[Severe refractory pulmonary hypertension after liver transplantation for hepatitis C liver cirrhosis]. / Therapierefraktäre schwere pulmonal-arterielle Hypertonie nach Lebertransplantation bei HCV-Leberzirrhose.

BACKGROUND: We report the case of a 43-year-old male with liver cirrhosis based on a chronically active hepatitis C. CASE REPORT: Before liver transplantation right-ventricular pressure values of 36 mmHg (+ central venous pressure) were measured whereas, after transplantation, he developed severe pulmonary hypertension with pressure values up to 90 mmHg. These elevated pressure values correlated inversely with graft function. Given the diagnosis of portopulmonary hypertension, we initiated treatment with intravenous epoprostenol and inhalative iloprost but both treatments were not tolerated because of systemic side effects. A combined heart-lung transplantation was considered but the patient died from insufficient cardiac function. CONCLUSIONS: The case report discusses the present diagnostic and therapeutic state of the art in portopulmonary hypertension and reveals basic problems of the present screening strategy.

Influence of biliary cirrhosis on the detoxification and elimination of a food derived carcinogen.

BACKGROUND AND AIMS: The liver is the central organ for the detoxification of numerous xenobiotics, including carcinogens. We studied the influence of cholestasis and biliary cirrhosis on the detoxification, elimination, and tissue distribution of a model compound and food derived carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). METHODS: Wistar rats were injected with (14)C-PhIP into the portal vein one or six weeks after common bile duct ligation (CBDL). Bile flow was reconstituted, bile and urine were collected over 120 minutes, and metabolites were analysed using high performance liquid chromatograpy. Total tissue radioactivity levels in several organs as well as tissue bound (ethanol insoluble tissue fraction) radioactivity levels were determined. RESULTS: Significant downregulation of the transport proteins multidrug resistance associated protein 2 and breast cancer resistance protein was observed in biliary cirrhosis. Biliary excretion of radioactivity was significantly reduced in cholestasis and biliary cirrhosis compared with controls (15 (2.9)% and 3.2 (1)% of the dose v 36.5 (2)%, respectively). Phase II metabolism was severely reduced in cirrhotic rats, resulting in a twofold increase in tissue radioactivity levels in the liver, kidney, and colon. Biliary cirrhosis increased tissue binding of reactive metabolites, as expressed in cpm/100 mg tissue in the liver and the colon (3267 (1218) v 1191 (429) in the liver, 3044 (1913) v 453 (253) in the colon). CONCLUSIONS: Biliary cirrhosis induced by CBDL causes impaired metabolism and elimination of PhIP, and leads to higher tissue levels of potentially genotoxic metabolites in the liver and colon of rats. These data may explain the increased incidence of hepatic and extrahepatic cancers in cholestasis and liver cirrhosis.

[Gastrointestinal stromal tumors: a broad clinical spectrum from incidental -discovery to acute gastrointestinal bleeding]. / Gastrointestinale Stromatumoren (GIST): variable klinische Manifestationen vom Zufallsbefund bis zur akuten gastrointestinalen Blutung.

Three cases of gastrointestinal stromal tumors (GIST) are reported as typical examples of the broad clinical spectrum in which these rare tumors can be detected. The first case describes an 82-year-old patient with a hemorrhagic shock due to upper gastrointestinal bleeding from a GIST of the stomach. GIST most frequently present with either gastrointestinal bleeding, abdominal pain or a detectable mass on physical examination or by ultrasound imaging. Clinically asymptomatic tumor growth also occurs as demonstrated by the second case of a 44-year-old -woman with an incidental finding of GIST during surgery of the esophagus. The cases are used to discuss the consequences for therapy and prognosis resulting from the heterogeneity of this tumor entity; the relevant immunohistochemical markers used to distinguish between various tumor subtypes of gastrointestinal mesenchymal tumors (GIMT) are listed. Since gastrointestinal stromal tumors (GIST) represent the most common subgroup of GIMT, we focus on the clinicopathological prognostic factors of GIST. The third case of a 40-year-old patient with a malignant GIST recurrence after surgery and exhibiting secondary resistance after one year of successful therapy with the receptor tyrosine kinase inhibitor imatinib (Gleevec), antagonizing pathogenetically relevant constitutive c-KIT activation, illustrates the potential and limitations of the only effective drug treatment for advanced GIST.

Constitutive rat multidrug-resistance protein 2 gene transcription is down-regulated by Y-box protein 1.

BACKGROUND/AIMS: Molecular mechanisms underlying transcriptional rat multidrug-resistance protein 2 (Mrp2, Abcc2) gene regulation are mostly unclear. Given the presence of putative binding sites for the Y-box binding protein YB-1 in the regulatory sequence, its trans-regulatory influence was analyzed. METHODS: Reporter assays in HepG2 cells with various Mrp2 deletion constructs in the absence and presence of co-transfected YB-1 were performed. DNA binding studies with recombinant YB-1 protein and nuclear extracts obtained from HepG2 cells and rat liver tissue were carried out. RESULTS: The minimal promoter sequence was confined to the proximal 186 bp. A YB-1 responsive element, Mrp2 YRE-1, was mapped at -186/-157, which exhibits specific YB-1 binding. YB-1 acts as a potent repressor of Mrp2 promoter activity in vitro. CONCLUSIONS: Constitutive Mrp2 gene expression is conferred through the proximal -186 bp. YB-1 acts as a repressor in vitro by specific binding to a defined element in the proximal promoter sequence.

Effect of glutathione depletion and hydrophilic bile acids on hepatic acute phase reaction in rats with extrahepatic cholestasis.

BACKGROUND: Extrahepatic cholestasis by biliary obstruction induces an acute phase reaction in the liver. It is a complex process involving cytokines, hormones and growth factors. To determine whether the regulation of acute phase proteins (APP) in cholestasis depends on glutathione (GSH), the effect of buthionine sulfoximine-induced (BSO-induced) GSH depletion on the expression of various APP was studied. In addition, we determined the influence of hepatoprotective bile acids on hepatic APP and underlying cytokine events. METHODS: Liver samples of bile-duct-ligated or sham-operated rats were examined. mRNA expression was quantified by densitometric analysis of Northern blots. RESULTS: Expression of APP increased 2-5-fold in bile-duct-ligated rats as compared to sham-operated controls. This acute phase reaction remained similar independently of whether cholestasis occurred for 5 days or 3 weeks. In contrast to alpha2-macroglobulin and tissue inhibitor of metalloproteinases-1 (TIMP-1), mRNA levels of both beta-fibrinogen and haptoglobin were significantly up-regulated after GSH depletion by BSO in cholestasis. Feeding of ursodeoxycholic and iso-ursodeoxycholic acid markedly down-regulated alpha2-macroglobulin and TIMP-1 expression in cholestasis but did not affect overexpression of beta-fibrinogen and haptoglobin. Cholestasis leads to an increased APP expression accompanied by an increased expression of inflammatory cytokines (IL-6, TNF-alpha). After feeding of hydrophilic bile acids, increases in inflammatory cytokines were abrogated. CONCLUSIONS: We show that GSH is involved in the acute phase reaction during obstructive cholestasis. In addition, bile acids might selectively ameliorate the acute phase response by reducing expression of the APP not affected by GSH depletion (alpha2-macroglobulin and TIMP-1).

[Neurologic complications in inflammatory bowel diseases]. / Neurologische Komplikationen bei entzündlichen Darmerkrankungen.

Some inflammatory diseases of the gastrointestinal system are associated with neurological symptoms which, in rare cases, may precede the onset of the gastrointestinal manifestation of the disease. Celiac sprue is characterized by an intolerance to the wheat protein gluten. The typical neurological manifestation of celiac sprue is cerebellar ataxia. The ataxia as well as the gastrointestinal symptoms can be treated with a strictly gluten-free diet. The neurological symptoms of Whipple's disease, a disorder caused by gram-positive bacilli, are variable. Typical symptoms of cerebral Whipple's disease include dementia, ophthalmoplegia, movement disorders, and signs of hypothalamic dysfunction. Nowadays, the diagnosis of cerebral Whipple's disease is made by PCR detection of the bacilli in the CSF. Crohn's disease and ulcerative colitis are associated with neurological symptoms to a similar proportion. Various forms of polyneuropathies have been observed. The CNS manifestations of inflammatory bowel diseases include thromboembolic stroke, cerebral venous thrombosis, and cerebral vasculitis.

Expression of isoforms and splice variants of the latent transforming growth factor beta binding protein (LTBP) in cultured human liver myofibroblasts.

BACKGROUND/AIMS: The activation of hepatic stellate cells (HSC) to extracellular matrix (ECM) producing myofibroblasts (MFB) is the key pathogenetic event in human liver fibrogenesis. Latent transforming growth factor beta binding protein (LTBP), a component of the profibrogenic large latent transforming growth factor (TGF)-beta complex, is suggested to be important for secretion, latency, storage and activation of TGF-beta in the ECM. This study was performed to identify the expression profile of all hitherto known LTBP isoforms and LTBP splice variants in conjunction with that of TGF-beta isoforms in cultured human liver MFB. METHODS: Cultured human MFB were analyzed for TGF-beta and LTBP using reverse-transcription polymerase chain reaction (RT-PCR), sequence analysis, immunofluorescence staining, metabolic labeling, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). RESULTS: Transcripts of all three TGF-beta isoforms, of all four LTBP isoforms and of nearly all splice variants of LTBP-1 and LTBP-4 so far known were detected. Metabolic labeling followed by immunoprecipitation with anti-LTBP-1 antibody revealed the synthesis of LTBP proteins. Secretion of free LTBP and LTBP integrated into the large latent TGF-beta complex was demonstrated by size-exclusion chromatography. Co-localization of LTBP-1 and -2 with fibronectin and collagen type I was observed by double immunofluorescence staining. CONCLUSION: The expression of a complete profile of hitherto known LTBP proteins by cultured human MFB suggests a role in modulating the bioactivity of TGF-beta in the diseased liver.

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.

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.

Molecular regulation of sinusoidal liver bile acid transporters during cholestasis.

Impairment of the hepatic transport of bile acids and other organic anions will result in the clinically important syndrome of cholestasis. Cloning of a number of specific hepatic organic anion transporters has enabled studies of their molecular regulation during cholestasis. The best characterized transport system is a 50-51 kDa sodium-dependent taurocholate cotransporting polypeptide (ntcp), which mediates the sodium-dependent uptake of conjugated bile acids at the sinusoidal plasma membrane of hepatocytes. Under physiologic conditions and after depletion of biliary constituents, ntcp remains constitutively expressed throughout the liver acinus. However, both function and expression of ntcp are rapidly down-regulated in rat liver in various models of experimental cholestasis, such as cholestasis induced by common bile duct ligation, estrogen, endotoxin or cytokine treatment. In addition to ntcp, the sinusoidal organic anion transporting polypeptide oatp-1 is also down-regulated at the protein and steady-state mRNA levels in estrogen-cholestasis, but does not affect sodium-independent uptake of taurocholate. The regulation of a recently cloned member of the organic anion transporter family (oatp-2), which is highly expressed in liver, remains to be studied under cholestatic conditions.

Endotoxin impairs biliary glutathione and HCO3- excretion and blocks the choleretic effect of nitric oxide in rat liver.

Cholestasis in patients with sepsis has been attributed to the effects of endotoxin (lipopolysaccharides, LPS) and LPS-induced cytokines, which are also potent stimulators of systemic and hepatic nitric oxide (NO) synthesis. NO donors stimulate bile acid-independent bile flow in normal rat liver, but the effects of LPS-induced NO on bile formation remain unclear. To address this question we examined the effects of NO and its mediator guanosine 3',5'-cyclic monophosphate (cGMP) on bile flow and biliary HCO3- and glutathione excretion in isolated perfused rat livers (IPRL) from LPS-treated rats. Portal and systemic NO2- + NO3- plasma levels were increased 47-fold in LPS-treated rats and were also elevated in perfusate (6-fold) and bile (9-fold) after isolating and perfusing livers from these animals. Bile flow, HCO3-, and glutathione output were decreased by 33%, 25%, and 81% in these IPRL, respectively. Stimulation of NO synthesis with L-arginine or inhibition of inducible NO synthesis with aminoguanidine did not change bile flow, although pretreatment with aminoguanidine inhibited NO production by 85%. Moreover, the choleretic effects of infusions of the NO donors sodium nitroprusside (SNP) and S-nitroso-acetyl-penicillamine were markedly reduced in endotoxemic IPRL compared with normal controls, and SNP-induced HCO3- and glutathione excretion were reduced by 61% and 86%, respectively. SNP-induced cyclic GMP production was 2.3-fold lower than in normals, but the choleretic effect of dibutyryl cGMP was only slightly reduced in endotoxemic livers. These findings indicate that LPS reduces bile acid-independent bile flow primarily by inhibiting biliary excretion of glutathione and to a lesser extent HCO3-, whereas LPS-induced NO does not modulate bile formation in endotoxemia. Thus, impairment of the major determinants of bile acid-independent bile flow by LPS may contribute significantly to the pathogenesis of the cholestasis of sepsis.

Expression of the rat liver Na+/taurocholate cotransporter is regulated in vivo by retention of biliary constituents but not their depletion.

Expression and function of the hepatic Na+/taurocholate cotransporter (ntcp) are down-regulated in several models of experimental cholestasis. To test whether retention and/or depletion of biliary constituents are involved in ntcp regulation, ntcp expression was quantified in several animal models with altered levels of these constituents. In choledochocaval fistula rats (CCF) (retention model), ntcp mRNA expression specifically declined after 1 and 3 days by 76 +/- 4% (P < .005) and 31 +/- 9% (P < .05), respectively, returning to control levels by 7 days. However, protein expression as assessed by Western blotting remained unchanged for up to 7 days of CCF. In rats with bile fistulas (depletion model) for 0.5, 1, 2, 4, and 7 days, both ntcp protein and mRNA expression remained unaltered. Infusion of either taurocholate or taurochenodeoxycholate for 12 hours also did not effect ntcp mRNA expression in intact animals, probably because of its inability to increase serum and intrahepatic bile acid levels. In rats with selective bile duct ligation (SBDL), ntcp mRNA levels were down-regulated by 40 +/- 10% (P < .05) only after 12 and 24 hours in ligated lobes, and mRNA levels returned to control values in these lobes after 2 and 4 days. ntcp mRNA expression remained unchanged in the nonobstructed lobes at any time. When data from CCF and SBDL rats were combined, serum bile acids correlated linearly with ntcp mRNA (r = .62, P < .0005) over a 0 to 110-micromol/L range. Our results indicate that ntcp is constitutively expressed and remains uneffected by either depletion or increased flux of biliary constituents. However, retention of biliary constituents results in rapid down-regulation of ntcp mRNA, consistent with the concept that hepatocytes may be protected from bile acid toxicity during cholestasis by this mechanism.

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.

Ethinyl estradiol cholestasis involves alterations in expression of liver sinusoidal transporters.

The mechanisms involved in ethinyl estradiol-induced cholestasis are controversial. Basal bile flow was reduced by ethinyl estradiol administration, with a half time (t1/2) of 12.5 +/- 0.6 h. In contrast, initial taurocholate uptake was not significantly reduced until 3 days to 59% of control and to 13 and 10% of control at 5 and 7 days, respectively. The t1/2 was 4.3 +/- 0.1 days. These physiological changes were correlated with measurement of protein mass and steady-state mRNA for Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase), Na(+)-dependent taurocholate transporter, organic anion transporters, and membrane lipid fluidity. Ethinyl estradiol significantly decreased Na(+)-K(+)-ATPase activity and membrane fluidity. However, neither Na(+)-K(+)-ATPase alpha-subunit nor beta-subunit mass was altered by ethinyl estradiol administration. In contrast, protein content of the Na(+)-dependent taurocholate transporter was significantly reduced to 21% of control (P < 0.001) at 5 days. The Na(+)-dependent taurocholate transporter was identified in sinusoidal membrane fractions as a doublet with a molecular size estimated to be 51 and 56 kDa. Although both bands were reduced with ethinyl estradiol treatment, the 56-kDa band was decreased more rapidly and to a greater extent than the 51-kDa band. The estimated t1/2 of 4.8 +/- 0.6 days for the doublet was similar to that for Na(+)-dependent taurocholate uptake. The organic anion transporter protein mass was similarly reduced with time of ethinyl estradiol administration to 21% of control (P < 0.01) at 5 days. Ethinyl estradiol also rapidly decreased the steady-state mRNA levels of Na(+)-dependent and organic anion transporters to approximately 50% and 15% of control at 5 days, respectively. These studies indicate early generalized abnormalities of the sinusoidal membrane lipid fluidity, Na(+)-K(+)-ATPase activity, and bile acid transport protein content.

Down-regulation of expression and function of the rat liver Na+/bile acid cotransporter in extrahepatic cholestasis.

BACKGROUND & AIMS: The molecular regulation of hepatic bile acid transporters during cholestasis is largely unknown. Cloning of complementary DNAs for the sinusoidal sodium-dependent taurocholate cotransporting polypeptide (ntcp), the cytosolic bile acid-binding protein 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), and a putative canalicular bile acid transporter Ca2+, Mg(2+)-ecto-adenosine triphosphatase, now facilitates such studies. METHODS: Protein mass, steady-state messenger RNA (mRNA) levels, and gene transcription were assessed in rat livers after common bile duct ligation (CBDL) from 1-7 days, and taurocholate uptake was determined in isolated hepatocytes. RESULTS: After CBDL, Na(+)-dependent taurocholate uptake (Vmax) declined by 70%. The levels of ntcp protein were reduced by more than 90%, and 3 alpha-HSD levels decreased by 66% by 7 days. Expression and canalicular localization of the ecto-adenosine triphosphatase remained unchanged. mRNA levels for both ntcp and 3 alpha-HSD diminished by about 60% 1 day after CBDL and remained unchanged up to 7 days. Transcriptional activity was decreased 1 day after CBDL only for ntcp. CONCLUSIONS: Extrahepatic cholestasis results in rapid down-regulation of Na(+)-dependent taurocholate uptake, ntcp transcription, and posttranscriptional regulation of both ntcp and 3 alpha-HSD mRNA. This selective decline of ntcp may represent a protective feedback mechanism in cholestasis to diminish uptake of potentially hepatotoxic bile acids.

The subcellular localization of a bile acid glucosyltransferase in rat liver.

Formation of bile acid glucosides occurs in rat liver homogenate with a specific enzyme activity of 0.014 +/- 0.001 nmol per min per mg protein. Subcellular fractionation of rat liver by differential centrifugation revealed an enrichment of bile acid glucosyltransferase activity both in the mitochondrial-lysosomal fraction and in microsomes with a recovery of 38.8 +/- 4.6% and 37.7 +/- 1.7%, respectively, of enzyme activity in the homogenate. Subfractionation of the mitochondrial-lysosomal fraction after treatment of rats with Triton WR 1339 showed an almost exclusive association of bile acid glucosyltransferase activity with purified lysosomes ("tritosomes"). After subfractionation of microsomes by analytical gradients, bile acid glucosyltransferase was bimodally distributed with peaks at modal densities of 1.09 g/cm3 and 1.16 g/cm3, respectively. If microsomes were pretreated with pyrophosphate, a membrane perturbant known to strip ribosomes, only the peak of bile acid glucosyltransferase at higher density (1.16 g/cm3) and UDP-glucuronosyltransferase (marker of endoplasmic reticulum) shifted to a similar lower equilibrium density. Both enzymes were unaffected in their distribution by pretreatment of microsomes with digitonin. In contrast, markers of plasma membranes (5'-nucleotidase) and the Golgi-complex (galactosyltransferase) shifted to higher equilibrium densities after digitonin treatment, but were unaltered in their distribution after pyrophosphate. Bile acid glucosyltransferase activity in the lower density range with a peak at 1.09 g/cm3 did not show any association with the density distributions of known marker enzymes. In purified microsomal fractions obtained by preparative gradients, bile acid glucosyltransferase activity was enriched in enzyme activity by 1.4-fold in rough and by 2.3-fold in smooth endoplasmic reticulum, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)