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Background: Fistulae represent an important clinical complication of Crohn's disease (CD). The fistula tracts are covered by flat, myofibroblast-like cells with an epithelial origin (transitional cells, TC). We recently demonstrated a role of epithelial mesenchymal transition (EMT) in the pathogenesis of CD-associated fistulae. EMT is associated with an increased migratory and invasive potential of epithelial cells in different tissues. Here we investigated whether cytokines or growth factors as well as EMT-associated SNAIL family transcription factors are expressed in CD fistulae. Methods: By immunohistochemistry we analyzed seven perianal fistulae from seven CD and two perianal fistulae from two non-inflammatory bowel disease (IBD) control patients. Hematoxylin and eosin staining or immunohistochemistry for the expression of tumor necrosis factor (TNF), TNF-receptor I (TNF-RI), SNAIL1, SLUG, fibroblast growth factors (FGF) 1, 2, 4, 7, epidermal growth factor (EGF), and TWIST were performed using standard techniques. Results: Immunohistochemical staining of surgical specimens from CD patients revealed a strong expression of TNF and TNF-RI in and around fistula tracts. While SNAIL1 was also heavily expressed in the nuclei of TC, indicative of transcriptionally active protein, SLUG, FGF-1, and FGF-2 were detected rather in the fibrotic periphery of CD fistulae than in TC. In contrast, we did not detect considerable protein staining for FGF-4 and FGF-7 nor of EGF or the transcription factor, TWIST. Conclusions: Our data demonstrate that SNAIL1 and TNF are strongly expressed in TC of CD-associated fistulae. These observations support our previous data and indicate the onset of EMT-associated events in the pathogenesis of CD fistulae. (Inflamm Bowel Dis 2010;17:1907–1916)

BCL-2 modifying factor (BMF) is a sentinel considered to register damage at the cytoskeleton and to convey a death signal to B-cell lymphoma 2. B-cell lymphoma 2 is neutralized by BMF and thereby facilitates cytochrome C release from mitochondria. We investigated the role of BMF for intestinal epithelial cell (IEC) homeostasis. Acute colitis was induced in Bmf-deficient mice (Bmf(-/-)) with dextran sulfate sodium. Colonic crypt length in Bmf(-/-) mice was significantly increased as compared with WT mice. Dextran sulfate sodium induced less signs of colitis in Bmf(-/-) mice, as weight loss was reduced compared with the WT. Primary human IEC exhibited increased BMF in the extrusion zone. Quantitative PCR showed a significant up-regulation of BMF expression after initiation of anoikis in primary human IEC. BMF was found on mitochondria during anoikis, as demonstrated by Western blot analysis. RNAi mediated knockdown of BMF reduced the number of apoptotic cells and led to reduced caspase 3 activity. A significant increase in phospho-AKT was determined after RNAi treatment. BMF knockdown supports survival of IEC. BMF is induced in human IEC by the loss of cell attachment and is likely to play an important role in the regulation of IEC survival.

Objective Epithelial to mesenchymal transition (EMT) seems to play an important role in the pathogenesis of fistulae, a common clinical complication of Crohn9s disease (CD). TGFβ and interleukin-13 (IL-13) have been correlated with the onset of EMT-associated organ fibrosis and high levels of TGFβ have been shown in transitional cells (TCs) lining CD fistula tracts. This study investigated whether IL-13 could be involved in the pathogenesis of CD-associated fistulae. Design Protein or mRNA levels in HT29 intestinal epithelial cells (IECs) or colonic lamina propria fibroblasts (CLPFs) were studied by western blotting or real-time PCR. CLPFs were isolated from non-inflammatory disease controls or patients with CD with or without fistulae and IL-13 levels were analysed in surgically removed fistula specimens by immunohistochemistry. Results TGFβ induced IL-13 secretion in CLPFs from patients with fistulising CD. In fistula specimens high levels of IL-13 were detected in TCs covering fistula tracts. In HT29 IEC monolayers, IL-13 induced SLUG and β6-integrin mRNA, which are associated with cell invasion. HT29 spheroids completely disintegrated when treated with TGFβ for 7 days, whereas IL-13-treated spheroids did not show morphological changes. Here, TGFβ induced mRNA expression of SNAIL1 and IL-13, whereas IL-13 elevated SLUG and β6-integrin mRNA. An anti-IL-13 antibody was able to prevent IL-13-induced SLUG expression in HT29 IECs. Conclusions TGFβ induces IL-13 expression and an EMT-like phenotype of IECs, while IL-13 promotes the expression of genes associated with cell invasion. These findings suggest that TGFβ and IL-13 play a synergistic role in the pathogenesis of fistulae and inhibition of IL-13 might represent a novel therapeutic approach for fistula treatment.

Background: The first identified susceptibility gene for Crohn’s disease, NOD2, acts as a sensor for the bacterial-wall peptidoglycan fragment muramyl dipeptide (MDP) and activates the transcription factor nuclear factor-ĸB (NF-ĸB). Upon NF-ĸB activation, intestinal macrophages (IMACs) induce expression of macrophage inflammatory protein (MIP)-3α to attract memory T lymphocytes. We therefore investigated the influence of NOD2 ligation of IMAC differentiation and functional MIP-3α induction. Methods: Human embryonal kidney HEK293 cells were transfected with NOD2 wild-type (NOD2WT) and the NOD2 SNP13 variant (NOD2L1007fsinsC) and stimulated with MDP. Recruitment of CD45R0+ and Th17 cells was determined by immunohistochemistry. Results: Endogenous NOD2 stimulation was followed by a dose-dependent increase in MIP-3α secretion in MONO-MAC-6 (MM6) cells. MIP-3α mRNA was also significantly (* p < 0.05) induced in HEK293 transfected with NOD2WT via MDP ligation. In vivo cell-cell contacts between IMACs and CD45R0+ memory T cells as well as recruitment of Th17 cells in patients of NOD2 variants were unchanged as compared to wild-type patients. Conclusion: Our data demonstrate a dose-dependent increase in MIP-3α secretion in the human myeloid cell line MM6 upon MDP. However, MIP-3α-driven recruitment of Th17 cells or CD45R0+ memory T lymphocytes is not affected in patients carrying heterozygous NOD2 variants., OA-Komponente aus Allianzlizenz

Background: In normal mucosa, intestinal lamina propria macrophages (IMACs) maintain tolerance against food antigens and the commensal bacterial flora. Several mechanisms have been identified that mediate tolerance. The ubiquitin-proteasome system (UPS) is a large multiprotein complex that degrades cellular proteins. As the UPS may modulate immune functions of IMACs, we performed a detailed investigation of UPS expression and function under normal conditions and in cells derived from patients suffering from inflammatory bowel disease (IBD). Methods: IMACs were isolated from intestinal mucosa. mRNA expression of macrophages differentiated in vitro (i.v. MACs) and IMACs was compared by Affymetrix® oligonucleotide arrays. Quantitative Taqman-PCR was performed on five exemplary proteasomal and five ubiquitinylation genes each. Proteins were analyzed by immunohistochemistry and Western blotting. Proteasome function was assessed by a fluorimetric test. Results: Affymetrix analysis showed downregulation of mRNA expression of almost all represented proteasomal and of 22 ubiquitination-associated genes in IMACs as compared to i.v. MACs and monocytes. By quantitative PCR, up to tenfold higher mRNA expression of 10 exemplary genes of the UPS (UBE2A, UBE2D2, UBE2L6, USP14, UBB and ATPase2, β2, β5, β2i/MECL-1, β5i/LMP7) was demonstrated in i.v. MACs as compared to IMACs. Immunohistochemistry and Western blots confirmed these findings in intestinal mucosa of controls and patients suffering from diverticulitis. In contrast, a significant increase in protein amounts was found in mucosa of patients with IBD. Conclusion: Reduced expression of subunits of the UPS in IMACs of normal mucosa supports the concept of the presence of a nonreactive, anergic macrophage phenotype in the gut under normal conditions. Reinduction in IMACs of IBD mucosa reflects activated IMACs which can present antigenic peptides and thus support inflammation., OA-Komponente aus Allianzlizenz

Background: Autophagy is a process of central importance for maintaining cell homeostasis, survival, and the regulation of inflammation. Recent studies associated variants within the gene loci, encoding protein tyrosine phosphatase nonreceptor type 2 (PTPN2), and autophagy genes, such as autophagy-related 16-like 1 (ATG16L1), with chronic inflammatory disorders, such as Crohn's disease (CD). We show that PTPN2 regulates autophagy in human intestinal epithelial cells (IEC) and primary colonic lamina propria fibroblasts (CLPF). Methods: Protein analysis in IEC and CLPF was performed by western blotting. Autophagososme formation was assessed by LC3B immunofluorescence or immunohistochemistry. Human intestinal tissue samples were obtained from noninflammatory bowel disease (IBD) control or from CD patients and genotyped for disease-associated PTPN2 or ATG16L1 variations. Results: Knockdown of PTPN2 causes impaired autophagosome formation and dysfunctional autophagy resulted in increased levels of intracellular Listeria monocytogenes (LM) and elevated IEC apoptosis in response to tumor necrosis factor (TNF) and interferon gamma (IFN-?). Similar findings were observed in primary CLPF derived from CD patients carrying the CD-associated PTPN2 variant. Presence of the ATG16L1 variant prevented the cytokine-induced rise in PTPN2 protein, finally resulting in impaired LC3B-II levels in IEC. Actively inflamed intestinal biopsies from CD patients carrying either ATG16L1 or PTPN2 genetic variants revealed aberrant LC3B expression patterns when compared with samples from non-IBD control patients. Conclusions: Our results demonstrate that PTPN2 regulates autophagosome formation in human intestinal cells. We provide a model of how a dysfunction of the CD susceptibility genes, PTPN2 and/or ATG16L1, may contribute to the onset and perpetuation of chronic intestinal inflammation.

Background The sphingolipid sphingomyelin is a constituent in food derived from animals. Digestive breakdown of sphingomyelin results in ceramide, recently suggested to be involved in activation of cathepsin D as a novel mediator of apoptosis. Damage of the epithelial barrier was detected in patients with inflammatory bowel disease (IBD) due to increased rates of intestinal epithelial cell (IEC) apoptosis. Methods Acute colitis was induced in C57-BL/6 mice with 2.0% dextran sulfate sodium (DSS) over 7 days. Spontaneous colitis was developed in B6-IL10tm1Cgn (interleukin 10-negative (IL-10 –/– )) mice. Mice received 4 or 8 mg sphingomyelin/day by oral gavage. IECs were isolated ex vivo. Apoptosis was determined by propidium iodide (PI) and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining. Execution of apoptosis was confirmed by analysis of active cathepsin D, caspase-3 and caspase-9 with western blot and immunohistochemistry (IHC). Results Following DSS-mediated colitis, fluorescence-activated cell sorting (FACS) analysis indicated increased apoptosis of IECs under dietary sphingomyelin. The mean sub-G 1 portion increased from 8.7±2.5% under a normal diet to 14.0±3.1% under dietary sphingomyelin. Cathepsin activity was significantly increased in isolated IECs after gavage of 4 mg of sphingomyelin per day. Western blot and IHC revealed execution of the apoptotic cascade via activated caspase-3 and caspase-9. Dietary sphingomyelin in the IL-10 –/– model confirmed aggravation of mucosal inflammation. Conclusion Apoptosis of IEC induced by dietary sphingomyelin is mediated via ceramide and cathepsin D activation. This shortens the physiological life cycle of IECs and impairs crucial functions of the intestinal mucosa: barrier, defence and nutrient absorption. The findings provide evidence that dietary sphingomyelin may increase intestinal inflammation.

Background: Recently we found that migration of colonic lamina propria fibroblasts in Crohn's disease patients (CD-CLPF) from inflamed mucosa is significantly reduced as compared to control-CLPF. The behavior of CD-CLPFs isolated from fistulae and strictures was now investigated in detail. Methods: Initially migration assays for all CLPF cultures (CD-CLPF, fibrosis-CLPF, and fistula-CLPF) were performed in the modified 48-well Boyden chamber. Subsequently, for a migration assay more resembling the in vivo situation a 3D matrix model was developed. After seeding of cells into the 3D matrix the CLPF layer was wounded by an ERBIUM:YAG laser leading to circular cell rupture without effect on the extracellular matrix. Results: In the modified Boyden chamber migration of fistula-CLPF was significantly reduced compared to CD-CLPF. This was correlated with a decrease in FAK-protein expression, whereas in migrating fibrosis-CLPF an increase in FAK-protein expression, -autophosphorylation and migratory potential was found. This was confirmed in the 3D matrix wounding assay: Fistula-CLPF migrated less than CD-CLPF, whereas fibrosis-CLPF migrated significantly more in the 3D matrix wounding assay. Between 1 to 36 hours incubation time fibrosis-CLPF always displayed increased migration ability as compared to CD-CLPF. In contrast, fistula-CLPF migratory potential was always below that of CD-CLPF. Conclusions: Myofibroblasts isolated from inflamed, fibrostenotic, or fistulized CD mucosa differ in their migratory potential both in the modified Boyden chamber as well as in a 3D matrix model. These different migratory behaviors could be an explanation for impaired or excess wound healing and subsequently for fistula and fibrosis formation. (Inflamm Bowel Dis 2011;)

Background/Aims: The non-lysosomal glucosylceramidase, β-glucosidase (Gba2), hydrolyzes glucosylceramide to glucose and ceramide (Cer). Cer is a potent second-messenger lipid that plays an important role in signaling cascades involved in apoptosis. The aim of this study was to investigate whether Gba2 knock-out (Gba2–/–) affects the extent of dextran sulfate sodium (DSS)-induced colitis in mice. Methods: Acute colitis was induced in wild-type (WT) and Gba2–/– mice by administration of 2% DSS in drinking water. After 7 days, mice underwent colonoscopy and were sacrificed. Results: Both DSS-treated WT (n = 10) and Gba2–/– (n = 12) mice showed elevated histological and endoscopic scores compared to respective H2O controls (n = 9 each). However, no significant differences between the DSS groups were detected. Flow cytometric analysis of propidium iodide staining, cleavage of caspases-3 and -8, indicative for apoptosis, as well as Cer levels were not altered in DSS-treated WT or Gba2–/– mice. Gba2–/– resulted in slightly decreased expression of glucocerebrosidase (Gba1) as well as in upregulation of proteins being involved in cellular regeneration, such as STAT3 (signal transducer and activator of transcription), JNK and iNOS, upon DSS treatment. Conclusion: We demonstrate that Gba2–/– does not affect the extent of DSS-induced inflammation in mice, however, it might be involved in tissue regeneration in response to toxic agents.

Objective The Crohn9s disease (CD) susceptibility gene, protein tyrosine phosphatase N2 (PTPN2), regulates interferon γ (IFNγ)-induced signalling and epithelial barrier function in T 84 intestinal epithelial cells (IECs). The aim of this study was to investigate whether PTPN2 is also regulated by tumour necrosis factor α (TNFα) and if PTPN2 controls TNFα-induced signalling and effects in IECs. Methods T 84 IECs were used for all cell studies. Protein levels were assessed by western blotting, mRNA levels by reverse transcription–PCR (RT–PCR) and cytokine levels by ELISA. PTPN2 knock-down was induced by small interfering RNA (siRNA). Imaging was performed by immunohistochemistry or immunofluorescence. Results TNFα treatment elevated PTPN2 mRNA as well as nuclear and cytoplasmic protein levels and caused cytoplasmic accumulation of PTPN2. Biopsy specimens from patients with active CD showed strong immunohistochemical PTPN2 staining in the epithelium, whereas samples from patients with CD in remission featured PTPN2 levels similar to controls without inflammatory bowel disease (IBD). Though samples from patients with active ulcerative colitis (UC) revealed more PTPN2 protein than non-IBD patients and patients with UC in remission, their PTPN2 expression was lower than in active CD. Samples from patients with CD in remission and responding to anti-TNF treatment also showed PTPN2 levels that were similar to those in control patients. Pharmacological inhibition of nuclear factor-κB (NF-κB) by BMS-345541 prevented the TNFα-induced rise in PTPN2 protein, independent of apoptotic events. PTPN2 knock-down revealed that the phosphatase regulates TNFα-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 phosphorylation, without affecting c-Jun N-terminal kinase (JNK), inhibitor of κB (IκB) or NF-κB phosphorylation. Loss of PTPN2 potentiated TNFα-induced secretion of interleukin 6 (IL-6) and IL-8. In TNFα- and IFNγ-co-treated cells, loss of PTPN2 enhanced protein expression of inducible nitric oxide synthase (iNOS). Conclusions TNFα induces PTPN2 expression in IECs. Loss of PTPN2 promotes TNFα-induced mitogen-activated protein kinase signalling and the induction of inflammatory mediators. These data indicate that PTPN2 activity could play a crucial role in the establishment of chronic inflammatory conditions in the intestine, such as CD.

Background: Environmental factors are thought to play an important role in the development of Crohn’s disease (CD). Immune responses against auto-antigens or food antigens may be a reason for the perpetuation of inflammation. Methods: In a pilot study, 79 CD patients and 20 healthy controls were examined for food immunoglobulin G (IgG). Thereafter, the clinical relevance of these food IgG antibodies was assessed in a double-blind cross-over study with 40 patients. Based on the IgG antibodies, a nutritional intervention was planned. The interferon (IFN)γ secretion of T cells was measured. Eosinophil-derived neurotoxin was quantified in stool. Results: The pilot study resulted in a significant difference of IgG antibodies in serum between CD patients and healthy controls. In 84 and 83% of the patients, respectively, IgG antibodies against processed cheese and yeast were detected. The daily stool frequency significantly decreased by 11% during a specific diet compared with a sham diet. Abdominal pain reduced and general well-being improved. IFNγ secretion of T cells increased. No difference for eosinophil-derived neurotoxin in stool was detected. Conclusion: A nutritional intervention based on circulating IgG antibodies against food antigens showed effects with respect to stool frequency. The mechanisms by which IgG antibodies might contribute to disease activity remain to be elucidated.

Background Cholangiocarcinoma (CC) is a malignant neoplasm of the bile ducts or the gallbladder. Targeting of growth factor receptors showed therapeutic potential in palliative settings for many solid tumors. The aim of this study was to determine the expression of seven growth factor receptors in CC cell lines and to assess the effect of blocking the EGFR receptor in vitro. Methods Expression of EGFR (epithelial growth factor receptor), HGFR (hepatocyte growth factor receptor) IGF1R (insulin-like growth factor 1 receptor), IGF2R (insulin-like growth factor 2 receptor) and VEGFR1-3 (vascular endothelial growth factor receptor 1-3) were examined in four human CC cell lines (EGI-1, HuH28, OZ and TFK-1). The effect of the anti-EGFR-antibody cetuximab on cell growth and apoptosis was studied and cell lines were examined for KRAS mutations. Results EGFR, HGFR and IGFR1 were present in all four cell lines tested. IGFR2 expression was confirmed in EGI-1 and TFK-1. No growth-inhibitory effect was found in EGI-1 cells after incubation with cetuximab. Cetuximab dose-dependently inhibited growth in TFK-1. Increased apoptosis was only seen in TFK-1 cells at the highest cetuximab dose tested (1 mg/ml), with no dose-response-relationship at lower concentrations. In EGI-1 a heterozygous KRAS mutation was found in codon 12 (c.35G>A; p.G12D). HuH28, OZ and TFK-1 lacked KRAS mutation. Conclusion CC cell lines express a pattern of different growth receptors in vitro. Growth factor inhibitor treatment could be affected from the KRAS genotype in CC. The expression of EGFR itself does not allow prognoses on growth inhibition by cetuximab.

Background: A frequent complication of Crohn's disease (CD) is the formation of strictures and stenoses. Strictures are characterized by a fibrosis of the bowel wall, induced by abnormal wound healing. Functional changes of colonic lamina propria fibroblasts (CLPF) reflected by increased proliferation and collagen synthesis, increased contractility or reduced migratory potential, indicate a change of the phenotype. We aimed to investigate differences in gene expression profiles between CLPF isolated from normal, inflamed and strictured areas of CD patients. Methods: We applied two methods of gene expression analysis, subtractive hybridisation and Affimetrix® microarrays to find differences in mRNA expression patterns. Findings were verified by dot blot analysis. Results: Using subtractive screening and dot blot analysis 74 clones could be confirmed to be differentially expressed in CD CLPF from nonstrictured areas compared to control CLPF. Fibronectin (transcript variant 1, NM_002026) could be confirmed as being upregulated in CD with a ratio of 143. Collagen (type I, NM_000089) was upregulated in CD with a ratio of 17. 41 clones could be confirmed as differentially expressed in CD CLPF derived from strictures compared to control CLPF. Five clones were identified as chitinase 3-like 1 (cartilage glycoprotein-39) and confirmed with dot blot with a ratio of 2.1. In an independent approach, microarray analysis showed upregulation of chitinase 3-like 1 (signal log ratio 1.9) in CD CLPF from strictures compared to control CLPF thus confirming subtractive hybridization. Conclusions: In the light of the current literature a number of interesting candidates resulted from the multiplicity of identified genes. In regard to the functional changes of CLPF during stenosis and other dysfunctions some proteins might represent a therapeutic target.

Summary Haem oxygenase-1 (HO-1) up-regulation was suggested to reduce mucosal tissue damage in inflammatory bowel disease (IBD) and an up-regulation of HO-1 expression in patients with Crohn's disease (CD) and ulcerative colitis (UC) was demonstrated. A HO-1 gene promoter microsatellite (GT)n dinucleotide repeat polymorphism was associated with regulation of HO-1 in response to inflammatory stimuli. We therefore hypothesized that IBD patients might segregate into phenotypes with high or low HO-1 inducibility. Ethylenediamine tetraacetic acid blood samples were obtained from 179 CD patients, 110 UC patients and 56 control patients without inflammation. Genomic DNA was purified and the 5′-flanking region of the HO-1 gene containing the (GT)n dinucleotide repeat was amplified. Polymerase chain reaction (PCR) products were purified and the length of the PCR fragments was analysed. The number of (GT)n repeats in the population studied ranged from 13 to 42. The distribution of the allele frequencies was comparable in patients and controls for both the short and the long alleles. The frequencies of short-, middle- and long-sized alleles were not changed among the groups studied. No correlation was found between IBD and microsatellite instability detected in five individals. Our data indicate that (GT)n dinucleotide repeats of the HO-1 promotor region have no significance for the pathophysiology and disease course of IBD.

IL-15, a T-cell growth factor, has been shown to be increased in inflammatory bowel disease (IBD). It has been suggested that neutralization of IL-15 could protect from T cell-dependent autoimmune inflammation. On the other hand, an anti-apoptotic effect of IL-15 has been demonstrated in kidney epithelial cells during nephritis. We therefore tested the role of IL-15 in two different experimental models of colitis in vivo, and in models of intestinal epithelial cell (IEC) apoptosis in vitro. IL-15 blockade in chronic dextran sulphate sodium-induced colitis resulted in aggravation of the disease with a significantly 2.1-fold increased epithelial damage score compared to controls. TUNEL staining clearly revealed increased apoptosis. IL-6, TNF and IFN-gamma secretion by mesenteric lymph node cells were increased. In the T cell-dependent SCID transfer model of colitis IL-15 neutralization reduced the inflammatory infiltration and proinflammatory cytokine production. Despite that, the intestinal epithelial damage was not reduced. In vitro, IL-15 pre-incubation prevented up to 75% of CH11 antibody-induced apoptosis in SW-480 cells and reduced caspase-3 activity. According to this, endogenously produced IL-15 in chronic colitis does not only act as a proinflammatory cytokine but has at the same time the potential to reduce mucosal damage by preventing IEC apoptosis.

Background: The heat shock protein gp96 is an endoplasmic reticulum chaperone for multiple protein substrates. We showed that a lack of gp96 in intestinal macrophages of Crohn's Disease (CD) patients is correlated with loss of tolerance against the host gut flora, leading to chronic inflammation. A recent manuscript suggested gp96 to be the major chaperone for TLRs in murine macrophages. Therefore, we studied the impact of gp96 knockdown on TLR function in the monocytic cell line Mono Mac 6 (MM6). Methods: For stable gp96 knockdown a lentiviral system was used. Stimulations were performed with lipopolysaccharide (LPS), a ligand of TLR 4. Internalization was shown using FITC-labeled LPS. TLR folding and functionality was investigated by Western blotting and flow cytometric analysis. IL-8 secretion upon stimulation with LPS was determined by ELISA. Results: Flow cytometric analysis of TLR 2 and 4 showed similar patterns on the cell surface of gp96knockdown cells as well as on control MM6 cells. Internalization of LPS by gp96-knockdown cells was shown using a FITC-labeled derivative. No reduction by gp96 knockdown was observed. Western blot analysis of phospho-IκB/IκB and phospho-NFκB/NFκB ratios did not reveal a significant difference in TLR mediated NFκB signaling between gp96 knockdown and control cells. Induction of IL-8 secretion upon stimulation with LPS was comparable between knockdown and control cells. Conclusions: In contrast to recent reports loss of gp96 does not have pivotal effects on functionality of TLRs in human MM6 cells. These findings indicate that the loss of tolerance against commensal gut flora is caused by different mechanisms yet to be identified.

Background: Mutations within the gene loci, encoding protein tyrosine phosphatase nonreceptor type 2 (PTPN2) and nucleotide oligomerization domain 2 (NOD2), have been associated with Crohn's disease (CD). We have recently shown that PTPN2 limits proinflammatory effects in human intestinal epithelial cells (IEC) and monocytes. A dysregulated immune response to bacterial antigens, such as the NOD2-ligand, muramyl-dipeptide (MDP), has been strongly implicated in the pathogenesis of CD. The transcription factor T-bet is upregulated in NOD2-deficient mice, activated during CD and controls the secretion of proinflammatory cytokines, such as IFNγ. Here, we studied whether PTPN2 controls NOD2mediated effects in human THP-1 monocytes. Methods: Protein analysis was performed by Western blotting, mRNA analysis by real-time PCR, cytokine secretion by ELISA and visual imaging by immunofluorescence (IF) studies. PTPN2 or NOD2 knock-down was induced by siRNA. Primary intestinal colonic lamina propria fibroblasts (CLPF) were isolated from surgical specimens derived from patients with active CD (n=5) and were genotyped for the CD-associated PTPN2 variant. Results: MDP (100 ng/ml) increased PTPN2 mRNA (p

DSS-induced histopathology of individual (or aggregate) scores for inflammation, crypt damage, ulcerations indicates less mucosal damage in A3AR mice than WT (p

Introduction: CC is a rare carcinoma of the biliary epithelium. Standard chemotherapy has little effect on CC. Growth factors and their receptors play an important role in tumor growth and metastasis. Neutralizing antibodies targeting growth factors and their receptors have been shown to be therapeutically effective. We therefore determined the expression of 7 growth factors in 4 human CC cell lines. The effect of monoclonal EGFR antibody on two human CC cell lines was tested. Methods: mRNA expression was studied for EGFR, VEGFR1, VEGFR2, VEGFR3, IGF1R, IGF2R and HGFR in the four human CC cell lines TFK-1, EGI-1, OZ and HuH28. Protein expression of these molecules was investigated by Western blot and immunohistochemistry. The effects of 0.1, 1.0, 10, 100 and 1000 mg/ml monoclonal anti-EGFR antibody (cetuximab, ErbituxO, Merck) were tested In Vitro in the two cell lines TFK-1 and EGI-1. Cell growth was analyzed by cell counting, cell metabolism by XTT assay and apoptosis by FACS. Results: mRNA and protein expression of all seven growth factor receptors was found in all four human CC cell lines by PCR, Western blot and immunohistochemistry. Cetuximab significantly inhibited cell growth of TFK-1 in a dose dependent manner (p < 0.05) but had no influence on EGI-1 cells. The metabolic activity of TFK-1 cells treated with cetuximab was increased dose dependently, however, decreased significantly over time. For EGI-1 cells metabolic activity remained largely unchanged. TFK-1 but not EGI-1 showed increased rates of apoptosis (p < 0.05). Results: Cetuximab has different major effects on cell growth, cell metabolism and apoptosis on human CC cell lines TFK-1 and EGI-1. Cetuximab inhibits TFK-1 cell growth and increased apoptosis. These effects are not related to the expression of growth factor receptors.

BACKGROUND: The single nucleotide polymorphism (SNP) rs2542151 within the gene locus region encoding protein tyrosine phosphatase non receptor type 2 (PTPN2) has been associated with Crohn's disease (CD) ulcerative colitis (UC) type I diabetes and rheumatoid arthritis. We have previously shown that PTPN2 regulates mitogen activated protein kinase (MAPK) signaling and cytokine secretion in human THP 1 monocytes and intestinal epithelial cells (IEC). Here we studied whether intronic PTPN2 SNP rs1893217 regulates immune responses to the nucleotide oligomerization domain 2 (NOD2) ligand muramyl dipeptide (MDP). MATERIALS AND METHODS: Genomic DNA samples from 343 CD and 663 non IBD control patients (male and female) from a combined German Swiss and Polish cohort were genotyped for the presence of the PTPN2 SNPs rs2542151 and rs1893217. PTPN2 variant rs1893217 was introduced into T(84) IEC or THP 1 cells using a lentiviral vector. RESULTS: We identified a novel association between the genetic variant rs1893217 located in intron 7 of the PTPN2 gene and CD. Human THP 1 monocytes carrying this variant revealed increased MAPK activation as well as elevated mRNA expression of T bet transcription factor and secretion of interferon ? in response to the bacterial wall component MDP. In contrast secretion of interleukin 8 and tumor necrosis factor were reduced. In both T(84) IEC and THP 1 monocytes autophagosome formation was impaired. CONCLUSIONS: We identified a novel CD associated PTPN2 variant that modulates innate immune responses to bacterial antigens. These findings not only provide key insights into the effects of a functional mutation on a clinically relevant gene but also reveal how such a mutation could contribute to the onset of disease. (Inflamm Bowel Dis 2011;).