Quantitative Trait Locus Analysis Implicates CD4⁺/CD44high Memory T Cells in the Pathogenesis of Murine Autoimmune Pancreatitis.

The mouse strain MRL/MpJ is prone to spontaneously develop autoimmune pancreatitis (AIP). To elucidate the genetic control towards the development of the phenotype and to characterize contributions of immunocompetent cell types, MRL/MpJ mice were interbred with three additional strains (BXD2/TYJ, NZM2410/J, CAST/EIJ) for four generations in an advanced intercross line. Cellular phenotypes were determined by flow cytometric quantification of splenic leukocytes and complemented by the histopathological evaluation of pancreatic lesions. An Illumina SNP array was used for genotyping. QTL analyses were performed with the R implementation of HAPPY. Out of 41 leukocyte subpopulations (B cells, T cells and dendritic cells), only three were significantly associated with AIP: While CD4+/CD44high memory T cells and CD4+/CD69+ T helper (Th) cells correlated positively with the disease, the cytotoxic T cell phenotype CD8+/CD44low showed a negative correlation. A QTL for AIP on chromosome 2 overlapped with QTLs for CD4+/CD44high and CD8+/CD44high memory T cells, FoxP3+/CD4+ and FoxP3+/CD8+ regulatory T cells (Tregs), and CD8+/CD69+ cytotoxic T cells. On chromosome 6, overlapping QTLs for AIP and CD4+/IL17+ Th17 cells and again FoxP3+/CD8+ Tregs were observed. In conclusion, CD4+/CD44high memory T cells are the only leukocyte subtype that could be linked to AIP both by correlation studies and from observed overlapping QTL. The potential role of this cell type in the pathogenesis of AIP warrants further investigations.

Identification of quantitative trait loci for murine autoimmune pancreatitis.

BACKGROUND AND AIMS: Autoimmune pancreatitis (AIP) represents a rare but clinically relevant cause of pancreatic inflammation. Using MRL/Mp mice as a model of spontaneous AIP, the genetic basis of the disease was studied. METHODS: To identify quantitative trait loci (QTL) of AIP, an advanced intercross line was studied, originating from MRL/MpJ parental mice and the following three mouse strains: Cast (healthy controls), BXD2 (susceptible to collagen induced arthritis), and NZM (a model of lupus erythematosus). This concept was chosen to identify both general autoimmune disease associated loci and AIP specific QTL. Therefore, generation G4 of outbred intercross mice was characterised phenotypically by scoring histopathological changes of the pancreas and genotyped with single nucleotide polymorphism (SNP) arrays. Data were analysed with the R implementation of HAPPY. RESULTS: Five QTLs, correlating with the severity of AIP, were identified. Two of them mapped to chromosome 4 and one to chromosomes 2, 5, and 6, respectively. The QTL on chromosome 6 displays the highest LOD score (5.4) and contains the C-type lectin domain family 4 member a2 in its peak region, which encodes a receptor protein of dendritic cells that has previously been implicated in autoimmune diseases such as Sjogren's syndrome. AIP candidate genes of other QTL's include heterogeneous nuclear ribonucleoprotein A3; nuclear factor, erythroid derived 2, like 2; Sjogren syndrome antigen B; and ubiquitin protein ligase E3 component n-recognin 3. CONCLUSIONS: This study has identified QTLs and putative candidate genes of murine AIP. Their functional role and relevance to human AIP will be studied further.

Synergistic growth inhibitory effects of the dual endothelin-1 receptor antagonist bosentan on pancreatic stellate and cancer cells.

Pancreatic stellate cells (PSC) play a key role in pancreatic fibrosis. Activation of PSC occurs in response to pro-fibrogenic stimuli and is maintained by autocrine loops of mediators, such as endothelin (ET)-1. Here, we have evaluated effects of the dual ET receptor antagonist bosentan in models of pancreatic fibrogenesis and cancer. Cell culture studies revealed that PSC and DSL6A pancreatic cancer cells expressed both ET-1 and ET receptors. Bosentan efficiently inhibited proliferation of both cell types and collagen synthesis in PSC. Expression of the myofibroblastic marker alpha-smooth muscle actin, connective tissue growth factor, and ET-1 itself in PSC was reduced, while expression of matrix metalloproteinase-9 was enhanced. Like PSC, DSL6A cells secrete less ET-1 when cultured with bosentan. In a rat model of pancreatic fibrosis, chronic pancreatitis induced by dibutyltin dichloride, a tendency towards a diminished disease progression was observed in a subgroup of rats with less severe disease. Together, our results indicate that bosentan exerts antifibrotic and antitumor effects in vitro. Its efficiency in vivo warrants further investigation.

Injectable liver: a novel approach using fibrin gel as a matrix for culture and intrahepatic transplantation of hepatocytes.

Cell transplantation and tissue engineering with liver cells are currently under investigation as experimental therapies for certain liver diseases. In this study we evaluated a fibrin-based gel matrix as carrier for hepatocytes in culture. Furthermore, a novel technique for direct intrahepatic injection of fibrin gel-immobilized hepatocytes was developed and evaluated in a rat model. Hepatocytes were harvested from rats. Fibrin matrix was generated with modified fibrin sealant. Cells, in medium containing epidermal growth factor and insulin, were seeded in a drop of fibrin matrix onto plastic culture dishes. Cell numbers were assessed by DNA content. Hepatocyte differentiation was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistology (IH) for cytokeratin (CK)-18 and albumin. PKH26-labeled fibrin gel-immobilized hepatocytes were transplanted into liver by direct injection underneath the capsule. Fluorescence microscopy of explanted liver was performed to identify PKH26+ donor cells. Neotissue was characterized by IH for the markers CK-18, ED1, and desmin. Culture in a fibrin matrix allowed stable cell numbers and three-dimensional neotissue formation. RT-PCR and IH showed preservation of liver-specific markers CK-18 and albumin in vitro. Transplanted cells were identified by fluorescence microscopy after 2 and 7 days. CK-18 and desmin staining showed integration of hepatocytes and hepatic stellate cells into the host liver. Fibrin matrix is an appropriate environment for hepatocytes in culture. Direct intrahepatic injection of fibrin gel-immobilized hepatocytes is technically feasible. We conclude that fibrin gel immobilization is an attractive tool for the development of tissue engineering-based liver support systems.

Hepatic lineages isolated from developing rat liver show different ways of maturation.

Immunocytochemical analysis revealed that different hepatic cell types exist during liver development: (i). cells co-expressing the stem-cell marker Thy1 and the hepatic lineage marker CK-18 and (ii). cells only expressing CK-18 (hepatoblasts). In this study we separated the different hepatic cells and analyzed gene-expression and phenotype. Fetal rat livers were digested by collagenase solution. OX43- and OX44-positive hematopoietic cells were depleted and Thy1-positive cells were enriched using Magnetic cell sorting. The different cell compartments were analyzed by RT-PCR and immunocytochemistry for Thy1, CK-18, AFP, and albumin. Hepatoblasts expressed albumin at all times and AFP in the early stages. Thy1-enriched cells expressed CK-18 at all times, albumin in the early, and AFP in the late stages. Thy1-positive cells from fetal livers express liver specific genes. The data suggest that Thy1-positive hepatic cells develop towards hepatic stem cells, and hepatoblasts develop towards mature hepatocytes of the adult liver.