Plm Jansen

Hepatocanalicular bile salt export pump deficiency in patients with progressive familial intrahepatic cholestasis

BACKGROUND & AIMS Progressive familial intrahepatic cholestasis (PFIC), an inherited liver disease of childhood, is characterized by cholestasis and either normal or increased serum gamma-glutamyltransferase activity. Patients with normal gamma-glutamyltransferase activity have mutations of the FIC1 locus on chromosome 18q21 or mutations of the BSEP gene on chromosome 2q24. Also, patients with bile acid synthesis defects have low gamma-glutamyltransferase activity. We investigated expression of the bile salt export pump (BSEP) in liver samples from patients with a PFIC phenotype and correlated this with BSEP gene mutations. METHODS BSEP and multidrug resistance protein 2 (MRP2) expressions were studied by immunohistochemistry in liver specimens of 28 patients and BSEP gene mutation analysis in 19 patients. Bile salt kinetics were studied in 1 patient. RESULTS Sixteen of 28 liver samples showed no canalicular BSEP staining. Staining for MRP2 showed a normal canalicular pattern in all but 1 of these samples. Ten of 19 patients showed BSEP gene mutations; BSEP protein expression was lacking in all 10 patients. No mutations were found in 9 of 19 patients, and in all except 1, BSEP protein expression was normal. Bile salt concentration in bile of BSEP-negative/MRP2-positive PFIC patients was 0.2 +/- 0.2 mmol/L (n = 9; <1% of normal) and in BSEP-positive PFIC patients 18.1 +/- 9.9 mmol/L (n = 3; 40% of normal). The kinetic study confirmed the dramatic decrease of bile salt secretion in BSEP-negative patients. CONCLUSIONS The findings show a close correlation between BSEP gene mutations and canalicular BSEP expression. Biliary secretion of bile salts is greatly reduced in BSEP-negative patients.

Expression of nitric oxide synthases and formation of nitrotyrosine and reactive oxygen species in inflammatory bowel disease

Nitric oxide (NO) and reactive oxygen species (ROS) are important mediators in the pathogenesis of inflammatory bowel disease (IBD). NO in IBD can be either harmful or protective. NO can react with superoxide anions (O2.−), yielding the toxic oxidizing agent peroxynitrite (ONOO−). Peroxynitrite induces nitration of tyrosine residues (nitrotyrosine), leading to changes of protein structure and function. The aim of this study was to identify the cellular source of inducible nitric oxide synthase (iNOS) and to localize superoxide anion‐producing cells in mucosal biopsies from patients with active IBD. Additional studies were performed to look at nitrotyrosine formation as a measure of peroxynitrite‐mediated tissue damage. For this, antibodies against iNOS, endothelial NOS (eNOS), and nitrotyrosine were used. ROS‐producing cells were detected cytochemically. Inflamed mucosa of patients with active IBD showed intense iNOS staining in the epithelial cells. iNOS could not be detected in non‐inflamed mucosa of IBD patients and control subjects. eNOS was present in blood vessels, without any difference in the staining intensity between IBD patients and control subjects. ROS‐producing cells were increased in the lamina propria of IBD patients; a fraction of these cells were CD15‐positive. Nitrotyrosine formation was found on ROS‐positive cells. These results show that iNOS is induced in epithelial cells from patients with active ulcerative colitis or Crohns disease. Nitration of proteins was detected only on the ROS‐producing cells at some distance from the iNOS‐producing epithelial cells. These findings indicate that tissue damage during active inflammation in IBD patients is probably more related to ROS‐producing cells than to NO. One may speculate that NO has a protective role when during active inflammation other mucosal defence systems are impaired. Copyright

Assignment of the human UDP glucuronosyltransferase gene (UGT1A1) to chromosome region 2q37

UDP glucuronosyltransferases (UGTs) comprise a multigene family of drug-metabolizing enzymes. The sub-family of UGTs that conjugate bilirubin and phenolic compounds with glucuronic acid has been termed UGT1A1. In man, UGT1A1 isoforms are encoded by a single gene, UGT1A1. Protein isoforms encoded by UGT1A1 originate by alternative splicing. In the present study, we used the cDNA of UGT1A1*4, a bilirubin-conjugating isoform, to localize the UGT1A1 locus in the human genome. The UGT1A1 gene was assigned by in situ hybridization to chromosome region 2q37.

ATP binding cassette transporter gene expression in rat liver progenitor cells

Background and aim: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are cytoprotective efflux pumps that may contribute to the preservation of these cells. The aim of this study was to determine the ABC transporter phenotype of HPCs. Methods: HPC activation was studied in rats treated with 2- acetylaminofluorene (2-AAF) followed by partial hepatectomy (PHx). ABC transporter gene expression was determined by real time detection reverse transcription-polymerase chain reaction in isolated HPCs, hepatocytes, cholangiocytes, and cultured progenitor cell-like RLF ϕ 13 cells and by immunohistochemistry of total liver samples. ABC transporter efflux activity was studied in RLF ϕ 13 cells by flow cytometry. Results: 2-AAF/PHx treated animals showed increased hepatic mRNA levels of the genes encoding multidrug resistance proteins Mdr1b, Mrp1, and Mrp3. Immunohistochemistry demonstrated expression of Mrp1 and Mrp3 proteins in periportal progenitor cells and of the Mdr1b protein in periportal hepatocytes. Freshly isolated Thy-1 positive cells and cultured RLF ϕ 13 progenitor cells highly expressed Mrp1 and Mrp3 mRNA while the hepatocyte specific transporters Mdr2, Bsep, Mrp2, and Mrp6 were only minimally expressed. Blocking Mrp activity by MK-571 resulted in accumulation of the Mrp specific substrate carboxyfluorescein in RLF ϕ 13 cells. Conclusion: HPCs express high levels of active Mrp1 and Mrp3. These may have a cytoprotective role in conditions of severe hepatotoxicity.

Increased Expression of Inducible Nitric Oxide Synthase in Circulating Monocytes from Patients with Active Inflammatory Bowel Disease

Background: Inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) synthesis are increased in epithelial cells and in tissue macrophages of the inflamed mucosa from patients with inflammatory bowel disease (IBD). Since tissue macrophages are derived from circulating monocytes, we studied iNOS expression in circulating monocytes and related this expression to disease activity. In view of the possible role of NO in monocyte function, we also studied iNOS expression in relation to markers of monocyte activation. Methods: The expression of iNOS in circulating monocytes from 15 patients with active IBD, 6 patients who went into remission and 18 healthy controls was quantified by flow cytometry and correlated with surface markers (CD63, CD11b, HLA-DR) for monocyte activation. In addition, iNOS expression in circulating monocytes was assessed by Western blotting, immunocytochemistry and measurement of the NO metabolites nitrite and nitrate in plasma. Results: The expression of iNOS in circulating monocytes and the percentage of iNOS-positive monocytes were increased in patients with active IBD compared to healthy controls (fluorescence index: 1.3 (0.1-6.3) versus 0.8 (0.0- 1.8); P < 0.05; percentage of iNOS positive monocytes: 37.3 (1.0-77.0)% versus 5.3 (0.0-43.3)%; P < 0.01). The six patients who went into remission all had a marked reduction of iNOS expression. iNOS expression was confirmed by Western blotting and immunocytochemistry. Plasma nitrite and nitrate levels were elevated in three patients with active IBD. The surface markers for monocyte activation, CD63 and CD11b, were not elevated. HLA-DR expression was decreased on circulating monocytes from patients with active ulcerative colitis. Conclusions: iNOS is increased in circulating monocytes from patients with active IBD and this increased expression correlates with disease activity. Considering the decreased HLA-DR expression and absence of monocyte activation markers, NO produced by iNOS may have a function in suppressing systemic monocyte activation.

IBD and genetics: new developments.

BACKGROUND Inflammatory bowel disease (IBD) is a complex disorder with an aetiology that is only partly understood. Apart from environmental factors, inheritance contributes to IBD. REVIEW Family studies show an increased risk among family members of a patient with IBD, particularly among first-degree relatives. In twin studies, concordance for disease type and localization is observed. In genetically isolated groups there is a higher prevalence of IBD. For instance. Ashkenazi Jews carry the highest risk. Further evidence comes from animal species that spontaneously develop IBD. Unlike Mendelian inheritance, in complex genetic diseases like IBD, genes are expected to be low penetrant and therefore less prone to selection, which results in higher expected gene frequencies. NOD2/CARD15, the first gene associated with IBD, is a polymorphic gene involved in the innate immune system. The gene has over 60 variations. Three of these play a role in 27% of patients with CD, with a predilection for patients with ileal disease. CONCLUSION Genetics plays an important role in unravelling the pathogenesis of IBD leading to possible new therapeutic approaches.

Primary sclerosing cholangitis and liver transplantation

Primary sclerosing cholangitis is a chronic disease, strongly associated with ulcerative colitis and cholangiocarcinoma. Ulcerative colitis itself does not influence the liver transplant results. However, intensified screening after liver transplantation for carcinoma of the colon may be necessary. Cholangiocarcinoma, although incidentally found in hepatectomy specimens, has a bad prognosis. Initial reports in the literature indicate a far lower survival when liver transplantation is performed for PSC, in comparison to the results achieved in other transplant indications. This might have been due to surgical interventions which nowadays are avoided. Later reports show a better prognosis than the initial ones. We report on the results of liver transplantation for PSC, as indicated in the literature and on the results of the Groningen transplant centre.

Hepatitis B-Associated Liver Cirrhosis as an Indication for Liver Transplantation

Fourteen HBsAg-positive patients received a liver transplant in Groningen. Two were HBeAg-positive and 12 HBeAg-negative. No anti-HBs immunoglobulin was given at the time. Both HBeAg-positive and 9 of 12 of the HBeAg-negative patients became HBsAg-positive again after transplantation. Virus titers were tested in eight patients. Two HBeAg-negative patients were HBV-DNA-negative at transplantation and are still HBV-DNA-negative one-and-half-years after transplantation, both by the branched DNA hybridization technique and by PCR (cut-off values 0.7 x 10(6) and 10(3) HBV genomes/ml, respectively). One patient who had a low HBV-DNA titer at transplantation remained PCR-positive thereafter, but became HBsAg-negative. All other patients were HBV-DNA-positive and had a recurrence that rapidly led to high HBV titers. The liver histology was characterized by fibrosis and cirrhosis, centrilobular cholestasis and high expression of HBsAg and HBcAg, but with little inflammatory infiltrate. We conclude from these results that without anti-HBs immunoglobulin prophylaxis there is a high rate of HBV recurrence after transplantation. The current policy is that patients who test negative in the HBV-DNA dot-blot assay (< 10(7) genomes/ml) are transplantation candidates and are treated with high-dose anti-HBs immunoglobulin after transplantation. HBV-DNA-positive patients (> 10(7) genomes/ml) remain poor candidates for liver transplantation, even with anti-HBs immunoprophylaxis.

Nieuwe inzichten in de pathologie van progressieve familiaire intrahepatische cholestase

SummaryThe formation of bile is an osmotic process driven by the active secretion of biliary constituents out of the hepatocyte into the bile canalicular lumen. Several transport proteins, mainly atp-dependent proteins localized at the canalicular membrane, play roles in bile formation. Recently it has become clear that an increasing number of genetic cholestatic liver diseases in humans can be ascribed to mutations in genes encoding specific transporters. Progressive familial intrahepatic cholestasis (pfic) can now be divided into at least 4 subgroups. In this article, the responsible genes and characteristics of pfic type 1, 2, 3 and 4 are discussed. New diagnostic and therapeutic strategies for cholestatic diseases will be available in the near future.SamenvattingDe vorming van gal is een osmotisch proces dat berust op actieve uitscheiding van galcomponenten uit de leverparenchymcel naar het lumen van het galkanaaltje. Diverse transporteiwitten, voornamelijk atp-afhankelijke transporters in de canaliculaire membraan, spelen een rol bij de galvorming. Recent is duidelijk geworden dat aangeboren cholestatische aandoeningen bij de mens kunnen berusten op mutaties in genen coderend voor specifieke transporteiwitten. Het ziektebeeld progressieve familiare intrahepatische cholestase (pfic) blijkt tenminste 4 subgroepen te omvatten. In dit artikel worden de verantwoordelijke genen en karakteristieken van pfic type 1, 2, 3 en 4 besproken. In de naaste toekomst komen nieuwe diagnostische en therapeutische strategieën voor cholestatische leverziekten ter beschikking.

Upregulation of the multidrug resistance genes mrp1 and mdr1b and downregulation of the organic anion transporter mrp2 and bile salt transporter spgp in endotoxemic rat liver

Endotoxin-induced cholestasis is mainly caused by an impaired canalicular secretion. Mrp2, the canalicular multispecific organic anion transporter, is strongly down-regulated in this situation, and canalicular bile salt secretion is also reduced. We hypothesized that other adenosine triphosphate-binding cassette (ABC) transporters may compensate for the decreased transport activity to protect the cell from cytokine-induced oxidative damage. Therefore, we examined the expression of ABC-transport proteins in membrane fractions of whole liver and of isolated hepatocytes of endotoxin-treated rats and performed reverse-transcriptase polymerase chain reaction (RT-PCR) on mRNA isolated from these livers. In addition, the localization of these transporters was examined using confocal scanning laser microscopy. By 6 hours after endotoxin administration, we found a clear increase of mrp1 mRNA and protein, whereas mrp2 mRNA and protein were decreased. This was confirmed in isolated hepatocytes. In addition, mdr1b mRNA was strongly increased, whereas mdr1a and mdr2 mRNA did not change significantly. Both the mRNA and protein levels of the sister of P-glycoprotein (spgp), the recently cloned bile salt transporter, decreased. After endotoxin treatment, the normally sharply delineated canalicular staining of mrp2 and spgp had changed to a fuzzy pattern, suggesting localization in a subapical compartment. We conclude that endotoxin-induced cholestasis is caused by decreased mrp2 and spgp levels, as well as an abnormal localization of these proteins. The simultaneous up-regulation of mrp1 and mdr1b may confer resistance to hepatocytes against cytokine-induced metabolic stress.