Borut Klopcic

Tumor necrosis factor-like weak inducer of apoptosis is a mitogen for liver progenitor cells

Liver progenitor cells (LPCs) represent the cell compartment facilitating hepatic regeneration during chronic injury while hepatocyte‐mediated repair mechanisms are compromised. LPC proliferation is frequently observed in human chronic liver diseases such as hereditary hemochromatosis, fatty liver disease, and chronic hepatitis. In vivo studies have suggested that a tumor necrosis factor family member, tumor necrosis factor–like weak inducer of apoptosis (TWEAK), is promitotic for LPCs; whether it acts directly is not known. In our murine choline‐deficient, ethionine‐supplemented (CDE) model of chronic liver injury, TWEAK receptor [fibroblast growth factor‐inducible 14 (Fn14)] expression in the whole liver is massively upregulated. We therefore set out to investigate whether TWEAK/Fn14 signaling promotes the regenerative response in CDE‐induced chronic liver injury by mitotic stimulation of LPCs. Fn14 knockout (KO) mice showed significantly reduced LPC numbers and attenuated inflammation and cytokine production after 2 weeks of CDE feeding. The close association between LPC proliferation and activation of hepatic stellate cells in chronic liver injury prompted us to investigate whether fibrogenesis was also modulated in Fn14 KO animals. Collagen deposition and expression of key fibrogenesis mediators were reduced after 2 weeks of injury, and this correlated with LPC numbers. Furthermore, the injection of 2‐week‐CDE‐treated wildtype animals with TWEAK led to increased proliferation of nonparenchymal pan cytokeratin–positive cells. Stimulation of an Fn14‐positive LPC line with TWEAK led to nuclear factor kappa light chain enhancer of activated B cells (NFκB) activation and dose‐dependent proliferation, which was diminished after targeting of the p50 NFκB subunit by RNA interference. Conclusion: TWEAK acts directly and stimulates LPC mitosis in an Fn14‐dependent and NFκB‐dependent fashion, and signaling via this pathway mediates the LPC response to CDE‐induced injury and regeneration. (HEPATOLOGY 2010)

Identification of nestin-positive putative mammary stem cells in human breastmilk.

Stem cells in mammary tissue have been well characterised by using the mammary stem cell marker, cytokeratin (CK) 5 and the mature epithelial markers CK14, CK18 and CK19. As these markers have never been reported in cells from breastmilk, the aim of this study has been to determine whether mammary stem cells are present in expressed human breastmilk. Cultured cells from human breastmilk were studied by using immunofluorescent labelling and reverse transcription/polymerase chain reaction (RT-PCR). We found a heterogeneous population of cells with differential expression of CK5, CK14, CK18 and CK19. Further, by using the multipotent stem cell marker, nestin, we identified cells in culture that were positive only for nestin or double-positive for CK5/nestin, whereas no co-staining was observed for CK14, CK18 and CK19 with nestin. When cells isolated from breastmilk were analysed by using RT-PCR prior to culture, only nestin and CK18 were detected, thereby indicating that breastmilk contained differentiated epithelial and putative stem cells. Furthermore, fluorescence-activated cell-sorting analysis demonstrated, in breastmilk, a small side-population of cells that excluded Hoechst 33342 (a key property of multipotent stem cells). When stained for nestin, the cells in the side-population were positive, whereas those not in the side-population were negative. The presence of nestin-positive putative mammary stem cells suggests that human breastmilk is a readily available and non-invasive source of putative mammary stem cells that may be useful for research into both mammary gland biology and more general stem cell biology.

SPARC, FOXP3, CD8 and CD45 Correlation with Disease Recurrence and Long-Term Disease-Free Survival in Colorectal Cancer

Background SPARC is a matricellular protein involved in tissue remodelling, cell migration and angiogenesis, while forkhead box P3 (FOXP3) protein functions as a transcription factor involved in immune cell regulation. Both SPARC and FOXP3 can play an anti-tumorigenic role in cancer progression. The aim was to determine if SPARC, FOXP3, CD8 and CD45RO expression levels are associated with colorectal cancer (CRC) stage, disease outcome and long-term cancer-specific survival (CSS) in stage II and III CRC. Methods and Findings SPARC expression was initially assessed in 120 paired normal and stage I-IV CRCs. Subsequently, approximately 1000 paired patient samples of stage II or III CRCs in tissue microarrays were stained for SPARC, FOXP3, CD8 or CD45RO. Proportional hazards modelling assessed correlations between these markers and clinicopathological data, including disease outcome and cancer specific survival (CSS). Both SPARC and FOXP3 expression were significantly greater in CRC than normal colon (p<0.0001). High SPARC expression correlated with good disease outcome (≥60 mths without disease recurrence, p = 0.0039) and better long-term CSS in stage II CRC (<0.0001). In stage III CRC, high SPARC expression correlated with better long-term CSS (p<0.0001) and less adjuvant chemotherapy use (p = 0.01). High FOXP3 correlated with a good disease outcome, better long-term CSS and less adjuvant chemotherapy use in stage II (p<0.0037, <0.0001 and p = 0.04 respectively), but not in stage III CRC. High CD8 and CD45RO expression correlated with better disease outcome in stage II CRC, and better CSS, but the differences were not as marked as for SPARC and FOXP3. Conclusions These data suggest that high SPARC and FOXP3 are associated with better disease outcome in stage II CRC and may be prognostic indicators of CSS. Further assessment of whether these markers predict patients at high risk of recurrence with stage II CRC and functional studies of these effects are underway

Dietary iron enhances colonic inflammation and IL-6/IL-11-Stat3 signaling promoting colonic tumor development in mice

Chronic intestinal inflammation and high dietary iron are associated with colorectal cancer development. The role of Stat3 activation in iron-induced colonic inflammation and tumorigenesis was investigated in a mouse model of inflammation-associated colorectal cancer. Mice, fed either an iron-supplemented or control diet, were treated with azoxymethane and dextran sodium sulfate (DSS). Intestinal inflammation and tumor development were assessed by endoscopy and histology, gene expression by real-time PCR, Stat3 phosphorylation by immunoblot, cytokines by ELISA and apoptosis by TUNEL assay. Colonic inflammation was more severe in mice fed an iron-supplemented compared with a control diet one week post-DSS treatment, with enhanced colonic IL-6 and IL-11 release and Stat3 phosphorylation. Both IL-6 and ferritin, the iron storage protein, co-localized with macrophages suggesting iron may act directly on IL-6 producing-macrophages. Iron increased DSS-induced colonic epithelial cell proliferation and apoptosis consistent with enhanced mucosal damage. DSS-treated mice developed anemia that was not alleviated by dietary iron supplementation. Six weeks post-DSS treatment, iron-supplemented mice developed more and larger colonic tumors compared with control mice. Intratumoral IL-6 and IL-11 expression increased in DSS-treated mice and IL-6, and possibly IL-11, were enhanced by dietary iron. Gene expression of iron importers, divalent metal transporter 1 and transferrin receptor 1, increased and iron exporter, ferroportin, decreased in colonic tumors suggesting increased iron uptake. Dietary iron and colonic inflammation synergistically activated colonic IL-6/IL-11-Stat3 signaling promoting tumorigenesis. Oral iron therapy may be detrimental in inflammatory bowel disease since it may exacerbate colonic inflammation and increase colorectal cancer risk.

Secreted Protein Acidic and Rich in Cysteine (SPARC) Exacerbates Colonic Inflammatory Symptoms in Dextran Sodium Sulphate-Induced Murine Colitis

Background Secreted Protein Acidic and Rich in Cysteine (SPARC) is expressed during tissue repair and regulates cellular proliferation, migration and cytokine expression. The aim was to determine if SPARC modifies intestinal inflammation. Methods Wild-type (WT) and SPARC-null (KO) mice received 3% dextran sodium sulphate (DSS) for 7 days. Inflammation was assessed endoscopically, clinically and histologically. IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-12/IL23p40, TNF-α, IFN-γ, RANTES, MCP-1, MIP-1α, MIP-1β, MIG and TGF-β1 levels were measured by ELISA and cytometric bead array. Inflammatory cells were characterised by CD68, Ly6G, F4/80 and CD11b immunofluorescence staining and regulatory T cells from spleen and mesenteric lymph nodes were assessed by flow cytometry. Results KO mice had less weight loss and diarrhoea with less endoscopic and histological inflammation than WT animals. By day 35, all (n = 13) KO animals completely resolved the inflammation compared to 7 of 14 WT mice (p<0.01). Compared to WTs, KO animals at day 7 had less IL1β (p = 0.025) and MIG (p = 0.031) with higher TGFβ1 (p = 0.017) expression and a greater percentage of FoxP3+ regulatory T cells in the spleen and draining lymph nodes of KO animals (p<0.01). KO mice also had fewer CD68+ and F4/80+ macrophages, Ly6G+ neutrophils and CD11b+ cells infiltrating the inflamed colon. Conclusions Compared to WT, SPARC KO mice had less inflammation with fewer inflammatory cells and more regulatory T cells. Together, with increased TGF-β1 levels, this could aid in the more rapid resolution of inflammation and restoration of the intestinal mucosa suggesting that the presence of SPARC increases intestinal inflammation.

Proteomics: An Overview

The application of genomics has established a firmfoundation for research into both human health and disease,and the development of proteomics was the next logical step,but what exactly is proteomics? The term proteomics wasfirst coined in 1995 from a combination of ‘‘protein’’ and‘‘genomics,’’ but it was far earlier in 1979 that the concept tofully characterize the human proteome was first proposed byNorman G. Anderson and N. Leigh Anderson in a submissionentitled the ‘‘Human Proteins Index Project.’’

High Dose Vitamin D supplementation alters faecal microbiome and predisposes mice to more severe colitis

Vitamin D has been suggested as a possible adjunctive treatment to ameliorate disease severity in human inflammatory bowel disease. In this study, the effects of diets containing high (D++, 10,000 IU/kg), moderate (D+, 2,280 IU/kg) or no vitamin D (D−) on the severity of dextran sodium sulphate (DSS) colitis in female C57Bl/6 mice were investigated. The group on high dose vitamin D (D++) developed the most severe colitis as measured by blinded endoscopic (p < 0.001) and histologic (p < 0.05) assessment, weight loss (p < 0.001), drop in serum albumin (p = 0.05) and increased expression of colonic TNF-α (p < 0.05). Microbiota analysis of faecal DNA showed that the microbial composition of D++ control mice was more similar to that of DSS mice. Serum 25(OH)D3 levels reduced by 63% in the D++ group and 23% in the D+ group after 6 days of DSS treatment. Thus, high dose vitamin D supplementation is associated with a shift to a more inflammatory faecal microbiome and increased susceptibility to colitis, with a fall in circulating vitamin D occurring as a secondary event in response to the inflammatory process.

Tu1934 SPARC Modifies Colonic Tissue Healing and Inflammation by Regulating Collagen and MMP Expression

OP07 Use of fecal calprotectin as marker of disease activity in patients under maintenance treatment with infliximab for ulcerative colitis M. De Vos1 *, J. Jahnsen2, J. Vandervoort3, G. D’Haens4, O. Dewit5, E. Louis6, D. Franchimont7, F. Baert8, R. Torp9, P. Potvin10, P. Van Hootegem11, M. Henriksen12, B. Vander Cruyssen1, S. Vermeire13, on behalf of BIRD14. 1Ghent University Hospital, Department of Gastroenterology, Gent, Belgium, 2Oslo University Hospital, Department of Gastroenterology, Aker, Norway, 3OLV Hospital, Department of Gastroenterology, Aalst, Belgium, 4Imelda Hospital, Department of Gastroenterology, Bonheiden, Belgium, 5Clinique Universitaire St Luc, Department of Gastroenterology, Brussel, Belgium, 6University of Liege and CHU Liege, Department of Gastroenterology, Liege, Belgium, 7Erasme Hospital, Department of Gastroenterology, Brussel, Belgium, 8H. Hart Hospital, Department of Gastroenterology, Roeselare, Belgium, 9Innlandet Hospital, Department of Gastroenterology, Hammar, Norway, 10St-Jozephs Hospital, Department of Gastroenterology, Bornem, Belgium, 11AZ St Lucas, Department of Gastroenterology, Brugge, Belgium, 12Ostfold Fredrikstad Hospital, Department of Gastroenterology, Fredrikstad, Norway, 13University Hospital Gasthuisberg, Department of Gastroenterology, Leuven, Belgium, 14, Belgium