Aigli G. Vakrakou

Decreased serum DNase1-activity in patients with autoimmune liver diseases

Abstract Deoxyribonuclease1 (DNase1) is involved in chromatin degradation of apoptotic cells. Its deficiency results in accumulation of self-DNA, which in turn may induce inflammation and autoimmunity. We assessed for the first time serum DNase1-activity in a large consecutive cohort of treatment-naïve patients with autoimmune liver diseases (ALD). DNase1-activity was determined by single radial enzyme-diffusion (SRED) at diagnosis of 224 patients with autoimmune hepatitis (AIH), 249 with primary biliary cirrhosis (PBC) and 36 with primary sclerosing cholangitis (PSC). Sera from 146 patients with chronic hepatitis B or C, 140 with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH) and 114 healthy individuals served as disease and healthy controls. Available serum samples during remission from 50 AIH and 39 PBC patients were also investigated by paired analyzes. DNase1-activity was significantly lower in AIH, PBC and PSC compared to viral hepatitis (p < 0.02, p < 0.001, p = 0.03), NAFLD/NASH (p < 0.001) and healthy (p < 0.001). No significant difference was found in between each specific ALD. In AIH, DNAse1-activity was positively correlated with aspartate aminotransferase (AST) (p < 0.02), bilirubin (p < 0.01) and increased IgG (>1400 mg/dl; p < 0.05); in PBC, with AST (p < 0.01), alanine aminotransferase (ALT) (p < 0.03) and anti-mitochondrial antibodies (AMA) (p = 0.008). In PSC, DNase1-activity was inversely associated with alkaline phosphatase (ALP) (p < 0.05). In AIH, complete responders were characterized by increased baseline DNase1-activity compared to partial responders, relapsers and non-responders (p < 0.02), whereas it was significantly increased after achievement of remission (p < 0.001). Serum DNase1-activity is significantly decreased in ALD patients, indicating its potential implication in their pathogenesis. Furthermore, DNase1-activity could be used as a new surrogate biomarker for predicting response to AIH treatment.

A6.30 Activation of inflammasome correlating with the presence of non-degraded cell-free DNA in the peripheral blood and the salivary glands of patients with severe primary SjÖgren’s syndrome

Background and objectives Inflammasomes are intracellular multiprotein complexes that sense pathogenic microorganisms, as well as DNA and other molecules released after tissue injury. Such activation of inflammasome leads to the upregulation of various inflammasome-related molecules and the release of pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and IL-18. We have recently shown that patients with primary Sjögren’s syndrome (SS) manifest increased serum levels of circulating nucleosomes and cell-free genomic DNA (CF-DNA) owing to impaired DNA degradation. Herein, we assessed whether the inflammasome is activated in the peripheral blood and salivary glands (SG) of SS patients and whether serum CF-DNA represents a novel IL-1β stimulating agent. Materials and methods The mRNA expression of inflammasome-related molecules ASC, NLRP3, AIM2 and IL-1β were evaluated by RT-PCR in PBMC (15 SS, 9 non-SS-controls). Sera were studied for the levels of circulating CF-DNA (by RT-PCR; 8 SS and 5 non-SS-controls) and ASC protein (by ELISA; 39 SS, 21 non-SS-controls). SG biopsies were examined by confocal microscopy for the presence of CF-DNA (anti-dsDNA/TO-PRO-3/membranous-WAG immunostaining; 3 SS, 2 non-SS-controls) and the expression of ASC protein (15 SS, 5 non-SS-controls). To evaluate the immunostimulatory capacity of CF-DNA, healthy PBMC were treated with 5 SS sera-derived CF-DNA and IL-1β produced was measured by ELISA. Results Significantly high mRNA expression of ASC, IL-1β, NLRP-3 was detected in the PBMC of SS patients (all for p < 0.05, vs. controls), with ASC highly positively correlating with disease activity scores (ESSDAI; p = 0.007). SS patients manifested high serum levels of ASC protein (p < 0.0001, vs. controls) that were particularly high among SS patients who had developed lymphoma (p = 0.009). High serum levels of CF-DNA were also detected in SS patients (p < 0.05, vs. controls) that correlated positively with serum levels of ASC (r = 0.654, p = 0.015). The SG specimens of SS patients (but not non-SS-controls) manifested ample evidence of CF-DNA, as well as strong ASC protein expression by both epithelial and infiltrating cells, mainly CD68+ macrophages. The serum CF-DNA of SS patients induced high IL-1β secretion by healthy PBMC (8-fold induction over mock-treated cells). Conclusions SS patients manifest evidence of chronic inflammasome activation in the peripheral blood and the salivary glands, likely due to the presence of lingering non-degraded CF-DNA. As supported by its correlation with disease activity and lymphoma development, inflammasome activation in SS patients likely participates in the pathogenesis of the adverse clinical outcomes of the disorder and may specify novel disease biomarkers.

OP0085 The Patients with Primary Sjögren's Syndrome Display Inflammasome Activation in the Peripheral Blood and the Salivary Glands that Correlates With Deficient Degradation of DNA

Background Inflammasomes are intracellular multiprotein complexes that sense pathogenic microorganisms, as well as DNA and other molecules released after tissue injury. Such activation of inflammasome leads to the upregulation of various inflammasome-related molecules and the release of pro-inflammatory cytokines, such as interleukin-1β (IL-1β). We have recently shown that patients with primary Sjögrens syndrome (SS) manifest increased serum levels of circulating nucleosomes and cell-free genomic DNA (CF-DNA) owing to impaired DNA degradation. Objectives Herein, we assessed whether the inflammasome is activated in the peripheral blood and salivary glands (SG) of SS patients and whether serum CF-DNA represents a novel inflammasome-stimulating agent. Methods The mRNA expression of inflammasome-related molecules ASC, NLRP3, AIM2 and IL-1β were evaluated by RT-PCR in PBMC (15 SS, 9 non-SS controls). Sera were studied for DNase1 activity (by SRED assay), the levels of circulating CF-DNA (by RT-PCR; 8 SS and 5 non-SS controls), IL-1β and ASC protein (by ELISA; 39 SS patients, 21 controls). SG biopsies were examined for the presence of CF-DNA (by nucleic acids, cell membrane immunostaining; 3 SS patients, 2 non-SS controls) and the expression of ASC protein (by confocal microscopy; 15 SS patients, 5 non-SS controls). To evaluate the capacity of CF-DNA for inflammasome activation, healthy PBMC were treated with SS sera-derived CF-DNA and IL-1β produced was measured by ELISA. Results Significantly high mRNA expression of ASC, IL-1β, NLRP-3 was detected in the PBMC of SS patients (all for p<0.05, vs. controls), with ASC highly positively correlating with disease activity scores (ESSDAI; p=0.007). SS patients manifested high serum levels of ASC protein (p<0.0001, vs. controls) that were particularly high among SS patients who had developed lymphoma (p=0.009). High serum levels of CF-DNA were also detected in SS patients (p<0.05, vs. controls) that correlated inversely with DNase1 activity (r= -0.763 p<0.001) and positively with the levels of ASC (r=0.654, p=0.015) in serum. The SG specimens of SS patients (but not nonSS-controls) manifested ample evidence of CF-DNA, as well as strong ASC protein expression by both epithelial and infiltrating cells, mainly CD68+ macrophages. The serum CF-DNA of SS patients induced high expression of inflammasome-related genes and IL-1β secretion by healthy PBMC. Conclusions SS patients manifest evidence of chronic inflammasome activation in the peripheral blood and the salivary glands, likely due to the presence of lingering non-degraded CF-DNA. As supported by its correlation with disease activity and lymphoma development, inflammasome activation in SS patients likely participates in the pathogenesis of the adverse clinical outcomes of the disorder and may specify novel disease biomarkers. Disclosure of Interest None declared

A3.3 Inflammasome is activated in healthy salivary gland epithelial cell lines by necrotic cell debris, whereas it is constitutively active in the epithelial cells of Sjögren’s syndrome patients

Background and Objectives In our laboratory, recent transcriptome expression analysis of unstimulated cultured non-neoplastic salivary gland epithelial cell lines (SGEC) from Sjögren’s syndrome (SS) patients and non-SS controls had indicated the aberrant expression of various inflammatory genes in the SS-SGEC supporting their intrinsic activation status. Additionally, we have recently presented evidence of impaired removal of apoptotic cells and necrotic cell debris (SNEC) in SS patients. Here, we sought to investigate the effect of SNEC in cultured SGEC, as well as the expression of various inflammasome-related genes and proteins in salivary gland tissues and SGEC from SS patients and non-SS controls. Materials and Methods non-SS SGEC treated with SNEC or apoptotic cells were evaluated for the mRNA and protein expression of various activation markers. In addition, salivary gland (SG) tissues and SGEC from SS patients and non-SS controls were comparatively evaluated for the expression of various inflammasome-related genes and proteins. Results SGEC treatment (n = 3) with SNEC led to the induction of surface expression of inflammatory molecules, such as ICAM-1, MHC-I, CD86, Fas, TLR-2 (p<0.05). In contrast to SNEC, apoptotic cells did not exhibit such an inflammatory effect, but rather suppressed the inflammatory responsiveness to TLR-3 triggering. SGEC treatment (n = 4) with apoptotic cells ameliorated polyI:C-induced MHC-I expression (35% reduction). SNEC stimulation also upregulated the mRNA levels of IFN-β and PYCARD/ASC in SGEC (6-fold and 4-fold induction at 24-hrs, respectively). Exposure of SGEC to SNEC led to inflammasome activation, as it was indicated by the analysis of SNEC stimulation of LPS-primed SGEC showing the induction of caspase-1 activation (intracellular p20 expression) and IL-1β secretion in the culture supernatant (4.8-fold and 2.5-fold increase, respectively), as well as by speckled ASC formation in immunofluorescence microscopy. Microarray gene profiling and validation analysis in SGEC revealed the aberrant expression of genes involved in the inflammasome signalling in SS-SGEC. Moreover, increased protein expression of IL-1β and ASC was observed in SG tissues of SS patients (5 SS, 5 non-SS). SS-SGEC (n = 9) were also found to secrete constitutively more IL-1β in their culture supernatant, compared to non-SS SGEC (n = 5) (mean ± SE; SS-SGEC: 2.3 pg/ml ± 0.4, non-SS SGEC: 0.8 pg/ml ± 0.3, p = 0.02) likely suggesting the constitutive activation of inflammasome in SS-SGEC cells. Conclusions These findings indicate that the aberrant exposure of the epithelial cells to necrotic debris may be a major cause of inflammatory reactions via the activation of inflammasome and may hold a key role in the pathogenesis of disorders associated with epithelial activation such as SS.

THU0038 Activation of Inflammasome in the Salivary Gland Epithelial Cells of SjÖGren's Syndrome Patients Imposed by Aberrant Dnase1 Expression and Extracellular DNA Accumulation

Background Evidence from this laboratory had indicated that the epithelia of primary Sjögrens syndrome (SS) patients, by virtue of an intrinsically activated cell status, may have a crucial pathogenetic role in this disorder. The degradation of necrotic cell debris by exonucleases, such as DNase1, is thought protective against the inflammagenic action of necrotic cell-derived substances; Objectives therefore, we comparatively investigated various tissues from SS patients and non-SS controls for the handling of and response to necrotic cell debris (secondarily necrotic cells; SNEC), including the activation of inflammasome. Methods The mRNA and protein expression of DNase1 was investigated in salivary gland (SG) tissues, peripheral blood mononuclear cells (PBMC) and long-term cultured non-neoplastic salivary gland epithelial cell (SGEC) lines by real-time PCR, immunohistochemistry and immunoblotting. DNase1 activity was measured in cytosolic extracts of cultured SGEC and in SGEC culture supernatants by single radial enzyme diffusion (SRED assay). The mRNA and protein expression of various inflammasome-related genes were compared in SG tissues from SS patients and non-SS controls. The constitutive, as well as the SNEC-induced expression of inflammasome-related and cell activation genes were also comparatively studied in cultured SGEC. The presence of extracellular DNA (exDNA) in SG tissues and cultured SGEC was examined by confocal microscopy, using monoclonal anti-dsDNA antibody, DAPI and Ribogreen staining. Results The SG tissues of SS patients with intense lymphocytic infiltrates (≥2 infiltrations/4mm2) manifested significantly reduced DNase1 mRNA levels, compared to non-SS controls (p=0.023). Significantly decreased DNase1 mRNA and protein expression (both for p<0.01), as well as impaired cytosolic DNase1 activity (p=0.019) were also observed in SGEC lines (but not the PBMC) of SS patients, compared to controls. In line to these findings, significant amounts of exDNA (DNA strands) were observed in SG tissues of SS patients (particularly around epithelial ductal cells), as well as in the extracellular space of SS-SGEC (but not nonSS-SGEC) cultures. High expression of the inflammasome-related molecules PYCARD/ASC (1.6-fold increase) and AIM2 (5.8-fold increase), which are involved in inflammasome activation though DNA-sensing, was detected in the epithelial cells of SG tissues and constitutively in cultured SGEC lines from SS patients. SGEC treatment with SNEC was found to induce the expression of PYCARD/ASC, AIM2, several cell activation markers (ICAM-1, MHC-I, CD86, Fas, IFN-β) and DNase1, as well as the activity of DNase1 in SGEC culture supernatants. The SNEC-induced activation of inflammasome in SGEC was also demonstrated by detection of caspase-1 activation (intracellular p20 expression; 4.8-fold increase) and of IL-1β secretion in culture supernatants (2.5-fold increase). Conclusions These findings provide first evidence that the salivary epithelia of SS patients manifest impaired endogenous DNase1 activity, as well as constitutive activation of inflammasome that likely owes to the exposure of cells to necrotic debris. Such aberrations may hold a key pathogenetic role in the tissue inflammatory reactions of SS and may also explain the “intrinsic activation status” that characterizes the epithelia of these patients. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.5771

A2.3 AnTI-Apoptotic IgG Antibodies from Patients with Primary Sjögren’s Syndrome and Systemic Lupus Erythematosus Inhibit the Phagocytosis of Apoptotic Cells

Background and Objectives Recent studies in our laboratory have revealed that a significant portion of SS patients manifests significantly impaired phagocytosis of apoptotic cells (ApoCell-phagocytosis), in a manner similar to SLE, a fact which probably leads to the inflammatory and autoimmune responses that characterise these two disorders. Furthermore, our data indicate that approximately 80% of sera from patients with SS and SLE, but not those with RA and healthy individuals, inhibit the clearance of early apoptotic cells from healthy monocytes. In the present study, we sought to investigate the role of IgG immunoglobulins from patients with SLE and SS in the phagocytosis of apoptotic cells. Materials and Methods Total IgG immunoglobulin was isolated from the serum of patients with SS (n = 24), SLE (n = 12) and RA (n = 8) and healthy donors (n = 11) using Melon Gel Resin columns. Apoptotic Jurkat cells (induction of apoptosis by UV-radiation) were incubated with purified IgG (50 µg/ml) in PBS/BSA 1%. The binding of IgG on the surface of apoptotic cells was assessed by flow cytometry and analysed with the binding index (% positive cells × mean fluorescence intensity). ApoCell-phagocytosis was assessed by flow cytometry using monocyte-derived macrophages (MDM) from healthy individuals (n = 3) that were incubated (90 min) with CFSE-labelled early apoptotic Jurkat cells which had been opsonised with IgG from patients or healthy individuals or with PBS (control). Results IgG from patients with SS and SLE exhibited high binding levels in apoptotic cells compared to healthy donors and patients with RA (all for p < 0.0001). Pre-incubation of apoptotic cells with IgG from patients with both SS and SLE had a significant inhibitory effect on the phagocytosis process (comparative ApoCell-phagocytosis: after pre-incubation with PBS [x = 100%], with IgG from healthy individuals [n = 11, x = 100%], with IgG from RA patients [n = 4, x = 100%], with IgG from SS patients [n = 17, x = 75%, p < 0.0001], with IgG from SLE patients [n = 10, x = 71%, p = 0.0002]). Finally, negative correlation was found between the apoptotic cell binding levels of IgG from patients and healthy individuals and the results of ApoCell-phagocytosis assays using apoptotic cells pre-incubated with the corresponding IgG (n = 42, p < 0.0001). Conclusions Our results indicate that the impaired phagocytosis of apoptotic cells observed in patients with SS and SLE is primarily due to the presence of antibodies reactive with the surface of apoptotic cells. Further studies need to focus on the mechanism by which immunoglobulins exert such blocking effect, as well as its immunological consequences.

A2.18 The Expression of the Transcription Factor PPAR-Gamma is Significantly Reduced in the Salivary Gland Epithelial Cells from Patients with Primary Sjögren’s Syndrome

Background and Objectives PPAR-gamma is an essential transcription factor that apart from participating in the regulation of genes associated with lipogenesis, it exerts significant anti-inflammatory actions. PPAR-gamma has been implicated in the pathogenesis of human autoimmune diseases that are characterised by the inflammatory damage of epithelial cells. More specifically, dramatically reduced expression of PPAR-gamma has been shown in the epithelial cells of the intestinal tract and of the biliary tree of patients with ulcerative colitis and primary biliary cirrhosis, respectively. Primary Sjögren’s syndrome (SS or autoimmune epithelitis) is characterised by chronic inflammatory lesions mainly affecting epithelial tissues and is associated with systemic autoimmune responses and the chronic intrinsic activation of salivary gland epithelial cells (SGEC). Thus, SGEC are probably both the target and the inducer of inflammatory responses. In this context, we aimed to investigate the levels of constitutive expression of PPAR-gamma in cultured non-neoplastic SGEC lines from SS patients and non-SS controls, as well as the patterns of PPAR-gamma expression following cellular activation. Materials and Methods To examine the levels of mRNA expression of PPAR-g, total RNA was isolated from long-term cultured non-neoplastic SGEC and from peripheral blood mononuclear cells (PBMCs) of 18 SS patients and 11 non-SS disease controls. The expression of PPAR-gamma was studied by Real-time PCR with primers specific for PPAR-gamma and the reporter gene HPRT1 and analysed by the ddCt method. To evaluate the effect of epithelial activation in the expression of PPAR-g, SGEC from non-SS controls were stimulated with specific ligands of TLR-3 (Polyinosinic-polycytidylic acid, PolyI:C, 5 mg/ml), TLR-4 (lipopolysaccharide, LPS, 1 mg/ml) receptor and with the cytokine IFN-gamma (500 U/ml). Results PPAR-gamma mRNA expression was significantly reduced in the SGEC from patients with SS, compared to controls (p = 0.0001). In contrast, no difference was found in PPAR-gamma expression in PBMCs between patients and controls. The activation of cultured SGEC by stimulation with PolyI:C, LPS and IFN-g resulted in a significant down-regulation of PPAR-gamma mRNA expression (in all cases; by ≈80% at 12 hours, p = 0.0001). Conclusions Our findings indicate that the expression of PPAR-gamma in human epithelial cells is significantly reduced following activation via TLRs and the Th1 cytokine INF-gamma. Furthermore, the present study demonstrates for the first time the significantly reduced expression of PPAR-gamma in the SGEC of SS patients. This finding likely owes to the chronic intrinsic activation, which characterises the epithelia of SS patients.

A2.10 Increased Immunologic Exposure to Necrotic Cell Remnants in Patients with Primary SJögren’s Syndrome Owing to Defective Dnase-I Activity and The Presence of Opsonizing IgG Autoantibodies in Serum

Background and Objectives Our recent experiments have suggested that similarly to SLE, patients with primary Sjögren’s syndrome (SS) manifest significantly increased phagocytosis of necrotic cell debris (secondary necrotic cell material, SNEC). This pheno-menon has been attributed to serological aberrations of these patients, as indicated by the capacity of patients’ sera to promote the uptake of SNEC by healthy phagocytes. In this study, we comparatively investigated the role of serum DNAse-I activity and IgG immunoglobulins from SS, SLE and RA patients in the promotion of SNEC-phagocytosis by healthy monocytes. Materials and Methods The activity of DNase-I was assessed by single radial enzyme-diffusion assay (SRED) in the serum of patients with SS (n = 60), SLE (n = 22) and RA (n = 14) and healthy donors (HBD, n = 52). Total IgG immunoglobulins were isolated by negative selection from the serum of patients and controls using Melon Gel Resin columns. SNEC were prepared by heat-induced necrosis of normal lymphocytes and labelling with propidium iodide. The influence of serum components on SNEC-phagocytosis was assessed by flow cytometry in admixture experiments using normal phagocytes and SNEC pre-incubated with whole sera or purified serum IgG from patients or HBD. Results Serum DNase-I activity in patients with SS and SLE was found significantly reduced compared to HBD and RA patients (p < 0.0001) and correlated inversely with the ability of these sera to promote SNEC-phagocytosis by healthy monocytes (p = 0.0003). The capacity of HBD sera to promote SNEC-phagocytosis by normal monocytes was significantly increased (by 90%) following the addition of the DNase-I-specific inhibitor G-actin (800 µg/ml), supporting the important physiological role of DNA degradation by serum DNase-I in the prevention of SNEC-phagocytosis. SNEC opsonised with IgG isolated from autoimmune patients or from HBD were found to be similarly ingested by normal monocytes. However, in the presence of normal serum, the opsonisation of SNEC with IgG isolated from SS or SLE sera was found to induce significantly increased SNEC-phagocytosis, compared to that observed with SNEC opsonised with IgG isolated from HBD sera (p = 0.001). Conclusions Our results indicate that, in a manner similar to SLE, SS patients are characterised by deficient serum DNase-I activity. Such reduced serum capacity for degradation of nucleic acids, in conjunction with the opsonisation of SNEC by serum autoantibodies appears to lead to increased exposure of the immune system of these patients to necrotic cell debris, to enhanced SNEC-phagocytosis and consequently to the inflammatory responses that characterise the disorder.

THU0049 Impaired Constitutive Expression of PPAR-Gamma in Salivary Gland Epithelial Cell Lines of Primary Sjogren’s Syndrome Patients

Background PPAR-gamma is an essential transcription factor that participates in the regulation of lipogenesis, whereas it also exerts significant anti-inflammatory actions. Reduced expression of PPAR-g has been described in human autoimmune diseases that are characterized by inflammatory damage of epithelial cells (e.g. ulcerative colitis, primary biliary cirrhosis). Primary Sjogren’s syndrome (SS) is characterized by chronic inflammatory lesions of epithelial tissues and experimental evidence has indicated the occurrence of chronic intrinsic activation of salivary gland epithelial cells (SGEC). Thus, SGEC are probably both the target and the inducer of inflammatory responses. Objectives To investigate the constitutive expression of PPAR-g in cultured non-neoplastic SGEC lines from SS patients and non-SS controls, as well as to define potential cellular activators that affect PPAR-g expression. Methods Total RNA was isolated from long-term cultured non-neoplastic SGEC and from peripheral blood mononuclear cells (PBMC) of 19 SS patients and 11 non-SS disease controls. The expression of PPAR-g was studied by Real-time PCR with primers specific for PPAR-g and the reporter gene HPRT1 and analyzed by the ddCt method. The PPAR-g protein expression was examined by immunoblotting of cellular extracts. To evaluate the effect of epithelial activation in the mRNA expression of PPAR-g, SGEC from non-SS controls were stimulated with specific ligands of TLR-3 (Polyinosinic-polycytidylic acid, PolyI:C, 5μg/ml), TLR-4 (lipopolysaccharide, LPS, 1μg/ml) receptor and the cytokines IFN-gamma (500U/ml) and IL-1β (10ng/ml). Results The constitutive PPAR-g mRNA and protein expression was significantly reduced in the SGEC from SS patients, compared to controls (p=0.0002). In contrast, no difference was found in PPAR-g expression in PBMC between patients and controls. The activation of cultured SGEC by stimulation with PolyI:C, LPS, IFN-g and IL-1β resulted in significant down-regulation of PPAR-g mRNA expression (in all cases; by≈80-85% at 12 hours, p<0.05). Conclusions Our results indicate that PPAR-g expression in human SGEC is significantly reduced following activation via TLRs and the pro-inflammatory cytokines INF-g and IL-1β. Furthermore, the present study demonstrates for the first time the significantly reduced expression of PPAR-g in the SGEC of SS patients. This finding likely owes to the chronic intrinsic activation, which characterizes the epithelia of SS patients. References Manoussakis MN, Kapsogeorgou EK. The role of intrinsic epithelial activation in the pathogenesis of Sjögren’s syndrome. J Autoimmun. 35:219-24, 2010 Dubuquoy L. et al, Impaired expression of peroxisome proliferator-activated receptor gamma in ulcerative colitis. Gastroenterology 124:1265–1276, 2003. Harada K. et al, Th1 cytokine-induced downregulation of PPARgamma in human biliary cells relates to cholangitis in primary biliary cirrhosis. Hepatology 41:1329-38, 2005. Disclosure of Interest None Declared