Association of primary biliary cirrhosis with variants in the CLEC16A, SOCS1, SPIB and SIAE immunomodulatory genes.

We fine mapped two primary biliary cirrhosis (PBC) risk loci, CLEC16A (C-type lectin domain family 16 member A)-suppressor of cytokine signaling 1 (SOCS1) and Spi-B protein (SPIB) and sequenced a locus, sialic acid acetylesterase (SIAE), proposed to harbor autoimmunity-associated mutations. In all, 1450 PBC cases and 2957 healthy controls were genotyped for 84 single-nucleotide polymorphisms (SNPs) across the CLEC16A-SOCS1 and SPIB loci. All 10 exons of the SIAE gene were resequenced in 381 cases and point substitutions of unknown significance assayed for activity and secretion. Fine mapping identified 26 SNPs across the CLEC16A-SOCS1 and 11 SNPs across the SPIB locus with significant association to PBC, the strongest signals at the CLEC16A-SOCS1 locus emanating from a SOCS1 intergenic SNP (rs243325; P=9.91 × 10(-9)) and at the SPIB locus from a SPIB intronic SNP (rs34944112; P=3.65 × 10(-9)). Among the associated SNPs at the CLEC16A-SOCS1 locus, two within the CLEC16A gene as well as one SOCS1 SNP (rs243325) remained significant after conditional logistic regression and contributed independently to risk. Sequencing of the SIAE gene and functional assays of newly identified variants revealed six patients with functional non-synonymous SIAE mutations (Fisher's P=9 × 10(-4) vs controls) We demonstrate independent effects on risk of PBC for CLEC16A, SOCS1 and SPIB variants, while identifying functionally defective SIAE variants as potential factors in risk for PBC.

IgM anti-Fc gamma R autoantibodies trigger neutrophil degranulation.

Anti-Fc gamma R IgM monoclonal antibodies (mAbs) isolated from lipopolysaccharide-stimulated spleen cells from tightskin (TSK) mice were found to be polyspecific, reacting with a wide variety of molecules, including double-stranded DNA, topoisomerase, RNA polymerase, and different collagen types. Approximately 60% of the polyspecific IgM mAbs have anti-Fc gamma R specificity. These anti-Fc gamma R mAbs induce the release of hydrolases from both azurophil and specific granules of human neutrophils. 25-45% of the total cellular content (determined in Nonidet P-40 lysates) of neutrophil elastase, 10-25% of beta-glucuronidase, and 30-50% of alkaline phosphatase was released after incubation with the mAbs. The degranulation process was accompanied by dramatic morphological changes shown by scanning and transmission electron microscopy. The release of hydrolytic enzymes stimulated by the IgM anti-Fc gamma R mAbs was inhibited by preincubation of neutrophils with Fab fragments of either anti-human Fc gamma RII (IV.3) or anti-human Fc gamma RIII (3G8) mAbs. The binding of the anti-Fc gamma R TSK mAbs to human neutrophils was inhibited by Fab fragments of mAb 3G8. However, we found that the TSK anti-Fc gamma R mAbs do not bind to human Fc gamma RII expressed in either CHO cells or the P388D1 mouse macrophage cell line. Since the enzyme release could be inhibited by Fab fragments of mAb IV.3, we suggest that the signal transduction may require Fc gamma RII activation subsequent to crosslinking of the glycan phosphatidyl inositol-anchored Fc gamma RIII-1. These data demonstrate for the first time that polyspecific autoantibodies with Fc gamma R specificity can trigger neutrophil enzyme release via human Fc gamma RIII-1 in vitro and indicate a possible role for such autoantibodies in autoimmune inflammatory processes.

Regulation of phagocytosis and [Ca2+]i flux by distinct regions of an Fc receptor.

The binding of multivalent immunoglobulin G complexes to Fc receptors (Fc gamma Rs) on macrophages activates multiple immune functions. A murine macrophage cell line, but not a fibroblast cell line, that was transfected with human Fc gamma RIIA mediated phagocytosis and an intracellular Ca2+ concentration ([Ca2+]i) flux upon cross-linking of human Fc gamma RIIA. Transfected macrophages that expressed a truncated receptor lacking 17 carboxy-terminal amino acids phagocytosed small antibody complexes. However, only wild-type transfectants phagocytosed labeled erythrocytes and fluxed [Ca2+]i. Thus, the cytoplasmic domain of human Fc gamma RIIA contains distinct functional regions.

Bcl-2-dependent oxidation of pyruvate dehydrogenase-E2, a primary biliary cirrhosis autoantigen, during apoptosis.

The close association between autoantibodies against pyruvate dehydrogenase-E2 (PDC-E2), a ubiquitous mitochondrial protein, and primary biliary cirrhosis (PBC) is unexplained. Many autoantigens are selectively modified during apoptosis, which has focused attention on apoptotic cells as a potential source of "neo-antigens" responsible for activating autoreactive lymphocytes. Since increased apoptosis of bile duct epithelial cells (cholangiocytes) is evident in patients with PBC, we evaluated the effect of apoptosis on PDC-E2. Autoantibody recognition of PDC-E2 by immunofluorescence persisted in apoptotic cholangiocytes and appeared unchanged by immunoblot analysis. PDC-E2 was neither cleaved by caspases nor concentrated into surface blebs in apoptotic cells. In other cell types, autoantibody recognition of PDC-E2, as assessed by immunofluorescence, was abrogated after apoptosis, although expression levels of PDC-E2 appeared unchanged when examined by immunoblot analysis. Both overexpression of Bcl-2 and depletion of glutathione before inducing apoptosis prevented this loss of autoantibody recognition, suggesting that glutathiolation, rather than degradation or loss, of PDC-E2 was responsible for the loss of immunofluorescence signal. We postulate that apoptotic cholangiocytes, unlike other apoptotic cell types, are a potential source of immunogenic PDC-E2 in patients with PBC.

The features of mucosa-associated microbiota in primary sclerosing cholangitis.

BACKGROUND: Little is known about the role of the microbiome in primary sclerosing cholangitis. AIM: To explore the mucosa-associated microbiota in primary sclerosing cholangitis (PSC) patients across different locations in the gut, and to compare it with inflammatory bowel disease (IBD)-only patients and healthy controls. METHODS: Biopsies from the terminal ileum, right colon, and left colon were collected from patients and healthy controls undergoing colonoscopy. Microbiota profiling using bacterial 16S rRNA sequencing was performed on all biopsies. RESULTS: Forty-four patients were recruited: 20 with PSC (19 with PSC-IBD and one with PSC-only), 15 with IBD-only and nine healthy controls. The overall microbiome profile was similar throughout different locations in the gut. No differences in the global microbiome profile were found. However, we observed significant PSC-associated enrichment in Barnesiellaceae at the family level, and in Blautia and an unidentified Barnesiellaceae at the genus level. At the operational taxa unit level, most shifts in PSC were observed in Clostridiales and Bacteroidales orders, with approximately 86% of shifts occurring within the former order. CONCLUSIONS: The overall microbiota profile was similar across multiple locations in the gut from the same individual regardless of disease status. In this study, the mucosa associated-microbiota of patients with primary sclerosing cholangitis was characterised by enrichment of Blautia and Barnesiellaceae and by major shifts in operational taxa units within Clostridiales order.

A comprehensive assessment of environmental exposures among 1000 North American patients with primary sclerosing cholangitis, with and without inflammatory bowel disease.

BACKGROUND: The relationships between primary sclerosing cholangitis (PSC) and the environment are largely unknown. AIM: To validate associations reported in previous studies and to identify novel environmental exposures among PSC patients. METHODS: We performed a multicenter, case-control analysis utilising self-administered questionnaires. Responses between cases (n = 1000) and controls (n = 663) were compared using multivariable logistic regression adjusted for age and gender. The model was further stratified based on inflammatory bowel disease (IBD) status (with IBD n = 741 without IBD n = 259). RESULTS: Smoking was associated with PSC only when IBD was present (OR, 0.5; 95% CI 0.4-0.7) but not among those PSC patients without IBD (OR, 0.9; 95% CI 0.7-1.2). Compared to controls, women with PSC (irrespective of the presence of IBD) were less likely to have received hormone replacement therapy (HRT; OR, 0.5; 95% CI 0.4-0.7) and were more likely to have recurrent urinary tract infections (OR, 1.6; 95% CI 1.2-2.3). PSC patients regardless of gender or IBD status were less likely to eat fish (OR, 0.4; 95% CI 0.3-0.6) and grilled/barbecued meat (OR, 0.8; 95% CI 0.7-0.9). In contrast, PSC patients with and without IBD were more likely to consume steak/burgers that were more well done (OR, 1.3; 95% CI 1.2-1.5). CONCLUSIONS: IBD (rather than PSC) is associated with smoking. Women with PSC are more likely to have recurrent urinary tract infections and less likely to receive HRT. Dietary intake and methods of food preparation differ in PSC patients when compared to controls.

Lateral mobility of Fc gamma RIIa is reduced by protein kinase C activation.

The lateral mobility of membrane proteins can reflect the extent of various protein-protein interactions. Using the fluorescence recovery after photobleaching technique, we have studied the lateral mobility of human Fc gamma RIIa and some Fc gamma RIIa mutants expressed in either P388D1 cells, a mouse macrophage-like cell line, or in Chinese hamster ovary (CHO) cells [1]. After treatment with phorbol myristate acetate (PMA), only the Fc gamma RIIa molecules capable of mediating rapid endocytosis of immune complexes exhibited a reduced lateral diffusion coefficient with respect to untreated controls. Wild type Fc gamma RIIa expressed in CHO cells, and nonfunctional Fc gamma RIIa mutants expressed in P388D1 cells did not show any differences upon PMA treatment. This finding suggests that protein kinase C activation evokes additional protein-protein interactions with the cytoplasmic domain of functional Fc gamma RIIa, which reduced receptor lateral mobility. The identity of these putative interacting proteins and the nature of the interactions remain to be elucidated.

Effect of fibrosis on adverse events in patients with hepatitis C treated with telaprevir.

BACKGROUND: Data about adverse events are needed to optimise telaprevir-based therapy in a broad spectrum of patients. AIM: To investigate adverse events of telaprevir-based therapy in patients with and without advanced fibrosis or cirrhosis in a real-world setting. METHODS: Data on 174 hepatitis C-infected patients initiating telaprevir-based therapy at Mount Sinai and Montefiore medical centres were collected. Biopsy data and FIB-4 scores identified patients with advanced fibrosis. Multivariable fully adjusted models were built to assess the effect of advanced fibrosis on specific adverse events and discontinuation of treatment due to an adverse event. RESULTS: Patients with (n = 71) and without (n = 103) advanced fibrosis were similar in BMI, ribavirin exposure, gender, prior treatment history, haemoglobin and creatinine, but differed in race. Overall, 47% of patients completed treatment and 40% of patients achieved SVR. Treated patients with and without advanced fibrosis or cirrhosis had similar rates of adverse events; advanced fibrosis, however, was independently associated with ano-rectal discomfort (P = 0.03). Three patients decompensated and had advanced fibrosis. The discontinuation of all treatment medications due to an adverse event was significantly associated with older age (P = 0.01), female gender (P = 0.01) and lower platelets (P = 0.03). CONCLUSIONS: Adverse events were common, but were not significantly related to the presence of advanced fibrosis or cirrhosis. More critical monitoring in older and female patients with low platelets throughout treatment may reduce adverse event-related discontinuations.

Natural history of small duct primary sclerosing cholangitis: a case series with review of the literature.

BACKGROUND AND AIMS: Information about the natural history of small duct primary sclerosing cholangitis (SDPSC) remains scant despite literature suggesting that it constitutes 6-16% of all cases of primary sclerosing cholangitis (PSC). We combined clinical data on SDPSC cases from two tertiary care institutions with liver transplantation programs with the aim of studying the natural history of SDPSC. METHODS: Medical records of 25 individuals with SDPSC were reviewed. Diagnosis of SDPSC was based on liver biopsy findings consistent with PSC, a normal cholangiogram, and elimination of known causes of secondary sclerosing cholangitis. Demographic information, symptoms, past medical history, laboratory values, and histologic data were evaluated. Our primary outcome measure was liver transplantation or death. Secondary outcome measures included evidence of end-stage liver disease, development of cholangiocarcinoma, and/or the development of classic PSC on a repeat cholangiogram. RESULTS: Data on 25 individuals (13 males, 12 females; mean age 40 ± 15 years) diagnosed with SDPSC were analyzed. Upon presentation, 11 patients had symptoms including abdominal pain, fatigue, and pruritus. Inflammatory bowel disease was present in 14 patients (56%) at diagnosis. On initial liver biopsy, 60% had early-stage disease (I or II) and none had cirrhosis. On follow-up (1-168 months, median 17 months), malignancy or progression to classic large duct PSC was not noted. Two (8%) patients had evidence of varices and one of the two also developed ascites; one of these patients underwent liver transplantation and the other one died due to sepsis. CONCLUSIONS: SDPSC, a mild disease at presentation typically runs a benign course and likely is not an early stage of classic PSC. Further studies with a control group of classic PSC and longer follow-up are needed to study the natural history of SDPSC.

Specificity and class distribution of Fc gamma R-specific autoantibodies in patients with autoimmune disease.

To investigate the prevalence of autoantibodies directed against Fc gamma RII (CD32) and Fc gamma RIII (CD16), 151 serum samples from patients with different autoimmune diseases and 25 samples obtained from healthy individuals were assayed by ELISA on microtiter plates coated with recombinant truncated Fc gamma RII and Fc gamma RIII protein. Class specificity was defined with anti-IgG, anti-IgM, and anti-IgA reagents. High titers of circulating IgM autoantibodies reacting with both Fc gamma RII and Fc gamma RIII were characteristic for SLE and rheumatoid arthritis patients. Sera from patients with Raynaud's syndrome showed predominantly IgG reactivity with Fc gamma RIII. Sera from patients with progressive systemic sclerosis showed both IgG and IgM Fc gamma RII and Fc gamma RIII reactivity. Many patients diagnosed with degenerative osteoarthritis also had IgG autoantibodies, directed primarily against Fc gamma RII with lesser reactivity toward Fc gamma RIII. Further study is needed to correlate these findings to clinical characteristics of the different diseases.