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Background: Chronic pancreatitis is characterised clinically by early exocrine insufficiency, with diabetes mellitus occurring as a late phenomenon. This is mirrored pathologically by extensive acinar cell destruction and islet preservation. The mechanisms underlying this differential rate of cellular destruction are unknown. Aims: To test the hypothesis that acinar loss and islet preservation in chronic pancreatitis occurs due to differential epithelial kinetics and investigate the role of inflammatory cells and cell cycle associated molecules. Methods: Archival tissue from six chronic pancreatitis cases was compared with six normal controls using TUNEL and immunohistochemistry for CD3, CD20, CD68, MIB-1, Bcl-2, Bax, Fas, Fas ligand, retinoblastoma protein (Rb), and tissue inhibitor of metalloproteinases 1 (TIMP-1) and 2 (TIMP-2). Results: The acinar cell apoptotic index (AI) and proliferation index were higher in chronic pancreatitis than controls. T lymphocytes diffusely infiltrated fibrous bands and acini but rarely islets. Acinar Bcl-2 expression exceeded islet expression in chronic pancreatitis and controls while Bax was strongly expressed by a subset of islet cells and weakly by centroacinar cells. Islet Fas and Fas ligand expression exceeded acinar expression in chronic pancreatitis and controls. Acinar Rb expression was higher in chronic pancreatitis than in controls. Islets in chronic pancreatitis and controls showed intense TIMP-1 and TIMP-2 expression. Conclusion: Apoptosis plays a significant role in acinar loss in chronic pancreatitis. Acinar Bcl-2 and islet Bax expression indicates complex AI control. Increased acinar Rb expression in chronic pancreatitis may differentially promote acinar loss. Fas ligand expression may be restricted to islet cell membranes through TIMP-1 expression and inhibit islet damage by promoting apoptosis of cytotoxic T lymphocytes.

Background: Exocrine (acinar) destruction and endocrine (islet) preservation in chronic pancreatitis (CP) is related to differential apoptotic indices within these cellular compartments. Aim: To define the role of cell cycle regulatory proteins in the control of pancreatic epithelial cell apoptosis. Methods: Formalin-fixed, paraffin-embedded tissue from six cases of CP and eight normal controls (N) were studied using immunohistochemistry for BCl-2, Bax, retinoblastoma protein (Rb) and Fas ligand (Fas-L). Labelling patterns were assessed using semi-quantitative (intensity-proportion [I-P]) or fully quantitative (labelling index [LI]) analysis. Results: Rb protein was expressed at a higher level by acinar cells in CP than N (mean acinar cell Rb LI 1.45% in CP and 0.437% in N; p

Introduction EGFR pathway substrate 8 (Eps8) is an adapter protein that sits at the heart of a complex system regulating re-organisation of the actin cytoskeleton. Eps8 has been shown to be involved in cell motility and EGFR internalisation, via interactions with multiple intracellular proteins, including integrins. αvβ6 is an epithelial specific integrin, not present in healthy tissue but over-expressed in many carcinomas. αvβ6 promotes tumour invasion and is a key activator of TGFβ, which modulates tumour cell EMT and the desmoplastic response characteristically present in PC. The aim of this study was to investigate whether there is a link between Eps8 and αvβ6 in pancreatic cancer invasion. Methods We used immunochemistry to examine the expression of Eps8 and αvβ6 in normal pancreas and PC in vivo using tissue microarrays. PC cell motility was investigated using Transwell® assays. siRNA interference was used to down-regulate genes of interest. The ability of PC cells to activate TGFβ was measured using TGFβ-responsive mink lung epithelial cells. Cell surface αvβ6 levels were assessed with FACS analysis. Endocytosis of αvβ6 was suppressed using Clathrin siRNA to examine the role of endocytosis in αvβ6 functions. Pancreatic stellate cells (PSCs) were isolated from primary pancreatic resection tissue and used to optimise 3D pancreatic organotypic cultures. Results Eps8 and αvβ6 were up-regulated in > 70% PC in vivo but were not generally detected in normal pancreas. An organotypic model of pancreatic cancer was developed using primary PSCs to study αvβ6 functions. αvβ6 promoted PC cell invasion and TGFβ activation. Interestingly, Eps8 knockdown suppressed αvβ6-dependent motility but conversely, promoted αvβ6-dependent TGFβ activation. Although inhibition of αvβ6 endocytosis also increased TGFβ activation, this was not Eps8 dependent, nor did Eps8 knockdown affect total cell surface levels of the integrin. Conclusion αvβ6 and Eps8 are over-expressed in PC. αvβ6 promotes both invasion and TGFβ activation, and Eps8 appears to act as a molecular switch between these functions. How this relates to disease progression is unclear; in late stage disease, where canonical TGFβ signalling is dysregulated (e.g. SMAD4 mutations which are seen in > 50% of PCs) both αvβ6 functions are likely to be tumour promoting. However, in premalignancy when canonical TGFβ signalling acts as a tumour suppressor, we speculate that up-regulation of Eps8 may result in a pro-invasive phenotype and facilitate tumour development. Eps8 may therefore act as a master regulator of PC invasion by switching the cell function between motility and TGFβ activation at critical time points in PC progression. Disclosure of Interest None Declared

Introduction It is suggested that T-lymphocytes play a role in the pathogenesis of chronic pancreatitis (CP), but little is known about the composition of T-cell subsets in this disease and previous studies have been discordant. 1–4 We therefore aimed to characterise T-helper (Th) and T-regulatory (Treg) lymphocytes in CP tissue and peripheral blood. Methods Peripheral blood mononuclear cells were isolated from 15 patients with CP (all male, median age 48.2 years) and 14 controls (10 male, four female, median age 56.1 years). Mononuclear cells were also isolated from the pancreatic tissue of four CP patients (all male, median age 41 years) using enzymatic and mechanical digestion, followed by density gradient centrifugation. The mononuclear cells were stimulated with PMA and ionomycin (not Treg cells), and analysed using a FACSAria flow cytometer. Live CD3 + CD4 + Th1, Th2, Th17 and Treg cells were identified as IFN-γ + , IL-13 + , IL-17 + (IFN-γ ± ), and CD25 + FoxP3 + CD127 lo/− respectively. Statistical analysis was performed using a Mann–Whitney U Test. Results The peripheral blood of CP patients comprised a significantly higher percentage of Th1 cells (15.2% vs 8.11%; p=0.03), Th2 cells (2.00 % vs 1.17%; p=0.03), Th17 cells (1.23% vs 0.41%; p=0.003), dual secreting IFN-γ + IL-17 + Th17 cells (0.11% vs 0.03%; p=0.003) and Treg cells (6.30% vs 4.40%; p=0.05) compared to controls. CP patients who consume excess alcohol have significantly more Th1 cells than non-drinkers (23.7% vs 9.81%; p=0.01). The T-helper cell infiltrate in CP tissue was mainly composed of Th1 cells (26.1%–57.2%, median 41.5%). Th17 cells were also seen (0.95%–3.80%, median 2.1%), including IFN-γ + IL-17 + Th17 cells (0.22%–2.39%, median 0.37%). No discernible Th2 cells were identified and few or no Treg cells were seen in CP tissue. Conclusion This work is the first to demonstrate a significant increase in the number of Th17 cells in the peripheral blood of CP patients, and to clearly demonstrate that Th1 cells are the principal T-helper cell found in CP tissue along with appreciable numbers of Th17 cells. Interestingly there is no polarisation of the peripheral blood T-helper cell response in CP towards either a Th1 or Th2 phenotype. It appears therefore that the blood of CP patients is primed to respond non-specifically to inflammatory stimuli. Intriguingly CP patients who consume excess alcohol have more peripheral blood Th1 cells. Alcohol increases gut permeability causing high circulating levels of lipopolysaccharide which is known to generate Th1 cell responses. 5 These combined features may contribute to the pathogenesis of CP. Competing interests None declared. References 1. Hunger RE . Gastroenterology 1997. 2. Ebert MP . Am J Gastro 1998. 3. Miyoshi H . Pancreas 2008. 4. Schmitz-Winnenthal H . Gastroenterology 2010. 5. Bode C . J Hepatol 1987.

Introduction Mucosal-Associated Invariant T cells (MAIT) are recently described human T lymphocytes with possible functions in mucosal antibacterial host defence.1 MAIT express a highly conserved T cell receptor (TCR) alpha chain consisting of an invariant Vα7.2-Jα33 rearrangement.2 Recent evidence suggests a role for T lymphocytes in the pathogenesis of chronic pancreatitis (CP), but little is known about the composition of T cell subsets in this disease.34 Intestinal bacterial overgrowth is common in chronic pancreatitis patients.5 We hypothesised that this antigenic load may promote MAIT infiltration of the pancreas and used flow cytometry to study T lymphocytes in resected pancreatic tissue and blood of patients with chronic pancreatitis. Methods Fresh resected pancreatic tissue from five patients with CP (5 males, aged 35–62, median age 43) was diced finely, enzymatically digested and passed through a nylon mesh. Lymphocytes were then isolated using density gradient centrifugation. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of the CP patients and four control patients (2 male, 2 female, aged 31–75, median age 41) using widely established techniques. Isolated tissue lymphocytes and PBMCs were analysed with fluorochrome conjugated antibodies (anti- CD3; CD4; CD8; CD161; TCR Vα7.22 and a live/dead stain) according to standard techniques using a FACSAria. Results The ratio of CD4:CD8 T cells found in pancreatic tissue compared to blood in patients with CP trended towards a higher ratio in blood, however the trend was not statistically significant. Double negative (DN) and CD8 MAIT cells (TCR Vα7.2+ CD161) were identified in pancreatic tissue and blood in all patients. The proportion of DN MAIT cells was significantly higher in pancreatic tissue compared to blood in patients with CP: 19.4% (5.7–39.6%) versus 5.2% (1.9–11.1%); p Conclusion These preliminary data show that while the distribution of CD4 and CD8 lymphocytes in CP tissue and blood is similar, DN MAIT cells are over-represented in tissue. This suggests a specific role for DN MAIT cells in local immune responses in CP. Further analysis using a larger patient cohort and functional studies to assess the possible role of these cells in the regulation of pancreatic inflammation are required.

Introduction Pancreatic stellate cells (PSC) are exposed to supranormal concentrations of insulin via the islet–acinar portal system. We initially hypothesised that this might influence PSC numbers by effects on proliferation or apoptosis. This effect could be mediated via insulin receptors or via receptors for insulin-like growth factor (IGF-1). In this context, we examined the role of IGF-1 and insulin in the prevention of pancreatic stellate cell apoptosis. Following initial experiments, it became apparent that PSC can synthesise IGF-1, we therefore examined whether IGF-1 might be an autocrine growth factor in cultured stellate cells. The aims were to determine (1) the expression of IGF-1 and insulin receptor by PSC; (2) the secretion of IGF-1 by pancreatic stellate cells; (3) the effects of IGF-1 on PSC proliferation and apoptosis. Methods Culture-activated rat PSCs were used between passages 1 and 4 and serum deprived prior to all experiments. PSC proliferation was determined by [3H]-thymidine incorporation. Apoptosis was determined with acridine orange counting of apoptotic bodies and caspase 3 activity assays. Western blotting and immunocytochemistry were used to demonstrate the presence of IGF-1 and insulin receptors. Real-time quantitative PCR was used to detect receptor mRNA. A sandwich ELISA was used to determine the secretion of IGF-1, and real-time quantitative PCR use to confirm mRNA expression. Results Activated PSC express protein and mRNA for IGF-1 and insulin receptors. IGF-1 secretion is upregulated 2.40-fold following serum withdrawal compared with 16% FCS (see Abstract 001). This upregulation is also seen at the level of mRNA in response to serum withdrawal. Exogenous IGF-1 (100 ng/ml) and insulin (10 000 ng/ml) prevented cycloheximide-induced PSC apoptosis in a concentration-dependent manner, 63% at 24 h, and 71% at 24 h, respectively. There was moderate effect on PSC proliferation following exposure to IGF-1 but not with insulin. Conclusion These data suggest that IGF-1 has an autocrine role in reducing PSC apoptosis on serum withdrawal. Inhibiting IGF-1 expression or blocking the IGF-1 receptor may increase susceptibility to apoptosis. This represents a potential therapeutic target for prevention or treatment of pancreatic fibrosis.

BACKGROUND—Hepatitis E (HEV) is the primary cause of enterically transmitted non-A non-B hepatitis worldwide. Case reports of HEV in individuals in the United Kingdom relate to travel to endemic areas or contact with individuals who have visited these areas. CASE REPORTS—Four individuals presented with acute hepatitis to a hepatology clinic in a teaching hospital. Serology confirmed acute hepatitis E in all four. Investigation by the Communicable Disease Control Department established no links between the cases, no travel to an endemic area, and no contacts. CONCLUSION—Contrary to current belief, community acquired hepatitis E virus infection occurs sporadically in the United Kingdom and should be considered as a cause of seronegative hepatitis. Keywords: hepatitis E; viral hepatitis; epidemiology