Background & Aims: Currently, staging of fibrosis in preclinical rodent liver fibrosis models is achieved histologically. Many animals are used at multiple time-points to assess disease progression or therapeutic responses. Hepatic myofibroblasts promote liver fibrosis therefore quantifying these cells in vivo could assess disease or predict therapeutic responses in mice. We fluorescently labelled a single chain antibody (C1-3) that binds hepatic myofibroblasts to monitor fibrogenesis in vivo.Methods: CCl4 was used to induce acute liver injury in WT and cRel(-/-) mice. Bile duct ligation was used to model chronic fibrosis. Hepatic myofibroblasts were depleted using a liposome-drug delivery system or chemically with sulfasalazine. An IVIS (R) spectrum visualised fluorophore-conjugated C1-3 in vivo.Results: IVIS detection of fluorescently labelled-C1-3 but not a control antibody discriminates between fibrotic and nonfibrotic liver in acute and chronic liver fibrosis models. cRel(-/-) mice have a fibro-protective phenotype and IVIS signal is reduced in CCl4 injured cRel(-/-) mice compared to wild-type. In vivo imaging of fluorescently labelled-C1-3 successfully predicts reductions in hepatic myofibroblast numbers in fibrotic liver disease in response to therapy.Conclusions: We report a novel fluorescence imaging method to assess murine hepatic myofibroblast numbers in vivo during liver fibrosis and after therapy. We also describe a novel liposomal antibody targeting system to selectively deliver drugs to hepatic myofibroblasts in vivo. C1-3 binds human hepatic myofibroblast therefore imaging labelled-C1-3 could be used for clinical studies in man to help stage fibrosis, demonstrate efficacy of drugs that promote hepatic myofibroblast clearance or predict early therapeutic responses.Lay summary: In response to damage and injury scars develop in the liver and the main cell that makes the scar tissue is the hepatic myofibroblast (HM). C1-3 is an antibody fragment that binds to the scar forming HM. We have fluorescently labelled C1-3 and given it to mice that have either normal or scarred livers (which contain HM) and then used a machine called an in vivo imaging system (IVIS) that takes pictures of different wavelengths of light, to visualise the antibody binding to HM inside the living mouse. Using fluorescently labelled C1-3 we can assess HM numbers in the injured liver and monitor response to therapy. We have also used C1-3 to target drugs encapsulated in lipid carriers (liposomes) to the HM to kill the HM and reduce the liver disease. (C) 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Outcomes after islet transplantation continue to improve but etiology of graft failure remains unclear. De novo donor-specific human leukocyte antigen (HLA) antibodies (DSA) posttransplant are increasingly recognized as a negative prognostic marker. Specific temporal associations between DSA and graft function remain undefined particularly in programs undertaking multiple sequential transplants. Impact of de novo DSA on graft function over 12 months following first islet transplant was determined prospectively in consecutive recipients taking tacrolimus/mycophenolate immunosuppression at a single center. Mixed-meal tolerance test was undertaken in parallel with HLA antibody assessment pretransplant and 1-3 months posttransplant. Sixteen participants received a total of 26 islet transplants. Five (19%) grafts were associated with de novo DSA. Five (31%) recipients were affected: three post-first transplant; two post-second transplant. DSA developed within 4 weeks of all sensitizing grafts and were associated with decreased stimulated C-peptide (median [interquartile range]) at 3 months posttransplant (DSA negative: 613(300-1090); DSA positive 106(34-235) pmol/L [p=0.004]). De novo DSA directed against most recent islet transplant were absolutely associated with loss of graft function despite maintained immunosuppression at 12 months in the absence of a rescue nonsensitizing transplant. Alemtuzumab induction immunosuppression was associated with reduced incidence of de novo DSA formation (p=0.03).

OBJECTIVE:Relative contributions of reversible β-cell dysfunction and true decrease in β-cell mass in type 2 diabetes remain unclear. Definitive rodent lineage-tracing studies have identified β-cell dedifferentiation and subsequent reprogramming to α-cell fate as a novel mechanism underlying β-cell failure. The aim was to determine whether phenotypes of β-cell dedifferentiation and plasticity are present in human diabetes.RESEARCH DESIGN AND METHODS:Immunofluorescence colocalization studies using classical endocrine and mesenchymal phenotypic markers were undertaken using pancreatic sections and isolated islets from three individuals with diabetes and five nondiabetic control subjects.RESULTS:Intraislet cytoplasmic coexpression of insulin and vimentin, insulin and glucagon, and vimentin and glucagon were demonstrated in all cases. These phenotypes were not present in nondiabetic control subjects.CONCLUSIONS:Coexpression of mesenchymal and α-cell phenotypic markers in human diabetic islet β-cells has been confirmed, providing circumstantial evidence for β-cell dedifferentiation and possible reprogramming to α-cells in clinical diabetes.

Background: Despite intensive research and novel adjuvant therapies, there is currently no cure for metastatic melanoma. The chemokine receptor CXCR4 controls metastasis to sites such as the liver; however, the therapeutic blockade with the existing agents has proven difficult.Methods: AMD11070, a novel orally bioavailable inhibitor of CXCR4, was tested for its ability to inhibit the migration of melanoma cells compared with the commonly described antagonist AMD3100.Results: AMD11070 abrogated melanoma cell migration and was significantly more effective than AMD3100. Importantly for the clinical context, the expression of B-RAF-V600E did not the affect the sensitivity of AMD11070.Conclusion: Liver-resident myofibroblasts excrete CXCL12, which is able to promote the migration of CXCR4-expressing tumour cells from the blood into the liver. Blockade of this axis by AMD11070 thus represents a novel therapeutic strategy for both B-RAF wild-type and mutated melanomas.

This poster session discusses designing a physical activity intervention study for youth with Type 1 diabetes. It was presented at the Diabetes UK Professional Conference 2013, Manchester Central Convention Complex, Manchester, UK, 13-15 March 2013

Background. Inflammatory cell recruitment during allograft rejection is driven by a group of inflammatory cytokines termed chemokines. Chemokines are presented on the surface of the vascular endothelium where they ligate specific receptors expressed on the surface of leukocytes. Recently, a group of nonsignaling chemokine receptors have been described. These bind and internalize chemokines but do not drive leukocyte migration. It is believed that these compete with classical signaling receptors to modulate inflammation.Methods. This study describes the first examination of the human decoy chemokine receptor D6 during rejection; D6 binds at least 12 potent proinflammatory chemokines. The expression of D6 by graft infiltrating leukocytes was examined in cardiac allografts by confocal microscopy on biopsy sections (n = 19). Cytokine regulation of D6 was examined in vitro, and a chemokine scavenging assay was performed using the prototypical transplant-associated chemokine CCL5/RANTES.Results. D6 expression was found to be higher in the biopsies taken from more severe cardiac allograft rejection (P < 0.01) and was predominantly localized to graft infiltrating CD45(+)CD68(+) leukocytes. In vitro studies demonstrated that the transforming growth factor-beta strongly increased the expression of D6 by monocytes, which significantly enhanced D6-mediated chemokine scavenging (by 85%, P < 0.05).Conclusions. We present the first examination of the biology of D6 during rejection and identify a transplant-associated cytokine that is able to regulate its expression. These data suggest an exciting new mechanism for the antiinflammatory actions of transforming growth factor-beta. Understanding the expression patterns of D6 may provide important insight into the regulation and control of inflammatory cell recruitment during allograft rejection.

To the Editor: There is an increasing debate focused on the contribution made by direct epithelial to mesenchymal cell transition (EMT) during chronic fibrosis. This process has been defined recently in the murine liver by transgenic lineage tracing, which showed that hepatocytes can transform directly into fibroblasts as a consequence of injury with the fibrogenic agent, CCl4 (1). Our group is researching the role of EMT in human inflammatory liver disease (2, 3) and has recently completed a study that demonstrates a clear relationship between the stage of fibrosis in primary sclerosing cholangitis (PSC) and the number of epithelial cells expressing markers characteristic of EMT within both the bile ducts and the periportal ductular reaction. Seventeen biopsy specimens provided sections for examination (five from normal liver, three from Mayo stage 1 PSC, four from stage 2 and five from stage 3). Each section was labelled by two-colour immunohistochemistry to identify S100A4 [a fibroblast marker induced in epithelial cells at an early stage of EMT, termed FSP-1 in mice (4)] and cytokeratin 19 (CK-19; a marker of biliary epithelial cells that is lost at a later stage of EMT). Figure 1a demonstrates these proteomic features of EMT with acquisition of S100A4 and partial loss of CK-19 (black arrows) by the cells in two ductules, while an elongated, fibroblast-like cell (green arrow) shows simultaneous expression of CK-19 and S100A4 (3). Quantitative data, also presented in Figure 1, show, for normal and PSC liver, the total number of epithelial cells undergoing EMT per mm2 of the portal tract (Fig. 1b) and the number of these cells restricted to the ductular reaction (Fig. 1c). (a) Dual-labelling (S100A4, red; CK-19, brown) in a PSC grade 2 biopsy section. (b, c) Graphs representing quantitative data – see text. Analysis of the morphometric data (anova) demonstrated a significant increase in the number of cells co-expressing both CK-19 and S100A4 within the entire portal tract population (b; P<0.02) and the subset of biliary epithelial cells within the ductular reaction (c; P<0.01) in PSC liver; a general increase was also observed in the number of cells undergoing EMT from stage 1 to stage 3 fibrosis. This study provides strong evidence that biliary epithelial cells can be induced to undergo EMT, there by contributing to portal tract fibrogenesis in PSC. Furthermore, it is clear that this EMT is not limited to the cells that form the native bile ducts but is also a prominent feature of epithelial cells within the ductular reaction. The formation and expansion of this reaction during inflammatory diseases such as PSC could provide an increasingly large reservoir of epithelial cells capable of generating fibroblasts and myofibroblasts through the process of EMT. The definition of this pathogenetic mechanism is important as several studies have shown that EMT can be reversed to some extent by treatment with agents such as bone morphogenetic protein-7 (1, 5). This work could lead to exciting new strategies for the management of otherwise intractable and progressive cholestatic liver diseases.

The relationship between bile duct damage and portal fibrosis in chronic liver diseases remains unclear. This study was designed to show whether human intrahepatic biliary epithelial cells can undergo epithelial–mesenchymal cell transition, thereby directly contributing to fibrogenesis. Primary human cholangiocytes were stimulated with transforming growth factor- β (TGF β ) or TGF β -presenting T cells and examined for evidence of transition to a mesenchymal phenotype. Liver sections were labelled to detect antigens associated with biliary epithelial cells (cytokeratin 7 and 19 and E-cadherin), T cells (CD8), epithelial–mesenchymal transition (S100A4, vimentin and matrix metalloproteinase-2 (MMP-2)), myofibroblasts ( α -smooth muscle actin) and intracellular signal-transduction mediated by phosphorylated (p)Smad 2/3; in situ hybridisation was performed to detect mRNA encoding TGF β and S100A4. Stimulation of cultured cells with TGF β induced the expression of pSmad2/3, S100A4 and α -smooth muscle actin; these cells became highly motile. Although normal bile ducts expressed ALK5 (TGF β RI), low levels of TGF β mRNA and nuclear pSmad2/3, they did not express S100A4, vimentin or MMP-2. However, TGF β mRNA and nuclear pSmad2/3 were strongly expressed in damaged ducts, which also expressed S100A4, vimentin and MMP-2. Fibroblast-like cells which expressed S100A4 were present around many damaged bile ducts. Cells in the ‘ductular reaction’ expressed both epithelial and mesenchymal markers together with high levels of TGF β mRNA and pSmad2/3. In conclusion, the cells forming small- and medium-sized bile ducts and the ductular reaction undergo EMT during chronic liver diseases, resulting in the formation of invasive fibroblasts; this process may be driven by a response to local TGF β , possibly presented by infiltrating T cells.