Milan Jirsa

Gilbert syndrome and ischemic heart disease: a protective effect of elevated bilirubin levels.

BACKGROUND Oxidation processes play an important role in atherogenesis. Bilirubin IXalpha is recognised as a potent antioxidant. In the present study, we assessed the role of elevated serum bilirubin levels in the prevention of ischemic heart disease (IHD). METHODS The occurrence of IHD was determined in Gilbert syndrome (GS) patients above 40 years (n=50). The diagnosis was based on past medical history and ECG criteria. The occurrence was related to that of the comparable general population (n=2296). Serum biochemistry, including the total antioxidant status was evaluated in the GS subjects, IHD patients (n=38) and control subjects (n=38). RESULTS The prevalence of IHD in GS subjects (aged 49.7+/-9.0 years) was 2% (0.05-10.7%, 95% confidence interval), compared to 12.1% in a general population (P<0.05). Bilirubin, total antioxidant capacity and high density lipoprotein (HDL) cholesterol were found to be significantly higher in GS subjects compared to control groups (P<0.05). According to linear discriminant analysis, hyperbilirubinemia rather than elevation of HDL cholesterol levels seemed to be more important in protection from IHD. CONCLUSIONS In the present study, low prevalence of IHD in GS subjects was detected. It may be presumed that chronic hyperbilirubinemia prevent the development of IHD by increasing the serum antioxidant capacity.

Severe bile salt export pump deficiency : 82 different ABCB11 mutations in 109 families

BACKGROUND & AIMS Patients with severe bile salt export pump (BSEP) deficiency present as infants with progressive cholestatic liver disease. We characterized mutations of ABCB11 (encoding BSEP) in such patients and correlated genotypes with residual protein detection and risk of malignancy. METHODS Patients with intrahepatic cholestasis suggestive of BSEP deficiency were investigated by single-strand conformation polymorphism analysis and sequencing of ABCB11. Genotypes sorted by likely phenotypic severity were correlated with data on BSEP immunohistochemistry and clinical outcome. RESULTS Eighty-two different mutations (52 novel) were identified in 109 families (9 nonsense mutations, 10 small insertions and deletions, 15 splice-site changes, 3 whole-gene deletions, 45 missense changes). In 7 families, only a single heterozygous mutation was identified despite complete sequence analysis. Thirty-two percent of mutations occurred in >1 family, with E297G and/or D482G present in 58% of European families (52/89). On immunohistochemical analysis (88 patients), 93% had abnormal or absent BSEP staining. Expression varied most for E297G and D482G, with some BSEP detected in 45% of patients (19/42) with these mutations. Hepatocellular carcinoma or cholangiocarcinoma developed in 15% of patients (19/128). Two protein-truncating mutations conferred particular risk; 38% (8/21) of such patients developed malignancy versus 10% (11/107) with potentially less severe genotypes (relative risk, 3.7 [confidence limits, 1.7-8.1; P = .003]). CONCLUSIONS With this study, >100 ABCB11 mutations are now identified. Immunohistochemically detectable BSEP is typically absent, or much reduced, in severe disease. BSEP deficiency confers risk of hepatobiliary malignancy. Close surveillance of BSEP-deficient patients retaining their native liver, particularly those carrying 2 null mutations, is essential.

Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver

Bilirubin, a breakdown product of heme, is normally glucuronidated and excreted by the liver into bile. Failure of this system can lead to a buildup of conjugated bilirubin in the blood, resulting in jaundice. The mechanistic basis of bilirubin excretion and hyperbilirubinemia syndromes is largely understood, but that of Rotor syndrome, an autosomal recessive disorder characterized by conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic uptake of anionic diagnostics, has remained enigmatic. Here, we analyzed 8 Rotor-syndrome families and found that Rotor syndrome was linked to mutations predicted to cause complete and simultaneous deficiencies of the organic anion transporting polypeptides OATP1B1 and OATP1B3. These important detoxification-limiting proteins mediate uptake and clearance of countless drugs and drug conjugates across the sinusoidal hepatocyte membrane. OATP1B1 polymorphisms have previously been linked to drug hypersensitivities. Using mice deficient in Oatp1a/1b and in the multispecific sinusoidal export pump Abcc3, we found that Abcc3 secretes bilirubin conjugates into the blood, while Oatp1a/1b transporters mediate their hepatic reuptake. Transgenic expression of human OATP1B1 or OATP1B3 restored the function of this detoxification-enhancing liver-blood shuttle in Oatp1a/1b-deficient mice. Within liver lobules, this shuttle may allow flexible transfer of bilirubin conjugates (and probably also drug conjugates) formed in upstream hepatocytes to downstream hepatocytes, thereby preventing local saturation of further detoxification processes and hepatocyte toxic injury. Thus, disruption of hepatic reuptake of bilirubin glucuronide due to coexisting OATP1B1 and OATP1B3 deficiencies explains Rotor-type hyperbilirubinemia. Moreover, OATP1B1 and OATP1B3 null mutations may confer substantial drug toxicity risks.

5‐aminolaevulinic acid‐induced fluorescence cystoscopy during transurethral resection reduces the risk of recurrence in stage Ta/T1 bladder cancer

To assess the influence of 5‐aminolaevulinic acid‐induced fluorescence cystoscopy (FC) during transurethral resection (TUR) on the recurrence rate and the length of tumour‐free interval in stage Ta/T1 transitional cell carcinoma (TCC) of the urinary bladder.

Relation between hepatic expression of ATP-binding cassette transporters G5 and G8 and biliary cholesterol secretion in mice

BACKGROUND/AIM Mutations in genes encoding the ATP-binding cassette (ABC)-transporters ABCG5 and ABCG8 underlie sitosterolemia, which is characterized by elevated plasma levels of phytosterols due to increased intestinal absorption and impaired biliary secretion of sterols. The aim of our study was to correlate the expression levels of Abcg5 and Abcg8 to biliary cholesterol secretion in various (genetically-modified) mouse models. METHODS Bile was collected from genetically-modified mice fed a chow diet, or from mice fed either a chow diet, or chow supplemented with either 1% diosgenin, 0.1% simvastatin, or a synthetic liver X receptor agonist, for determination of biliary lipids. Livers and small intestines were harvested and expression levels of Abcg5, Abcg8 and Abcb4 were determined by real-time polymerase chain reaction. RESULTS Intestinal expression of Abcg5 and Abcg8 did not show much variation between the various models. In contrast, a linear correlation between hepatic expression levels of Abcg5 and Abcg8 and biliary cholesterol secretion rates was found. This relation was independent of Abcb4-mediated phospholipid secretion. However, in diosgenin-fed mice showing cholesterol hypersecretion, hepatic Abcg5 and Abcg8 expression levels remained unchanged. CONCLUSIONS Our results strongly support a role for Abcg5 and Abcg8 in regulation of biliary cholesterol secretion, but also indicate the existence of a largely independent route of cholesterol secretion.

Troy, a Tumor Necrosis Factor Receptor Family Member, Interacts With Lgr5 to Inhibit Wnt Signaling in Intestinal Stem Cells

BACKGROUND & AIMS The Wnt signaling pathway is required for maintenance of the intestinal epithelia; blocking this pathway reduces the proliferative capacity of the intestinal stem cells. However, aberrant Wnt signaling leads to intestinal cancer. We investigated the roles of the Wnt pathway in homeostasis of the intestinal epithelium and during malignant transformation in human cells and mice. METHODS We performed chromatin immunoprecipitation (ChIP) with DNA microarray analysis (ChIP-on-chip) to identify genes regulated by Wnt signaling in human colorectal cancer cells Colo320, DLD1, LS174T, and SW480. Formation of intestinal tumor was induced in C57BL/6J mice using azoxymethane and dextran sulfate. Intestinal tissues from these mice, as well as Apc(+/Min) and Apc(CKO/CKO)/Lgr5-EGFP-IRES-CreERT2 mice, were analyzed by immunohistochemistry and in situ hybridization. RESULTS We identified promoter regions of 960 genes that interacted with the Wnt pathway nuclear effector T-cell factor 4 in 4 different human colorectal cancer-derived cell lines; 18 of these promoters were present in all chromatin precipitates. Wnt signaling up-regulated a member of the tumor necrosis factor receptor superfamily called TROY. Levels of TROY messenger RNA were increased in human cells with deficiencies in the adenomatous polyposis coli (APC) gene and in cells stimulated with the Wnt3a ligand. Expression of Troy was significantly up-regulated in neoplastic tissues from mice during intestinal tumorigenesis. Lineage tracing experiments revealed that Troy is produced specifically by fast-cycling intestinal stem cells. TROY associated with a unique marker of these cells, leucine-rich repeat-containing G-protein coupled receptor (LGR) 5. In organoids established from the intestinal crypts, Troy suppressed signaling mediated by R-spondin, a Wnt agonist. CONCLUSIONS TROY is up-regulated in human colorectal cancer cell lines and in intestinal tumors in mice. It functions as a negative modulator of the Wnt pathway in LGR5-positive stem cells.

Photodynamic therapy of cutaneous metastases of breast cancer after local application of meso-tetra-(para-sulphophenyl)-porphin (TPPS4)

Nine patients with cutaneous metastases of breast cancer were treated using photodynamic therapy. Meso-tetra-(para-sulphophenyl)-porphin (TPPS4) was applied locally at a dose of 0.15-0.3 mg directly to the lesion. The light source was an argon dye laser emitting light of 630 nm at a fluence rate of 312-680 mW cm-2 and a fluence of 150 J cm-2. No signs of local or systemic side effects or toxicity of the photosensitizer were observed. Treatments were well tolerated with no photosensitivity of the skin. Complete destruction of the tumour was observed in three patients, reduction of the tumour size by more than 50% in two patients, reduction-of the tumour size by less than 50% in two patients and no regression in two patients. The advantages of this method include the high concentration of the photosensitizer in the tumour, the extremely low total dose and the lack of side effects. The disadvantages include the less homogeneous distribution of the photosensitizer in the tumour tissue compared with that obtained by intravenous application.

New insights in bilirubin metabolism and their clinical implications

Bilirubin, a major end product of heme breakdown, is an important constituent of bile, responsible for its characteristic colour. Over recent decades, our understanding of bilirubin metabolism has expanded along with the processes of elimination of other endogenous and exogenous anionic substrates, mediated by the action of multiple transport systems at the sinusoidal and canalicular membrane of hepatocytes. Several inherited disorders characterised by impaired bilirubin conjugation (Crigler-Najjar syndrome type I and type II, Gilbert syndrome) or transport (Dubin-Johnson and Rotor syndrome) result in various degrees of hyperbilirubinemia of either the predominantly unconjugated or predominantly conjugated type. Moreover, disrupted regulation of hepatobiliary transport systems can explain jaundice in many acquired liver disorders. In this review, we discuss the recent data on liver bilirubin handling based on the discovery of the molecular basis of Rotor syndrome. The data show that a substantial fraction of bilirubin conjugates is primarily secreted by MRP3 at the sinusoidal membrane into the blood, from where they are subsequently reuptaken by sinusoidal membrane-bound organic anion transporting polypeptides OATP1B1 and OATP1B3. OATP1B proteins are also responsible for liver clearance of bilirubin conjugated in splanchnic organs, such as the intestine and kidney, and for a number of endogenous compounds, xenobiotics and drugs. Absence of one or both OATP1B proteins thus may have serious impact on toxicity of commonly used drugs cleared by this system such as statins, sartans, methotrexate or rifampicin. The liver-blood cycling of conjugated bilirubin is impaired in cholestatic and parenchymal liver diseases and this impairment most likely contributes to jaundice accompanying these disorders.

Genetic background of cholesterol gallstone disease

Cholesterol gallstone formation is a multifactorial process involving a multitude of metabolic pathways. The primary pathogenic factor is hypersecretion of free cholesterol into bile. For people living in the Western Hemisphere, this is almost a normal condition, certainly in the elderly, which explains the very high incidence of gallstone disease. It is probably because the multifactorial background genes responsible for the high incidence have not yet been identified, despite the fact that genetic factors clearly play a role. Analysis of the many pathways involved in biliary cholesterol secretion reveals many potential candidates and considering the progress in unraveling the regulatory mechanisms of the responsible genes, identification of the primary gallstone genes will be successful in the near future.

Revised King's College score for liver transplantation in adult patients with Wilson's disease.

Fulminant Wilsons disease (WD) is almost invariably fatal, and liver transplantation is the only life‐saving treatment. Decompensated chronic WD usually responds to chelation therapy. Our aim was to validate 3 published scoring systems for deciding between chelation treatment and liver transplantation in patients with chronic decompensated and fulminant WD. Model for end‐stage liver disease (MELD) score, as well as WD prognostic index (WPI) and its recently revised version (RWPI) were evaluated as predictors of the safety for chelation therapy. A group of 14 adult patients with decompensated chronic WD who improved on penicillamine treatment were compared with 21 patients with fulminant WD. The diagnosis of WD was based on increased urinary copper excretion and confirmed by elevated liver copper content and/or mutation analysis of the WD gene. The MELD score, WPI, and RWPI were calculated for all patients with WD. The accuracy of the MELD score, WPI, and RWPI for prediction of response to chelation therapy in patients with decompensated chronic WD was 0.968, 0.980, and 0.993, respectively. None of the decompensated chronic WD patients had a MELD score >30, RWPI >11, or WPI >7. RWPI showed the highest accuracy and the lowest false negativity compared with WPI and MELD. In conclusion, our data indicate that RWPI, originally proposed for pediatric patients, is also useful for adults. Liver Transpl, 2007.