Altered placental macrophage numbers and subsets in pregnancies complicated with intrahepatic cholestasis of pregnancy (ICP) compared to healthy pregnancies.

INTRODUCTION: Intrahepatic cholestasis of pregnancy (ICP) can result in adverse outcomes for both mother and fetus. Inflammatory (M1 subset) or anti-inflammatory (M2 subset) macrophage polarisation is associated with various complications of pregnancy. However, the influence of ICP on macrophage numbers and polarisation remains unknown. This study analyses macrophage density and distribution in placentas of patients with ICP compared to controls. Clinical parameters were correlated to macrophage distribution and ursodeoxycholic acid use (UDCA). METHODS: This study included routinely collected placental tissue samples of 42 women diagnosed with ICP and of 50 control pregnancies. Immunohistochemical staining was performed on placental tissue using CD68 antibody as a pan-macrophage marker, CD206 antibody as an M2 and HLA-DR antibody as an M1 macrophage marker. Macrophage density (cells/mm2) and distribution (CD206+/CD68+ or CD206+/CD68+HLA-DR+) in both decidua (maternal tissue) and villous parenchyma (fetal tissue) were compared between groups. Macrophage density and distribution were correlated to clinical parameters for ICP patients. RESULTS: The density of CD68+ macrophages differed significantly between groups in villous parenchyma. In both decidua and villous parenchyma, CD206+/CD68+ ratio was significantly lower in ICP patients compared to controls (p = 0.003 and p=<0.001, respectively). No difference was found based on UDCA use or in CD68+HLA-DR+ cell density. Significant correlations were found between macrophage density and peak serum bile acids and liver enzymes. DISCUSSION: In ICP patients, an immune shift was observed in both decidual and villous tissue, indicated by a lower CD206+/CD68+ ratio. ICP seems to affect placental tissue, however more research is required to understand its consequences.

Case report: progressive familial intrahepatic cholestasis type 3 with compound heterozygous ABCB4 variants diagnosed 15 years after liver transplantation.

BACKGROUND: Progressive familial intrahepatic cholestasis (PFIC) type 3 is an autosomal recessive disorder arising from mutations in the ATP-binding cassette subfamily B member 4 (ABCB4) gene. This gene encodes multidrug resistance protein-3 (MDR3) that acts as a hepatocanalicular floppase that transports phosphatidylcholine from the inner to the outer canalicular membrane. In the absence of phosphatidylcholine, the detergent activity of bile salts is amplified and this leads to cholangiopathy, bile duct loss and biliary cirrhosis. Patients usually present in infancy or childhood and often progress to end-stage liver disease before adulthood. CASE PRESENTATION: We report a 32-year-old female who required cadaveric liver transplantation at the age of 17 for cryptogenic cirrhosis. When the patient developed chronic ductopenia in the allograft 15 years later, we hypothesized that the patient's original disease was due to a deficiency of a biliary transport protein and the ductopenia could be explained by an autoimmune response to neoantigen that was not previously encountered by the immune system. We therefore performed genetic analyses and immunohistochemistry of the native liver, which led to a diagnosis of PFIC3. However, there was no evidence of humoral immune response to the MDR3 and therefore, we assumed that the ductopenia observed in the allograft was likely due to chronic rejection rather than autoimmune disease in the allograft. CONCLUSIONS: Teenage patients referred for liver transplantation with cryptogenic liver disease should undergo work up for PFIC3. An accurate diagnosis of PFIC 3 is key for optimal management, therapeutic intervention, and avoidance of complications before the onset of end-stage liver disease.

Anti-inflammatory, anti-oxidative stress and novel therapeutic targets for cholestatic liver injury.

Cholestasis is a pathological process in which bile drainage is poor for a variety of reasons. Many studies have shown that cholestatic liver injury is a neutrophil-mediated inflammatory response, and oxidative stress induced by neutrophils is the main mechanism of liver cell death. The literature summarizes the bile acid signaling pathway, the neutrophil chemotaxis recruitment process during cholestasis, and the oxidative stress damage produced by neutrophil activation, summarizes the latest research progress. Sphingosine-1-phosphate receptor (S1PR) is a potential therapeutic target for cholestasis that reduces neutrophil aggregation without inhibiting systemic immune status. Early growth response factor 1 (Egr-1) may play a central role in the inflammation induced by cholestasis, and it is also a potential therapeutic target to inhibit the inflammation induced by cholestasis. Strengthening the antioxidant system of hepatocytes to cope with oxidative stress of neutrophils is a feasible treatment for cholestatic liver injury.

Alloimmunity and Cholestasis After Liver Transplantation in Children With Progressive Familial Intrahepatic Cholestasis.

OBJECTIVES: Bile salt export pump (BSEP) deficiency is an important reason for chronic cholestasis leading to liver transplantation (LT) in early childhood. The underlying pathology is a dysfunction of BSEP due to various mutations in the ABCB11 gene. Cases of clinical recurrence after LT due to alloantibodies directed against BSEP (antibody-induced BSEP deficiency [AIBD]) have been reported. Most of these patients could be controlled by intensified immunosuppression. METHODS: We here report on 3 children with BSEP-deficiency and end-stage liver disease, which developed AIBD after LT refractory to extensive immunosuppressive and immunomodulatory treatments; retransplantation was necessary in all 3 patients. In 1 patient, a stem cell transplantation was performed successfully. RESULTS: AIBD seems to be induced by triggering factors such as initial impaired graft function or infections after LT. CONCLUSIONS: The underlying mutation may play a role in this process. Intensifying immunosuppression may be able to control AIBD, but some cases seem to be refractory to treatment and require retransplantation. Stem cell transplantation may provide a new therapeutic option for cases refractory to conservative treatment.

Expression and significance of dendritic cells and Th17/Treg in serum and placental tissues of patients with intrahepatic cholestasis of pregnancy.

PURPOSE: Dendritic cells (DCs) are involved in immune system, which can also regulate the differentiation of T helper 17 (Th17) and regulatory T cells (Treg). DCs and Th17/Treg participate in preeclampsia and recurrent spontaneous abortion (RSA), but there is still lack of research in intrahepatic cholestasis of pregnancy (ICP). The aim was to evaluate the expression and significance of CD83+DCs, CD1a+DCs, interleukin-17 (IL-17) and IL-35 in serum and placental tissues of patients with ICP. METHODS: Thirty cases of mild ICP, 25 cases of severe ICP were selected, and 30 cases of normal pregnant women were selected as control group. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) were used to detect the expression of CD83+DCs, CD1a+DCs, IL-17 and IL-35 in serum and placenta tissues, respectively. RESULTS: There were more CD83+DCs, IL-17 expressed in placenta from women with ICP than in normal pregnancies, while the number of decidual CD1a+DCs, IL-35 was significantly lower in ICP than in normal pregnant women. The comparison within three groups had statistical difference (p < .05). Serum CD83+DCs and CD1a+DCs levels had no significance. IL-17 was higher in ICP, while IL-35 was lower. CONCLUSIONS: DCs are involved in damaging the maternal-fetal immune tolerance by changing the phenotype and mature state, which may affect the differentiation of Th17/Treg to cause ICP.

Cholestasis After Pediatric Liver Transplantation-Recurrence of a Progressive Familial Intrahepatic Cholestasis Phenotype as a Rare Differential Diagnosis: A Case Report.

INTRODUCTION: Nonobstructive cholestasis after pediatric liver transplantation is a common diagnostic and therapeutic dilemma. We describe a girl with neonatal cholestasis because of progressive familial intrahepatic cholestasis 2 (PFIC-2) and presence of a homozygous splice site mutation in the ABCB11 gene. Liver transplantation was performed because of end-stage liver disease at the age of 6. Cholestasis with normal gamma-glutamyl transferase (GGT) developed 8 years after liver transplantation. A liver biopsy showed canalicular cholestasis and giant cell hepatitis without evidence of rejection, mimicking PFIC-2. Immunofluorescence staining of normal human liver sections with patient's serum revealed reactivity toward a canalicular epitope, which could be identified as bile salt export pump (BSEP) using BSEP-yellow fluorescent protein (YFP) transfected cells. Our patient developed a recurrence of a PFIC-2 phenotype due to production of antibodies against BSEP (alloimmune BSEP disease [AIBD]). Intensification of immunosuppressive therapy as well as antibody treatment with plasmapheresis and Rituximab were initiated, leading to stabilization of the clinical condition and depletion of anti-BSEP antibodies in serum. However, after 1 year liver transplantation was necessary again because of end-stage liver insufficiency. Afterward, immunomodulatory treatment consisted of tacrolimus, mycophenolate mofetil, prednisone, immunoadsorption, and high-dose immunoglobulin therapy (1 g/kg/d). CONCLUSION: Cholestasis after liver transplantation may indicate an AIBD with a PFIC-2 phenotype. Besides enhancement of immunosuppressive therapy, an antibody depletion with plasmapheresis, immunoadsorption, immunoglobulins, and B-cell depletion represents a therapeutic option.

A Critical Evaluation of Liver Pathology in Humans with Danon Disease and Experimental Correlates in a Rat Model of LAMP-2 Deficiency.

Danon disease is a genetic deficiency in lysosome-associated membrane protein 2 (LAMP-2), a highly glycosylated constituent of the lysosomal membrane and characterized by a cardiomyopathy, skeletal muscle myopathy, and cognitive impairment. Patients, however, often manifest hepatic abnormalities, but liver function has not been well evaluated and the syndrome is relatively uncommon. Hence, we have taken advantage of a rat that has been deleted of LAMP-2 to study the relative role of LAMP-2 on liver function. Interestingly, rats deficient in LAMP-2 develop a striking increase in serum alkaline phosphatase (ALP) and a decrease in bile flow compared with wild-type littermates. Importantly and by ultrastructural analysis, deficient rats manifest dilated canaliculi that lack microvilli with evidence of bile-containing bodies. Moreover, following bile duct ligation, LAMP-2-deficient rats develop rapid and severe evidence of advanced cholestasis, with an increase in serum bilirubin, as early as 6 h later. In wild-type control rats, multidrug resistance-associated protein 2 (Mrp2) normally concentrates at the bile canalicular membranes to secrete conjugated bilirubin into bile. However, in LAMP-2y/- rats, Mrp2 was detected in hepatocytes compared with other canalicular proteins including P-glycoproteins, dipeptidyl peptidase IV (CD26), and aminopeptidase (CD13). Our data further suggest that LAMP-2 interacts with the membrane cytoskeletal proteins radixin and F-actin in determining the localization of integral membrane proteins.

Recurrence of Progressive Familial Intrahepatic Cholestasis Type 2 Phenotype After Living-donor Liver Transplantation: A Case Report.

BACKGROUND: Progressive familial intrahepatic cholestasis 2 (PFIC2) is the result of mutations in the ABCB11, which encodes for bile salt export pump (BSEP). An absence of BSEP in the canalicular membrane causes cholestasis and leads to the development of end-stage liver disease in the first decade of life. Liver transplantation (LT) has been considered curative for BSEP disease. However, patients with PFIC2 having undergone LT have recently been reported to develop recurrence of cholestasis together with the clinical and histological features of primary BSEP disease. CASE REPORT: We herein present a rare case of a patient with PFIC2 who developed post-transplantation recurrence of progressive intrahepatic cholestasis due to antibodies against BSEP after living-donor LT, which mimicked primary BSEP disease. The patient had mutations in the ABCB11 gene, resulting in the complete absence of BSEP in the native liver, explaining the lack of tolerance. Immunofluorescence staining of normal human liver sections with the patient's serum and using an anti-human immunoglobulin G antibody to detect serum antibodies showed reactivity to the BSEP epitope in the canalicular membrane. We suggest that the patients having undergone LT had been associated with a risk of autoantibody formation against the BSEP protein. The absence of primary tolerance for the BSEP epitopes may explain the formation of the anti-BSEP antibodies after LDLT.

Recovery from LCDD-associated Severe Liver Cholestasis: a Case Report and Literature Review.

Light chain deposition disease (LCDD) is a rare multisystemic disorder associated with plasma cell proliferation. It mainly affects the kidney, but liver and heart involvement may occur, sometimes mimicking the picture of systemic amyloidosis. Liver disease in LCDD is usually asymptomatic and exceptionally manifests with severe cholestatic hepatitis. We report the case of a 66-year-old female with κ-LCDD and cast nephropathy in the setting of symptomatic multiple myeloma who, after a first cycle of bortezomib-dexamethasone chemotherapy, developed severe and rapidly worsening intrahepatic cholestasis secondary to liver κ-light chain deposition. Intrahepatic cholestasis was attributed to LCDD on the basis of the liver histology and exclusion of possible diagnoses. Chemotherapy was maintained and resulted in progressive resolution of cholestasis. We report here an uncommon presentation of LCDD, with prominent liver involvement that fully recovered with bortezomib-based chemotherapy, and briefly review the relevant literature.

Bile salt export pump-reactive antibodies form a polyclonal, multi-inhibitory response in antibody-induced bile salt export pump deficiency.

UNLABELLED: 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.

Inflammation-associated microRNA-130b down-regulates cytochrome P450 activities and directly targets CYP2C9.

Expression of genes involved in absorption, distribution, metabolism, and excretion (ADME) of drugs is impaired in pathophysiologic conditions such as cholestasis and inflammation. The mechanisms of ADME gene down-regulation remain unclear. In our previous study, strongly elevated levels of microRNAs (miRNA) miR-21, miR-34a, and miR-130b in cholestatic liver and of miR-21 and miR-130b during inflammation were observed. Using HepaRG cells, which retain many functional characteristics of human hepatocytes, we investigated the potential of these miRNAs to down-regulate ADME genes. Cells were transfected with the corresponding miRNA mimics, chemically modified double-stranded RNAs that mimic endogenous miRNAs, followed by mRNA profiling by quantitative reverse-transcription polymerase chain reaction. Activities of six cytochrome P450 enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4) were determined with a liquid chromatography with tandem mass spectrometric cocktail assay. Although miR-21 and miR-34a showed few effects, transfection of miR-130b led to significantly lower expression of nuclear receptors constitutive androstane receptor (CAR) and farnesoid X receptor (FXRα), the CYPs 1A1, 1A2, 2A6, 2C8, 2C9, and 2C19, as well as GSTA2. Furthermore, miR-130b negatively affected activity levels of all measured P450s by at least 30%. Reporter gene assays employing the CYP2C9 3'-untranslated region (3'-UTR) confirmed direct regulation by miR-130b. These data support miR-130b as a potential negative regulator of drug metabolism by directly and/or indirectly affecting the expression of several ADME genes. This may be of relevance in pathophysiologic conditions such as cholestasis and inflammation, which are associated with increased miR-130b expression.

Autoimmune BSEP disease: disease recurrence after liver transplantation for progressive familial intrahepatic cholestasis.

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."

Placental gene-expression profiles of intrahepatic cholestasis of pregnancy reveal involvement of multiple molecular pathways in blood vessel formation and inflammation.

BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-associated liver disease with potentially deleterious consequences for the fetus, particularly when maternal serum bile-acid concentration >40 µM. However, the etiology and pathogenesis of ICP remain elusive. To reveal the underlying molecular mechanisms for the association of maternal serum bile-acid level and fetal outcome in ICP patients, DNA microarray was applied to characterize the whole-genome expression profiles of placentas from healthy women and women diagnosed with ICP. METHODS: Thirty pregnant women recruited in this study were categorized evenly into three groups: healthy group; mild ICP, with serum bile-acid concentration ranging from 10-40 µM; and severe ICP, with bile-acid concentration >40 µM. Gene Ontology analysis in combination with construction of gene-interaction and gene co-expression networks were applied to identify the core regulatory genes associated with ICP pathogenesis, which were further validated by quantitative real-time PCR and histological staining. RESULTS: The core regulatory genes were mainly involved in immune response, VEGF signaling pathway and G-protein-coupled receptor signaling, implying essential roles of immune response, vasculogenesis and angiogenesis in ICP pathogenesis. This implication was supported by the observed aggregated immune-cell infiltration and deficient blood vessel formation in ICP placentas. CONCLUSIONS: Our study provides a system-level insight into the placental gene-expression profiles of women with mild or severe ICP, and reveals multiple molecular pathways in immune response and blood vessel formation that might contribute to ICP pathogenesis.

Liver radiofrequency ablation compromises the biological gut barrier.

AIM: Liver radiofrequency ablation (RFA) has been shown to disrupt the mechanical component of the gut barrier. The aim of the present study was to investigate the consequences of liver RFA on the biological gut barrier in terms of the effects of bile production rate and bowel inflammatory state on intestinal microflora balance. METHOD: A total of 25 New Zealand rabbits were assigned to five groups (n = 5 per group): group CBD: subjected to common bile duct (CBD) extracorporeal bypass; group CBD-RFA: subjected to CBD bypass plus one session of open liver RFA; group RFA: subjected to liver RFA; group sham: subjected to sham operation; and group TBD: subjected to total bile deviation (TBD). In groups CBD and CBD-RFA, bile production rate was assessed for 48 h. In groups sham and RFA, measurement of biliary glycine conjugates of cholic and deoxycholic acid levels, histopathologic examination of the non-ablated liver tissue, morphometric analysis, and histopathologic examination of the terminal ileum and microbiological analysis of fecal and tissue samples collected from the jejunum and the cecum (and in group TBD) were performed at 48 h post-operation. RESULTS: One session of liver RFA resulted in ablation of 18.7 ± 2.7% of liver weight. Following liver RFA, bile production rate was reduced, while the levels of biliary bile salts were not affected. There was mild injury of the non-ablated liver parenchyma, mild intestinal wall inflammation, intestinal mucosa atrophy, and intestinal microbial population overgrowth. CONCLUSION: Reduced in bile production and mild bowel inflammation secondary to liver RFA impaired the biological gut barrier as manifested by intestinal microflora imbalance.

Role of macrophages in bile acid-induced inflammatory response of fetal lung during maternal cholestasis.

UNLABELLED: Infant respiratory distress syndrome (iRDS) in babies born from women with intrahepatic cholestasis of pregnancy (ICP) has been associated with intrauterine exposure to high bile acid levels. Here, we have investigated the role of macrophages in hypercholanemia-induced changes in maternal and fetal lung. Obstructive cholestasis in pregnant rats (OCP) was maintained from day 14 of gestation to term. Gene expression was determined by RT-QPCR, Western blot, and immunofluorescence. The maternal-fetal bile acid pool was radiolabelled using [(3)H]-taurocholate. OCP resulted in increased bile acids in maternal and fetal organs, including lungs. This was accompanied by structural changes in lung tissue, more marked in fetuses (peribronchial edema, collapse of alveolar spaces and deposits of hyaline material in the alveolar lumen), and infiltration of lung tissue by inflammatory cells. The abundance of macrophages and neutrophils in bronchoalveolar lavage fluid (BALF) was also increased in OCP group. Phospholipase A2-IIA (PLA2), the key enzyme in surfactant degradation, was mainly immunodetected in macrophages, which also expressed the bile acid receptor TGR5. The overall expression of PLA2 was markedly enhanced in maternal and fetal lungs of OCP group and in control maternal BALF cells incubated with bile acids. In neonates born from OCP mothers, the enhanced expression of erythropoietin suggested the presence of hypoxia due to iRDS. In conclusion, these results indicate that the accumulation of bile acids due to maternal cholestasis triggers an inflammatory response in the maternal and fetal lungs together with enhanced macrophage-associated PLA2 expression, which may play an important role in iRDS development. KEY MESSAGES: Maternal cholestasis causes respiratory distress syndrome in rat neonates. Cholestasis in pregnant rats causes bile acid accumulation in the fetal lung. This induces lung macrophages infiltration and inflammatory response. Alveolar macrophages co-express phospholipase A2-IIA and TGR5, but not FXR. Bile acid accumulation stimulates phospholipase A2-IIA, but not TGR5, expression.

Cholestatic liver disease after rituximab and adalimumab and the possible role of cross-reacting antibodies to Fab 2 fragments.

BACKGROUND: Millions of patients are treated with therapeutic monoclonal antibodies (Tmabs) for miscellaneous diseases. We investigated sera from six patients who received immune globulin, from one patient with refractory anti-neutrophil-cytoplasmic antibody (ANCA)-associated granulomatosis with polyangiitis (GPA) who developed two episodes of acute cholestatic liver disease, one after treatment with rituximab and a second after adalimumab and a healthy control group. METHODS: Three sera from the patient and six sera from patients who received immune globulin were analyzed for antibodies to rituximab and adalimumab by ELISA. Additionally, sera from the patients and from nine healthy blood donors were coated with the Fab fragment of an unrelated humanized monoclonal antibody, with human Fc proteins as well as a mouse IgG globulin. RESULTS: Viral serology for hepatitis A, B, C and autoantibodies specific for autoimmune liver disorders were negative. In all three sera from the patient antibodies to rituximab could be detected, but also antibodies to adalimumab were present even at time points when the patient had not yet received adalimumab, indicating cross reactivity between both substances. Testing against an unrelated human Fab fragment revealed positive results, indicating that the patient had antibodies against human Fab fragments in general. The Fc proteins were negative, and patients' sera did also not react with mouse IgG globulins. Remarkably, 2 out of 5 patients which were treated with immune globulin had antibodies against human Fab fragments in general whereas in none of the samples from healthy controls antibodies to Fab fragment could be detected. CONCLUSION: This is the first study demonstrating cholestatic liver disease induced by two different Tmabs. Cross - reacting antibodies to Fab2 fragments in general are probably involved. Further studies must show if these Fab2 antibodies in general are related with drug-induced side effects and accelerated drug clearance in patients on Tmab therapy.

Rituximab as therapy for the recurrence of bile salt export pump deficiency after liver transplantation.

Progressive familial intrahepatic cholestasis type 2 (PFIC2) results from recessive mutations in the adenosine triphosphate-binding cassette B11 gene, which encodes for bile salt export pump (BSEP). Liver transplantation (LT) is offered to PFIC2 patients with end-stage liver disease. Reports have described recurrent cholestasis in PFIC2 patients after transplantation, and this has been associated with immunoglobulin G antibodies to BSEP. High-titer anti-BSEP antibodies appear to correlate with episodes of cholestatic graft dysfunction. There is no established paradigm for treating antibody-mediated posttransplant BSEP disease. It appears to be refractory to changes in immunosuppressant medications that would typically be effective in treating allograft rejection. Taking what is known about its pathophysiology, we designed a treatment consisting of rituximab, a chimeric monoclonal anti-CD20 antibody, in combination with intravenous immunoglobulin and plasmapheresis. Using this approach, we report the successful management of 2 patients with antibody-mediated recurrence of PFIC2 after LT.

Effect of maternal cholestasis on TGR5 expression in human and rat placenta at term.

BACKGROUND & AIMS: TGR5 (Gpbar-1) is a plasma membrane-bound bile acid receptor expressed in several tissues, including liver, intestine and brain. High levels of TGR5 mRNA have been detected in human and rodent placenta, however, localization of the TGR5 protein has not been studied in this tissue. We aimed at characterizing TGR5 expression in placental tissue and investigated the effect of bile acids and progesterone metabolites, which accumulate during intrahepatic cholestasis of pregnancy (ICP), on receptor expression and localization. METHODS: TGR5 mRNA levels and cell-specific localization were determined by quantitative PCR and immunofluorescence, respectively. RESULTS: In human term placentas, TGR5 was mainly localized in fetal macrophages and to a lower extent in trophoblasts. In placentas from ICP patients and pregnant rats with obstructive cholestasis a marked down-regulation of TGR5 mRNA expression was observed. However, the cell-specific distribution of the TGR5 protein was unaffected. Besides bile acids, progesterone and its metabolites (5α-pregnan-3α-ol-20-one/5α-pregnan-3ß-ol-20-one), which increase in serum during ICP, were able to dose-dependently activate TGR5. In addition, progesterone metabolites but not their sulfated derivatives nor taurolithocholic acid, significantly down-regulated TGR5 mRNA and protein expression in isolated human macrophages and a macrophage-derived cell line. CONCLUSION: Since fetal macrophages and trophoblast cells are exposed to changes in the flux of compounds across the placental barrier, the expression of TGR5 in these cells together with its sensitivity to bile acids and progesterone metabolites regarding receptor activity and mRNA expression suggest that TGR5 may play a role in the effect of maternal cholestasis on the placenta.

Obstructive jaundice expands intrahepatic regulatory T cells, which impair liver T lymphocyte function but modulate liver cholestasis and fibrosis.

Although obstructive jaundice has been associated with a predisposition toward infections, the effects of bile duct ligation (BDL) on bulk intrahepatic T cells have not been clearly defined. The aim of this study was to determine the consequences of BDL on liver T cell phenotype and function. After BDL in mice, we found that bulk liver T cells were less responsive to allogeneic or syngeneic Ag-loaded dendritic cells. Spleen T cell function was not affected, and the viability of liver T cells was preserved. BDL expanded the number of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg), which were anergic to direct CD3 stimulation and mediated T cell suppression in vitro. Adoptively transferred CD4(+)CD25(-) T cells were converted into Treg within the liver after BDL. In vivo depletion of Treg after BDL restored bulk liver T cell function but exacerbated the degrees of inflammatory cytokine production, cholestasis, and hepatic fibrosis. Thus, BDL expands liver Treg, which reduce the function of bulk intrahepatic T cells yet limit liver injury.

[Expression of suppressor of cytokine signaling 3 and its significance in human placenta with pregnant intrahepatic cholestasis].

OBJECTIVE: To detect the expression and significance of suppressor of cytokine signaling 3(SOCS3) in the placentas of pregnant women with intrahepatic cholestasis of pregnancy (ICP). METHODS: Totally, 44 ICP gravidas, including 21 severe ICP and 23 mild ICP who delivered through cesarean section at the Second Xiangya Hospital of Central South University from April 2008 to January 2009, were selected as the ICP group, and another 25 healthy pregnant women were chosen as control. Placentas of the above gravidas were collected and the expression and localization of SOCS3 were determined by immunohistochemical peroxidase streptomyces-avidin link (SP) method (indicated by the percentage of positive cells and average gray scale) and the levels of interleukin-10 (IL-10) and interferon-γ (IFN-γ) from placenta homogenation were measured by enzyme linked immunoadsorbent assay (ELISA). RESULTS: (1) SOCS3 were expressed in placentas of both groups mainly in the intracytoplasma of trophocyte. However, weakly positive, positive, and strongly positive expressions were found in the severe ICP, mild ICP and the control group, respectively. Almost no expression was detected in membrane and nucleus of the trophoblasts. (2) The percentage of SOCS3 positive cells in the severe ICP group was significantly lower than in the control and the mild ICP group, respectively [(0.15 ± 0.08) % vs (0.69 ± 0.12) % and (0.42 ± 0.09) %, P < 0.01]. The average gay SOCS3 in placental tissue in the severe ICP group was significantly higher than that in control and mild ICP group, respectively (204 ± 7 vs 81 ± 7 and 147 ± 7, P < 0.01). (3) Significant lower level of IL-10 in placenta homogenation was found in the severe ICP group than in the control and mild ICP group [(1.16 ± 0.68) µg/L, vs (1.39 ± 0.08) µg/L and (1.22 ± 0.75) µg/L, P < 0.01]. (4) The opposite results were found in the level of IFN-γ in trophoblasts and the ratio of IFN-γ/IL-10 [severe ICP group: (16.8 ± 0.7) µg/L and 16.02 ± 2.79; control group: (10.5 ± 0.3) µg/L and 8.56 ± 0.14; mild ICP group: (13.4 ± 0.5) µg/L and 8.56 ± 0.14, P < 0.01]. (5) Negative correlation was shown between the percentage of SOCS3 positive cells in trophoblasts and the ratio of IFN-γ/IL-10 (r = -0.685 and -0.702, P < 0.01), and the average gay SOCS3 was positively correlated with the ratio of IFN-γ/IL-10 (r = 0.621 and 0.891, P < 0.01) in both mild and severe ICP group. CONCLUSIONS: SOCS3 may participate in the pathogenesis of ICP and its expression may affected by the severity of ICP, and SOCS3 may also play a role in the immunological regulation in ICP patients.