Severe cholestasis may result in end-stage liver disease with the need of liver transplantation (LTX). In children, about 10 % of LTX are necessary because of cholestatic liver diseases. Apart from ...bile duct atresia, three types of progressive familial intrahepatic cholestasis (PFIC) are common causes of severe cholestasis in children. The three subtypes of PFIC are defined by the involved genes: PFIC-1, PFIC-2, and PFIC-3 are due to mutations of P-type ATPase
ATP8B1
(familial intrahepatic cholestasis 1, FIC1), the ATP binding cassette transporter
ABCB11
(bile salt export pump, BSEP), or
ABCB4
(multidrug resistance protein 3, MDR3), respectively. All transporters are localized in the canalicular membrane of hepatocytes and together mediate bile salt and phospholipid transport. In some patients with PFIC-2 disease, recurrence has been observed after LTX, which mimics a PFIC phenotype. It could be shown by several groups that inhibitory anti-BSEP antibodies emerge, which most likely cause disease recurrence. The prevalence of severe BSEP mutations (e.g., splice site and premature stop codon mutations) is very high in this group of patients. These mutations often result in the complete absence of BSEP, which likely accounts for an insufficient auto-tolerance against BSEP. Although many aspects of this “new” disease are not fully elucidated, the possibility of anti-BSEP antibody formation has implications for the pre- and posttransplant management of PFIC-2 patients. This review will summarize the current knowledge including diagnosis, pathomechanisms, and management of “autoimmune BSEP disease.”
Bile Acid-Induced Suicidal Erythrocyte Death Lang, Elisabeth; Pozdeev, Vitaly I.; Gatidis, Sergios ...
Cellular physiology and biochemistry,
04/2016, Letnik:
38, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Background/Aims: In nucleated cells, bile acids may activate cation channels subsequently leading to entry of Ca2+. In erythrocytes, increase of cytosolic Ca2+ activity triggers eryptosis, the ...suicidal death of erythrocytes characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis is triggered by bile duct ligation, an effect partially attributed to conjugated bilirubin. The present study explored, whether bile acids may stimulate eryptosis. Methods: Phosphatidylserine exposing erythrocytes have been identified utilizing annexin V binding, cell volume estimated from forward scatter, cytosolic Ca2+ activity determined using Fluo-3 fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Results: The exposure of human erythrocytes to glycochenodesoxycholic (GCDC) and taurochenodesoxycholic (TCDC) acid was followed by a significant decrease of forward scatter and significant increase of Fluo-3 fluorescence, ceramide abundance as well as annexin V binding. The effect on annexin V binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusion: Bile acids stimulate suicidal cell death, an effect paralleled by and in part due to Ca2+ entry and ceramide. The bile acid induced eryptosis may in turn lead to accelerated clearance of circulating erythrocytes and, thus, may contribute to anemia in cholestatic patients.
AIM： To evaluate cut-off values and performance of acoustic radiation force impulse imaging （ARFI） using transient elastography FibroScan （FS） as a reference. METHODS： Six hundred and six patients ...were enrolled in this study.All patients underwent liver stiffness measurement with FS （FS-LS） and ARFI （with shear wave velocity quantification; ARFI-SWV） and the performance of ARFI in comparison to FS was determined. Sixtyeight patients underwent liver biopsy. RESULTS： Significantly higher success rates for the determination of liver stiffness were found using ARFI as compared to FS 604/606 （99.7%） vs 482/606 （79.5%）; P 〈 0.001. ARFI-SWV correlated significantly with FS-LS （r = 0.920, P 〈 0.001）. ARFI-SWV increased significantly with the stage of fibrosis （1.09 ± 0.13 m/s for patients with no significant fibrosis （FS-LS 〈 7.6 kPa）; 1.46 ± 0.27 m/s for patients with significant liver fibrosis （7.6 〈 FS-LS ≤ 13.0 kPa）; and 2.55 ± 0.77 m/s for patients with liver cirrhosis （FS-LS 〉 13.0 kPa））. ARFI-SWV cut-off values were identified for no significant fibrosis （1.29 m/s; sensitivity 91.4% and specificity 92.6%） and for liver cirrhosis （1.60 m/s; sensitivity 92.3% and specificity 96.5%）. The optimal cut-off value for predicting liver fibrosis （F ≥ 2） was 1.32 m/s （sensitivity 87.0% and specificity 80.0%） and for liver cirrhosis （F4） 1.62 m/s （sensitivity 100% and specificity 85.7%）, for patients who underwent liver biopsy. An excellent inter-and intraobserver reproducibility was observed for ARFI-SWV determinations. CONCLUSION： An ARFI-SWV cut-off value of 1.29 m/s seems to be optimal for patients with no significant liver fibrosis and 1.60 m/s for patients with liver cirrhosis.
Progressive familial intrahepatic cholestasis type 2 (PFIC‐2) is caused by mutations in ABCB11, encoding the bile salt export pump (BSEP). In 2009, we described a child with PFIC‐2 who developed ...PFIC‐like symptoms after orthotopic liver transplantation (OLT). BSEP‐reactive antibodies were demonstrated to account for disease recurrence. Here, we characterize the nature of this antibody response in 7 more patients with antibody‐induced BSEP deficiency (AIBD). Gene sequencing and immunostaining of native liver biopsies indicated absent or strongly reduced BSEP expression in all 7 PFIC‐2 patients who suffered from phenotypic disease recurrence post‐OLT. Immunofluorescence, western blotting analysis, and transepithelial transport assays demonstrated immunoglobulin (Ig) G‐class BSEP‐reactive antibodies in these patients. In all cases, the N‐terminal half of BSEP was recognized, with reaction against its first extracellular loop (ECL1) in six sera. In five, antibodies reactive against the C‐terminal half also were found. Only the sera recognizing ECL1 showed inhibition of transepithelial taurocholate transport. In a vesicle‐based functional assay, transport inhibition by anti‐BSEP antibodies binding from the cytosolic side was functionally proven as well. Within 2 hours of perfusion with antibodies purified from 1 patient, rat liver showed canalicular IgG staining that was absent after perfusion with control IgG. Conclusions: PFIC‐2 patients carrying severe BSEP mutations are at risk of developing BSEP antibodies post‐OLT. The antibody response is polyclonal, targeting both extra‐ and intracellular BSEP domains. ECL1, a unique domain of BSEP, likely is a critical target involved in transport inhibition as demonstrated in several patients with AIBD manifest as cholestasis. (Hepatology 2016;63:524–537)
The sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake transporter for bile salts into liver parenchymal cells, and PKC-mediated endocytosis was shown to regulate the number of ...Ntcp molecules at the plasma membrane. In this study, mechanisms of Ntcp internalization were analyzed by flow cytometry, immunofluorescence, and Western blot analyses in HepG2 cells. PKC activation induced endocytosis of Ntcp from the plasma membrane by ~30%. Endocytosis of Ntcp was clathrin dependent and was followed by lysosomal degradation. A dileucine motif located in the third intracellular loop of Ntcp was essential for endocytosis but also for processing and plasma membrane targeting, suggesting a dual function of this motif for intracellular trafficking of Ntcp. Mutation of two of five potential phosphorylation sites surrounding the dileucine motif (Thr225 and Ser226) inhibited PKC-mediated endocytosis. In conclusion, we could identify a motif, which is critical for Ntcp plasma membrane localization. Endocytic retrieval protects hepatocytes from elevated bile salt concentrations and is of special interest, because NTCP has been identified as a receptor for the hepatitis B and D virus.
We previously demonstrated that clinical administration of mobilized CD133
bone marrow stem cells (BMSC) accelerates hepatic regeneration. Here, we investigated the potential of platelets to modulate ...CD133
BMSC homing to hepatic endothelial cells and sequestration to warm ischemic livers. Modulatory effects of platelets on the adhesion of CD133
BMSC to human and mouse liver-sinusoidal- and micro- endothelial cells (EC) respectively were evaluated in in vitro co-culture systems. CD133
BMSC adhesion to all types of EC were increased in the presence of platelets under shear stress. This platelet effect was mostly diminished by antagonization of P-selectin and its ligand P-Selectin-Glyco-Ligand-1 (PSGL-1). Inhibition of PECAM-1 as well as SDF-1 receptor CXCR4 had no such effect. In a model of the isolated reperfused rat liver subsequent to warm ischemia, the co-infusion of platelets augmented CD133
BMSC homing to the injured liver with heightened transmigration towards the extra sinusoidal space when compared to perfusion conditions without platelets. Extravascular co-localization of CD133
BMSC with hepatocytes was confirmed by confocal microscopy. We demonstrated an enhancing effect of platelets on CD133
BMSC homing to and transmigrating along hepatic EC putatively depending on PSGL-1 and P-selectin. Our insights suggest a new mechanism of platelets to augment stem cell dependent hepatic repair.
The bile salt export pump BSEP mediates bile formation. Over 150 BSEP mutations are associated with progressive familial intrahepatic cholestasis type 2 (PFIC-2), with few characterised specifically. ...We examined liver tissues from two PFIC-2 patients compound heterozygous for the splice-site mutation c.150 + 3A > C and either c.2783_2787dup5 resulting in a frameshift with a premature termination codon (child 1) or p.R832C (child 2). Splicing was analysed with a minigene system and mRNA sequencing from patients' livers. Protein expression was shown by immunofluorescence. Using the minigene, c.150 + 3A > C causes complete skipping of exon 3. In liver tissue of child 1, c.2783_2787dup5 was found on DNA but not on mRNA level, implying nonsense-mediated mRNA decay (NMD) when c.2783_2787dup5 is present. Still, BSEP protein as well as mRNA with and without exon 3 were detectable and can be assigned to the c.150 + 3A > C allele. Correctly spliced transcripts despite c.150 + 3A > C were also confirmed in liver of child 2. In conclusion, we provide evidence (1) for effective NMD due to a BSEP frameshift mutation and (2) partial exon-skipping due to c.150 + 3A > C. The results illustrate that the extent of exon-skipping depends on the genomic and cellular context and that regulation of splicing may have therapeutic potential.
Summary Liver diseases are common in inhabitants and migrants of tropical countries, where the liver can be exposed not only to toxins but also to many viral, bacterial, fungal, and parasitic ...infections. Schistosomiasis—a common parasitic infection that affects at least 240 million people worldwide, mostly in Africa—is regarded as the most frequent cause of liver fibrosis worldwide. We present a case of a 19-year-old male refugee from Guinea with recurrent oesophageal variceal bleeding due to schistosomal liver fibrosis refractory to endoscopic therapy. This case was an indication for portosystemic surgery, which is a highly invasive non-reversible intervention. An alternative, less invasive, reversible radiological procedure, used in liver cirrhosis, is the placement of a transjugular intrahepatic portosystemic shunt (TIPS). After thorough considerations of all therapeutic options we placed a TIPS in our patient. In more than 3 years of observation, he is clinically well apart from one episode of hepatic encephalopathy related to an acute episode of viral gastroenteritis. Bleeding from oesophageal varices has not recurred. In this Grand Round, we review the diagnostic approaches and treatment options for portal hypertension due to schistosomal liver fibrosis.
Although there has been much research into the pathogenesis and treatment of hepatitis B virus (HBV) and hepatitis D virus (HDV) infections, we still do not completely understand how these pathogens ...enter hepatocytes. This is because in vitro infection studies have only been performed in primary human hepatocytes. Development of a polarizable, HBV-susceptible human hepatoma cell line and studies of primary hepatocytes from Tupaia belangeri have provided important insights into the viral and cellular factors involved in virus binding and infection. The large envelope (L) protein on the surface of HBV and HDV particles has many different functions and is required for virus entry. The L protein mediates attachment of virions to heparan sulfate proteoglycans on the surface of hepatocytes. The myristoylated N-terminal preS1 domain of the L protein subsequently binds to the sodium taurocholate cotransporting polypeptide (NTCP, encoded by SLC10A1), the recently identified bona fide receptor for HBV and HDV. The receptor functions of NTCP and virus entry are blocked, in vitro and in vivo, by Myrcludex B, a synthetic N -acylated preS1 lipopeptide. Currently, the only agents available to treat chronic HBV infection target the viral polymerase, and no selective therapies are available for HDV infection. It is therefore important to study the therapeutic potential of virus entry inhibitors, especially when combined with strategies to induce immune-mediated killing of infected hepatocytes.
AIM To investigate the relation of two different mutations to the outcome of partial external biliary diversion(PEBD)in severe bile salt export pump(BSEP) deficiency.METHODS Mutations in the gene ...encoding BSEP leading to severe BSEP deficiency in two unrelated patients were identified by genomic sequencing. Native liver biopsies and transiently transfected human embryonic kidney(HEK) 293 cells expressing either wild-type or mutated BSEP were subjected to immunofluorescence analysis to assess BSEP transporter localization. Bile acid profiles of patient and control bile samples were generated by ultra-performance liquid chromatographytandem mass spectrometry. Wild-type and mutant BSEP transport of ~3H-labeled taurocholate(TC) and taurochenodeoxycholate(TCDC) was assessed by vesicular transport assays.RESULTS A girl(at 2 mo) presented with pruritus, jaundice and elevated serum bile salts(BS). PEBD stabilized liver function and prevented liver transplantation. She was heterozygous for the BSEP deletion p.T919 del and the nonsense mutation p.R1235 X. At the age of 17 years relative amounts of conjugated BS in her bile were normal, while total BS were less than 3% as compared to controls. An unrelated boy(age 1.5 years) presenting with severe pruritus and elevated serum BS was heterozygous for the same nonsense and another missense mutation, p.G1032 R. PEBD failed to alleviate pruritus, eventually necessitating liver transplantation. BS concentration in bile was about 5% of controls. BS were mainly unconjugated with an unusual low amount of chenodeoxycholate derivatives(< 5%). The patients’ native liver biopsies showed canalicular BSEP expression. Both BSEP p.T919 del and p.G1032 R were localized in the plasma membrane in HEK293 cells. In vitro transport assays showed drastic reduction of transport by both mutations. Using purified recombinant BSEP as quantifiable reference, per-molecule transport rates for TC and TCDC were determined to be 3 and 2 BS molecules per wild-type BSEP transporter per minute, respectively.CONCLUSION In summary, our findings suggest that residual function of BSEP as well as substrate specificity influence the therapeutic effectiveness of PEBD in progressive familial intrahepatic cholestasis type 2(PFIC-2).
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