic equilibrium with those at the cell surface. The receptor‐antibody complexes formed at the cell surface appear to cycle between the cell surface and intracellular membranes. A fraction of the internalized antibodies dissociates from the receptors and is degraded after transfer into lysosomes. Complexing with Fab increases the concentration of the receptor in the lysosomes and decreases 2‐ to 3‐fold the half‐life of the receptor.

CC chemokine receptor 5 Δ32 polymorphism in two independent cohorts of hepatitis C virus infected patients without hemophilia

Recently CC chemokine receptor 5 (CCR5) related immune mechanisms and a functional mutation of the CCR5 gene have been implicated in hepatitis C virus (HCV) infection in a cohort of predominantly hemophiliac patients. The present study investigated the frequency and clinical consequences of the CCR5 Δ32 mutation in two genetically homogeneous populations of HCV infected patients with a different risk profile for infection. Genomic DNA samples from 333 German patients with chronic HCV infection were screened by PCR for the presence of the CCR5 Δ32 polymorphism. In-hospital patients admitted for other diseases than viral hepatitis but with a comparable risk for HCV exposure were used as control population (n=125). Allele frequencies of CCR5 Δ32 polymorphism did not differ significantly between the two groups (7.6% and 9.5%, respectively) and control subjects (10.4%), and did not deviate from Hardy-Weinberg equilibrium in any group. Furthermore, there were no major differences between patients with respect to HCV genotypes, viral loads, liver enzymes, or fibrosis scores in relation to the presence or absence of the heterozygous CCR5 Δ32 mutation. Differences in inflammatory scores in liver biopsy samples and response to antiviral therapy in CCR5 Δ32 heterozygotes in one cohort could neither be reproduced in the other group of patients nor when both cohorts were pooled. These results argue against a strong effect of the CCR5 Δ32 deletion regarding these phenotypes. In conclusion, we found no increased frequency of the CCR5 Δ32 polymorphism in two independent cohorts of patients with HCV infection but without hemophilia as the main risk factor for infection. As the major difference to investigations demonstrating an association between CCR5 Δ32 and HCV infection is the selection of cases and controls, our study emphasizes the importance of epidemiological criteria for association studies of HCV infection.