Jan Wehkamp

Publications of Jan Wehkamp

• Association of a Functional Variant in the Wnt Co-Receptor LRP6 with Early Onset Ileal Crohn's Disease.

  Authors: Maureen J Koslowski, Zora Teltschik, Julia Beisner, Elke Schaeffeler, Guoxing Wang, Irmgard Kübler, Michael Gersemann, Rachel Cooney, Derek Jewell, Walter Reinisch, Séverine Vermeire, Paul Rutgeerts, Matthias Schwab, Eduard F Stange, Jan Wehkamp

  PLoS genetics. 02/2012; 8(2):e1002523.

  Ileal Crohn's Disease (CD), a chronic small intestinal inflammatory disorder, is characterized by reduced levels of the antimicrobial peptides DEFA5 (HD-5) and DEFA6 (HD-6). Both of these α-defensinsIleal Crohn's Disease (CD), a chronic small intestinal inflammatory disorder, is characterized by reduced levels of the antimicrobial peptides DEFA5 (HD-5) and DEFA6 (HD-6). Both of these α-defensins are exclusively produced in Paneth cells (PCs) at small intestinal crypt bases. Different ileal CD-associated genes including NOD2, ATG16L1, and recently the β-catenin-dependant Wnt transcription factor TCF7L2 have been linked to impaired PC antimicrobial function. The Wnt pathway influences gut mucosal homeostasis and PC maturation, besides directly controlling HD-5/6 gene expression. The herein reported candidate gene study focuses on another crucial Wnt factor, the co-receptor low density lipoprotein receptor-related protein 6 (LRP6). We analysed exonic single nucleotide polymorphisms (SNPs) in a large cohort (Oxford: n = 1,893) and prospectively tested 2 additional European sample sets (Leuven: n = 688, Vienna: n = 1,628). We revealed an association of a non-synonymous SNP (rs2302685; Ile1062Val) with early onset ileal CD (OR 1.8; p = 0.00034; for homozygous carriers: OR 4.1; p = 0.00004) and additionally with penetrating ileal CD behaviour (OR 1.3; p = 0.00917). In contrast, it was not linked to adult onset ileal CD, colonic CD, or ulcerative colitis. Since the rare variant is known to impair LRP6 activity, we investigated its role in patient mucosa. Overall, LRP6 mRNA was diminished in patients independently from the genotype. Analysing the mRNA levels of PC product in biopsies from genotyped individuals (15 controls, 32 ileal, and 12 exclusively colonic CD), we found particularly low defensin levels in ileal CD patients who were carrying the variant. In addition, we confirmed a direct relationship between LRP6 activity and the transcriptional expression of HD-5 using transient transfection. Taken together, we identified LRP6 as a new candidate gene in ileal CD. Impairments in Wnt signalling and Paneth cell biology seem to represent pathophysiological hallmarks in small intestinal inflammation and should therefore be considered as interesting targets for new therapeutic approaches.

• Intestinal bacterial translocation in rats with cirrhosis is related to compromised paneth cell antimicrobial host defense.

  Authors: Zora Teltschik, Reiner Wiest, Julia Beisner, Sabine Nuding, Claudia Hofmann, Juergen Schoelmerich, Charles L Bevins, Eduard F Stange, Jan Wehkamp

  Hepatology (Baltimore, Md.). 11/2011;

  Liver cirrhosis is associated with bacterial translocation (BT) and endotoxemia. Most translocating bacteria belong to the common intestinal microbiota, suggesting a breakdown of intestinal barrierLiver cirrhosis is associated with bacterial translocation (BT) and endotoxemia. Most translocating bacteria belong to the common intestinal microbiota, suggesting a breakdown of intestinal barrier function. We hypothesized that diminished mucosal antimicrobial host defense could predispose to BT. Two rodent models of portal hypertension with increased BT were used, CCl(4) -induced ascitic cirrhosis and 2-day portal vein-ligated (PVL) animals. BT was assessed by standard microbiological techniques on mesenteric lymph nodes. Total RNA was isolated systematically throughout the intestinal tract, and expression of Paneth cell α-cryptdins and β-defensins was determined by real-time quantitative polymerase chain reaction (qPCR). To determine functional consequences, mucosal antimicrobial activity was assessed with a fluorescence-activated cell sorting assay. BT was detectable in 40% of rats with cirrhosis. Compared with the group without BT, these animals exhibited diminished intestinal Paneth cell α-cryptdin 5 and 7 expression. In contrast, PVL was associated with BT in all animals but did not affect antimicrobial peptides. The decrease in Paneth cell antimicrobials was most pronounced in the ileum and the coecum. Other antimicrobials showed no changes or even an induction in the case of BT at different sites. Antimicrobial activity toward different commensal strains was reduced, especially in the distal ileum and the cecum in experimental cirrhosis with BT (excluding PVL). Conclusion: Compromised Paneth cell antimicrobial host defense seems to predispose to BT in experimental cirrhosis. Understanding this liver-gut axis including the underlying mechanisms could help us to find new treatment avenues. (HEPATOLOGY 2011).

• Olfactomedin-4 is a glycoprotein secreted into mucus in active IBD.

  Authors: Michael Gersemann, Svetlana Becker, Sabine Nuding, Lena Antoni, German Ott, Peter Fritz, Naohide Oue, Wataru Yasui, Jan Wehkamp, Eduard F Stange

  Journal of Crohn's & colitis. 11/2011;

  BACKGROUND: Olfactomedin-4 (OLFM4) is a glycoprotein characteristic of intestinal stem cells and apparently involved in mucosal defense of the stomach and colon. Here we studied its expression,BACKGROUND: Olfactomedin-4 (OLFM4) is a glycoprotein characteristic of intestinal stem cells and apparently involved in mucosal defense of the stomach and colon. Here we studied its expression, regulation and function in IBD. METHODS: The expression of OLFM4, mucins Muc1 and Muc2, the goblet cell differentiation factor Hath1 and the proinflammatory cytokine IL-8 was measured in inflamed or noninflamed colon in IBD patients and controls. OLFM4 protein was located by immunohistochemistry, quantified by Dot Blot and its binding capacity to defensins HBD1-3 was investigated. The influence of bacteria with or without the Notch blocker dibenzazepine (DBZ) and of several cytokines on OLFM4 expression was determined in LS174T cells. RESULTS: OLFM4 mRNA and protein were significantly upregulated in inflamed CD (4.3 and 1.7-fold) and even more pronounced in UC (24.8 and 3.7-fold). OLFM4 expression was correlated to IL-8 but not to Hath1. In controls immunostaining was restricted to the lower crypts but in inflamed IBD it expanded up to the epithelial surface including the mucus. OLFM4 bound to HBD1-3 without profoundly inactivating these defensins. In LS174T-cells OLFM4 mRNA was significantly augmented after incubation with Escherichia coli K12, Escherichia coli Nissle and Bacteroides vulgatus. DBZ downregulated OLFM4 expression and blocked bacterial induction whereas IL-22 but not TNF-α was stimulatory. CONCLUSIONS: OLFM4 is overexpressed in active IBD and secreted into mucus. The induction is triggered by bacteria through the Notch pathway and also by the cytokine IL-22. OLFM4 seems to be of functional relevance in IBD as a mucus component, possibly by binding defensins.

• From intestinal stem cells to inflammatory bowel diseases.

  Authors: Michael Gersemann, Eduard Friedrich Stange, Jan Wehkamp

  World journal of gastroenterology : WJG. 07/2011; 17(27):3198-203.

  The pathogenesis of both entities of inflammatory bowel disease (IBD), namely Crohn's disease (CD) and ulcerative colitis (UC), is still complex and under investigation. The importance of theThe pathogenesis of both entities of inflammatory bowel disease (IBD), namely Crohn's disease (CD) and ulcerative colitis (UC), is still complex and under investigation. The importance of the microbial flora in developing IBD is beyond debate. In the last few years, the focus has changed from adaptive towards innate immunity. Crohn's ileitis is associated with a deficiency of the antimicrobial shield, as shown by a reduced expression and secretion of the Paneth cell defensin HD5 and HD6, which is related to a Paneth cell differentiation defect mediated by a diminished expression of the Wnt transcription factor TCF4. In UC, the protective mucus layer, acting as a physical and chemical barrier between the gut epithelium and the luminal microbes, is thinner and in part denuded as compared to controls. This could be caused by a missing induction of the goblet cell differentiation factors Hath1 and KLF4 leading to immature goblet cells. This defective Paneth and goblet cell differentiation in Crohn's ileitis and UC may enable the luminal microbes to invade the mucosa and trigger the inflammation. The exact molecular mechanisms behind ileal CD and also UC must be further clarified, but these observations could give rise to new therapeutic strategies based on a stimulation of the protective innate immune system.

• Reduction of disulphide bonds unmasks potent antimicrobial activity of human beta-defensin 1

  Authors: Bjoern O. Schroeder, Zhihong Wu, Sabine Nuding, Sandra Groscurth, Moritz Marcinowski, Julia Beisner, Johannes Buchner, Martin Schaller, Eduard F. Stange, Jan Wehkamp

  Nature. 01/2011; 469:419-423.

  Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal,Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal, reaching 99% of bacterial species in the colon. At colonic mucosa, oxygen partial pressure is below 25% of airborne oxygen content, moreover microbial metabolism causes reduction to a low redox potential of −200 mV to –300 mV in the colon. Defensins, characterized by three intramolecular disulphide-bridges, are key effector molecules of innate immunity that protect the host from infectious microbes and shape the composition of microbiota at mucosal surfaces. Human β-defensin 1 (hBD-1) is one of the most prominent peptides of its class but despite ubiquitous expression by all human epithelia, comparison with other defensins suggested only minor antibiotic killing activity. Whereas much is known about the activity of antimicrobial peptides in aerobic environments, data about reducing environments are limited. Herein we show that after reduction of disulphide-bridges hBD-1 becomes a potent antimicrobial peptide against the opportunistic pathogenic fungus Candida albicans and against anaerobic, Gram-positive commensals of Bifidobacterium and Lactobacillus species. Reduced hBD-1 differs structurally from oxidized hBD-1 and free cysteines in the carboxy terminus seem important for the bactericidal effect. In vitro, the thioredoxin (TRX) system is able to reduce hBD-1 and TRX co-localizes with reduced hBD-1 in human epithelia. Hence our study indicates that reduced hBD-1 shields the healthy epithelium against colonisation by commensal bacteria and opportunistic fungi. Accordingly, an intimate interplay between redox-regulation and innate immune defence seems crucial for an effective barrier protecting human epithelia.

• Reduction of disulphide bonds unmasks potent antimicrobial activity of human beta-defensin 1

  Authors: Bjoern O. Schroeder, Zhihong Wu, Sabine Nuding, Sandra Groscurth, Moritz Marcinowski, Julia Beisner, Johannes Buchner, Martin Schaller, Eduard F. Stange, Jan Wehkamp

  Keystone Symposium, Mucosal Biology: A Fine Balance between Tolerance and Immunity, Vancouver, British Columbia; 01/2011

• Reduction of disulphide bonds unmasks potent antimicrobial activity of human β-defensin 1.

  Authors: Bjoern O Schroeder, Zhihong Wu, Sabine Nuding, Sandra Groscurth, Moritz Marcinowski, Julia Beisner, Johannes Buchner, Martin Schaller, Eduard F Stange, Jan Wehkamp

  Nature. 01/2011; 469(7330):419-23.

  Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal,Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal, reaching 99% of bacterial species in the colon. At colonic mucosa, oxygen partial pressure is below 25% of airborne oxygen content, moreover microbial metabolism causes reduction to a low redox potential of -200 mV to -300 mV in the colon. Defensins, characterized by three intramolecular disulphide-bridges, are key effector molecules of innate immunity that protect the host from infectious microbes and shape the composition of microbiota at mucosal surfaces. Human β-defensin 1 (hBD-1) is one of the most prominent peptides of its class but despite ubiquitous expression by all human epithelia, comparison with other defensins suggested only minor antibiotic killing activity. Whereas much is known about the activity of antimicrobial peptides in aerobic environments, data about reducing environments are limited. Herein we show that after reduction of disulphide-bridges hBD-1 becomes a potent antimicrobial peptide against the opportunistic pathogenic fungus Candida albicans and against anaerobic, Gram-positive commensals of Bifidobacterium and Lactobacillus species. Reduced hBD-1 differs structurally from oxidized hBD-1 and free cysteines in the carboxy terminus seem important for the bactericidal effect. In vitro, the thioredoxin (TRX) system is able to reduce hBD-1 and TRX co-localizes with reduced hBD-1 in human epithelia. Hence our study indicates that reduced hBD-1 shields the healthy epithelium against colonisation by commensal bacteria and opportunistic fungi. Accordingly, an intimate interplay between redox-regulation and innate immune defence seems crucial for an effective barrier protecting human epithelia.

• Paneth's disease.

  Authors: Jan Wehkamp, Eduard F Stange

  Journal of Crohn's & colitis. 11/2010; 4(5):523-31.

  In about 70% of patients Crohn's disease (CD) affects the small intestine. This disease location is stable over time and associated with a genetic background different from isolated colonic disease.In about 70% of patients Crohn's disease (CD) affects the small intestine. This disease location is stable over time and associated with a genetic background different from isolated colonic disease. A characteristic feature of small intestinal host defense is the presence of Paneth cells at the bottom of the crypts of Lieberkühn. These cells produce different broad spectrum antimicrobial peptides (AMPs) most abundantly the α-defensins HD-5 and -6 (DEFA5 und DEFA6). In small intestinal Crohn's disease both these PC products are specifically reduced. As a functional consequence, ileal extracts from Crohn's disease patients are compromised in clearing bacteria and enteroadherent E. coli colonize the mucosa. Mechanisms for defective antimicrobial Paneth cell function are complex and include an association with a NOD2 loss of function mutation, a disturbance of the Wnt pathway transcription factor TCF7L2 (also known as TCF4), the autophagy factor ATG16L1, the endosomal stress protein XBP1, the toll-like receptor TLR9, the calcium mediated potassium channel KCNN4 as well as mutations or inactivation of HD5. Thus we conclude that small intestinal Crohn's disease is most likely a complex disease of the Paneth cell: Paneth's disease.

• Innate antimicrobial immunity in inflammatory bowel diseases.

  Authors: Julia Beisner, Eduard F Stange, Jan Wehkamp

  Expert review of clinical immunology. 09/2010; 6(5):809-18.

  Inflammatory bowel diseases are characterized by chronic intestinal inflammation at different sites. Data from animal models as well as human patients including gene-association studies suggest thatInflammatory bowel diseases are characterized by chronic intestinal inflammation at different sites. Data from animal models as well as human patients including gene-association studies suggest that different components of the innate barrier function are primarily defective. These recent advances support the evolving hypothesis that intestinal bacteria induce inflammation predominantly as a result of a weakened innate mucosal barrier in genetically predisposed individuals. This article discusses our current understanding of the primary events of disease. Together, these findings should result in new therapeutic avenues aimed at restoring antimicrobial barrier function to prevent a bacterial-triggered inflammatory response.

• Peroxisome proliferator-activated receptor gamma activation is required for maintenance of innate antimicrobial immunity in the colon.

  Authors: Laurent Peyrin-Biroulet, Julia Beisner, Guoxing Wang, Sabine Nuding, Sajit Thottathil Oommen, Denise Kelly, Erika Parmentier-Decrucq, Rodrigue Dessein, Emilie Merour, Philipe Chavatte, Teddy Grandjean, Aude Bressenot, Pierre Desreumaux, Jean-Frédéric Colombel, Béatrice Desvergne, Eduard F Stange, Jan Wehkamp, Mathias Chamaillard

  Proceedings of the National Academy of Sciences of the United States of America. 05/2010; 107(19):8772-7.

  Crohn's disease (CD), a major form of human inflammatory bowel disease, is characterized by primary immunodeficiencies. The nuclear receptor peroxisome proliferator-activated receptor gammaCrohn's disease (CD), a major form of human inflammatory bowel disease, is characterized by primary immunodeficiencies. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is essential for intestinal homeostasis in response to both dietary- and microbiota-derived signals. Its role in host defense remains unknown, however. We show that PPARgamma functions as an antimicrobial factor by maintaining constitutive epithelial expression of a subset of beta-defensin in the colon, which includes mDefB10 in mice and DEFB1 in humans. Colonic mucosa of Ppargamma mutant animals shows defective killing of several major components of the intestinal microbiota, including Candida albicans, Bacteroides fragilis, Enterococcus faecalis, and Escherichia coli. Neutralization of the colicidal activity using an anti-mDefB10 blocking antibody was effective in a PPARgamma-dependent manner. A functional promoter variant that is required for DEFB1 expression confers strong protection against Crohn's colitis and ileocolitis (odds ratio, 0.559; P = 0.018). Consistently, colonic involvement in CD is specifically linked to reduced expression of DEFB1 independent of inflammation. These findings support the development of PPARgamma-targeting therapeutic and/or nutritional approaches to prevent colonic inflammation by restoring antimicrobial immunity in CD.

• Paneth cell function--implications in pediatric Crohn disease.

  Authors: Julia Beisner, Eduard F Stange, Jan Wehkamp

  Gut microbes. 01/2010; 2(1):47-51.

  Defects in the intestinal barrier play a central role in disease pathogenesis. Recently we have demonstrated that children with ileal Crohn's disease show a reduced expression of small intestinalDefects in the intestinal barrier play a central role in disease pathogenesis. Recently we have demonstrated that children with ileal Crohn's disease show a reduced expression of small intestinal HD-5 at the age of onset suggesting that a compromised mucosal barrier function might be a key factor in the early disease pathogenesis. We also identified a disturbance of the Wnt signaling transcription factor TCF-4 as a major mechanism for this deficiency in children which might result in a compromised innate immune function of small intestinal Paneth cells via defensin secretion. Here we provide a summary on our recent findings and discuss the data in more detail especially focusing on the role of Paneth cell differentiation and function in the pathogenesis of pediatric ileal Crohn's disease.

• Antimicrobial peptides in gastrointestinal inflammation.

  Authors: Simon Jäger, Eduard F Stange, Jan Wehkamp

  International journal of inflammation. 01/2010; 2010:910283.

  Acute and chronic inflammations of mucosal surfaces are complex events in which the effector mechanisms of innate and adaptive immune systems interact with pathogenic and commensal bacteria. The roleAcute and chronic inflammations of mucosal surfaces are complex events in which the effector mechanisms of innate and adaptive immune systems interact with pathogenic and commensal bacteria. The role of constitutive and inducible antimicrobial peptides in intestinal inflammation has been investigated thoroughly over the recent years, and their involvement in various disease states is expanded ever more. Especially in the intestines, a critical balance between luminal bacteria and the antimicrobial peptides is essential, and a breakdown in barrier function by impaired production of defensins is already implicated in Crohn's disease. In this paper, we focus on the role of antimicrobial peptides in inflammatory processes along the gastrointestinal tract, while considering the resident and pathogenic flora encountered at the specific sites. The role of antimicrobial peptides in the primary events of inflammatory bowel diseases receives special attention.

• Defective Paneth Cell-Mediated Host Defense in Pediatric Ileal Crohn's Disease.

  Authors: Gøri Perminow, Julia Beisner, Maureen Koslowski, Lars Gustav Lyckander, Eduard Stange, Morten H Vatn, Jan Wehkamp

  The American journal of gastroenterology. 11/2009;

  OBJECTIVES:Adult ileal Crohn's disease (CD) is characterized by a specific decrease in ileal Paneth cell alpha-defensins. In addition to NOD2, we previously identified a disturbance of theOBJECTIVES:Adult ileal Crohn's disease (CD) is characterized by a specific decrease in ileal Paneth cell alpha-defensins. In addition to NOD2, we previously identified a disturbance of the Wnt-signaling transcription factor TCF-4 as a major mechanism for this deficiency. The aim of this study was to evaluate human alpha-defensin-5 (HD-5) and TCF-4 in an independent cohort of pediatric CD patients.METHODS:Expression levels of HD-5 and TCF-4 mRNA were quantified by real-time PCR in biopsies from newly diagnosed untreated pediatric CD patients (<18 years, n=36) and age-matched symptomatic non-inflammatory bowel disease controls with a histologically normal gut (n=29). To assess the influence of current inflammation, mucosal interleukin-8 (IL-8) and fecal calprotectin levels were determined.RESULTS:Small intestinal HD-5 and TCF-4 mRNA were significantly reduced in pediatric ileal CD (L1+L3) (P=0.022 and P=0.0005, respectively) and were significantly correlated (r=0.499; P=0.0001). In ileal but not colonic CD, TCF-4 was also reduced in the colon (P=0.005). Importantly, both HD-5 and TCF-4 were independent of inflammation, as measured by IL-8 expression or fecal calprotectin. In contrast to the small intestine, colonic Paneth cell HD-5 mRNA was significantly elevated in colonic CD (L2) (P=0.026) and was correlated with fecal calprotectin levels (r=0.481; P=0.020).CONCLUSIONS:In this study, we describe a specific decrease in HD-5 and TCF-4 mRNA expression levels in children with ileal CD. In the small intestine, this decrease was independent of current inflammation, whereas inflammation seems to induce Paneth cell metaplasia in the colon. Our data extend the hypothesis of an important role of antimicrobial host defense in pediatric CD patients.Am J Gastroenterol advance online publication, 10 November 2009; doi:10.1038/ajg.2009.643.

• Innate antimicrobial host defense in small intestinal Crohn's disease.

  Authors: Maureen J Koslowski, Julia Beisner, Eduard F Stange, Jan Wehkamp

  International journal of medical microbiology : IJMM. 10/2009;

  Paneth cells (PCs) are specialized epithelial cells predominantly found in the small intestinal crypts of Lieberkuehn. They produce different broad spectrum antimicrobial peptides most abundantly thePaneth cells (PCs) are specialized epithelial cells predominantly found in the small intestinal crypts of Lieberkuehn. They produce different broad spectrum antimicrobial peptides most abundantly the alpha-defensins HD-5 and -6 (DEFA5 und DEFA6). Both these PC products show a specific reduction in small intestinal Crohn's disease (CD) - a form of inflammatory bowel disease (IBD). Their decrease is independent of current inflammation and an association with a NOD2 frameshift mutation has been demonstrated. More recently, another independent and even more frequent mechanism has been found which is linked to diminished levels of the Wnt pathway transcription factor TCF7L2 (also known as TCF4). Besides regulating the expression of HD-5 and HD-6 as TCF4 target genes, the Wnt pathway also orchestrates Paneth cell differentiation and maturation and controls stem cell maintenance in the small intestine. Besides NOD2 (which is predominantly expressed in PC) and ATG16L1 (inter alia important in the exocytosis of PC products), TCF4 is the third gene which is associated with small intestinal CD and Paneth cell antimicrobial function. Thus, Paneth cells seem to be key player emphazising a paramount importance of antimicrobial host defense in small intestinal CD pathogenesis.

• Antibacterial activity of human defensins on anaerobic intestinal bacterial species: a major role of HBD-3.

  Authors: Sabine Nuding, Lutz T Zabel, Corinne Enders, Edith Porter, Klaus Fellermann, Jan Wehkamp, Holger A G Mueller, Eduard F Stange

  Microbes and infection / Institut Pasteur. 02/2009;

  Defensins are natural mucosal antimicrobial peptides and their broad spectrum activity against aerobic or facultative anaerobic bacteria has been well investigated. The aim of this study was toDefensins are natural mucosal antimicrobial peptides and their broad spectrum activity against aerobic or facultative anaerobic bacteria has been well investigated. The aim of this study was to systematically examine the antibacterial activity of the small intestinal Paneth cell-derived a-defensin HD5 and the major colonic beta-defensins HBD-1 - 3 against strict anaerobic intestinal bacteria. The antibacterial activity was assessed with a flow cytometric assay employing a membrane potential sensitive dye as marker for loss of cell viability. The majority of the tested strains belonging to the dominant anaerobe genera of the gut, Bacteroides and Parabacteroides, were only minimally affected by the constitutively expressed defensins HD5 and HBD1. The inducible defensin HBD-2 had a limited antibacterial effect, whereas the inducible HBD-3 exhibited potent activity against most strains. The effect of HBD-3 on Bacteroides sp. appeared to be dependent on the presence of oxygen. Bacteroides fragilis strains isolated from blood during bacteraemia or from extraintestinal infections were more resistant to HBD-3 than strains from the physiological gut flora. Thus, defensin resistance is not only species but also strain specific and may be clinically relevant in the host-bacterial interaction influencing mucosal translocation and systemic infection.

• Genetic variants of Wnt transcription factor TCF-4 (TCF7L2) putative promoter region are associated with small intestinal Crohn's disease.

  Authors: Maureen J Koslowski, Irmgard Kübler, Mathias Chamaillard, Elke Schaeffeler, Walter Reinisch, Guoxing Wang, Julia Beisner, Alexander Teml, Laurent Peyrin-Biroulet, Stefan Winter, Klaus R Herrlinger, Paul Rutgeerts, Séverine Vermeire, Rachel Cooney, Klaus Fellermann, Derek Jewell, Charles L Bevins, Matthias Schwab, Eduard F Stange, Jan Wehkamp

  PLoS ONE. 02/2009; 4(2):e4496.

  Reduced expression of Paneth cell antimicrobial alpha-defensins, human defensin (HD)-5 and -6, characterizes Crohn's disease (CD) of the ileum. TCF-4 (also named TCF7L2), a Wnt signalling pathwayReduced expression of Paneth cell antimicrobial alpha-defensins, human defensin (HD)-5 and -6, characterizes Crohn's disease (CD) of the ileum. TCF-4 (also named TCF7L2), a Wnt signalling pathway transcription factor, orchestrates Paneth cell differentiation, directly regulates the expression of HD-5 and -6, and was previously associated with the decrease of these antimicrobial peptides in a subset of ileal CD. To investigate a potential genetic association of TCF-4 with ileal CD, we sequenced 2.1 kb of the 5' flanking region of TCF-4 in a small group of ileal CD patients and controls (n = 10 each). We identified eight single nucleotide polymorphisms (SNPs), of which three (rs3814570, rs10885394, rs10885395) were in linkage disequilibrium and found more frequently in patients; one (rs3814570) was thereby located in a predicted regulatory region. We carried out high-throughput analysis of this SNP in three cohorts of inflammatory bowel disease (IBD) patients and controls. Overall 1399 healthy individuals, 785 ulcerative colitis (UC) patients, 225 CD patients with colonic disease only and 784 CD patients with ileal involvement were used to determine frequency distributions. We found an association of rs3814570 with ileal CD but neither with colonic CD or UC, in a combined analysis (allele positivity: OR 1.27, 95% CI 1.07 to 1.52, p = 0.00737), which was the strongest in ileal CD patients with stricturing behaviour (allele frequency: OR 1.32, 95% CI 1.08 to1.62, p = 0.00686) or an additional involvement of the upper GIT (allele frequency: OR 1.38, 95% CI 1.03 to1.84, p = 0.02882). The newly identified genetic association of TCF-4 with ileal CD provides evidence that the decrease in Paneth cell alpha-defensins is a primary factor in disease pathogenesis.

• Differences in goblet cell differentiation between Crohn's disease and ulcerative colitis.

  Authors: Michael Gersemann, Svetlana Becker, Irmgard Kübler, Maureen Koslowski, Guoxing Wang, Klaus Robert Herrlinger, Joscha Griger, Peter Fritz, Klaus Fellermann, Matthias Schwab, Jan Wehkamp, Eduard Friedrich Stange

  Differentiation; research in biological diversity. 01/2009; 77(1):84-94.

  Goblet cells are mucin-secreting intestinal cells forming the mucus layer that protects the mucosal surface. Ulcerative colitis (UC) has been associated with a defective colonic mucus layer and aGoblet cells are mucin-secreting intestinal cells forming the mucus layer that protects the mucosal surface. Ulcerative colitis (UC) has been associated with a defective colonic mucus layer and a reduced number of goblet cells. In experimental animals, colonic goblet cell differentiation is regulated by interacting transcription factors Hath1, KLF4 and the Notch, as well as Wnt pathways, whereas data in humans are limited. We investigated goblet cell differentiation factors and mucins in controls and in inflammatory bowel diseases (IBDs). We performed real-time PCR for Hath1, KLF4, several ligands, receptors and target genes of the Notch and Wnt pathways, as well as several mucins in biopsies from the sigmoid colon of controls (n=21), Crohn's disease (CD, n=48) and UC (n=40). In addition, Hath1 protein was quantitated with Western blot and localized with immunohistochemistry. Notably, the degree of inflammation as measured by IL-8 and histology was similar in both disease entities. The proportion of goblet cells was lowered in both IBDs, but specifically diminished in the upper third of the crypt in UC. Comparable levels of inflammation induced both Hath1 (2.0-fold, p<0.001) and KLF4 (1.8-fold for KLF4, p=0.031) mRNA expression in CD but not in UC (0.8-0.9-fold, ns). The differential induction was confirmed for Hath1 protein using Western blot. Hath1 immunostaining was found mostly in the lower half of the colonic crypts. Hath1, KLF4 and the Notch target gene Hes1 were significantly (p<0.001) and positively correlated. Moreover, both Hath1 and KLF4 were correlated (p<0.001) with MUC1, MUC2 as well as MUC4 in all control and IBD cohorts. The results indicate that both transcription factors are key regulators of goblet cell differentiation and mucin formation in the human colon. Conspicuously, inflammation is associated with an enhanced goblet cell differentiation in CD but not in UC, a defect possibly of pathogenic importance.

• Antimicrobial host defense in the upper gastrointestinal tract.

  Authors: Yoshio Hosaka, Maureen Koslowski, Sabine Nuding, Guoxing Wang, Miriam Schlee, Christian Schäfer, Katunori Saigenji, Eduard F Stange, Jan Wehkamp

  European journal of gastroenterology & hepatology. 01/2009; 20(12):1151-8.

  BACKGROUND: With the exception of fungi, microbial infections are rare in the oesophagus. Herein, we aimed to systematically assess the distribution and quantity of different antimicrobial hostBACKGROUND: With the exception of fungi, microbial infections are rare in the oesophagus. Herein, we aimed to systematically assess the distribution and quantity of different antimicrobial host factors as well as, for the first time, functional mucosal antimicrobial activity in the upper gastrointestinal tract. METHODS: We investigated biopsies from the healthy oesophagus, three different locations in the stomach and the duodenum in a total of 12 individuals. Using real-time PCR with external standards, we compared absolute expression of mRNA encoding antimicrobial peptides including defensins, cathelicidin, bactericidal/permeability-increasing protein, psoriasin, and elafin. In addition, we performed immunostaining for human-beta-defensin-1 (HBD1), elafin, and psoriasin. To test functional relevance, we assessed antimicrobial as well as antifungal activity of cationic extracts from biopsies against E. coli ATCC 25922 and a clinical isolate of Candida albicans. RESULTS: In contrast to HBD1 which was similarly expressed in all tissues, inducible beta-defensins in the healthy oesophagus were much higher compared with the stomach and duodenum (for HBD2-4: P<0.01). In addition, the antiproteases elafin and psoriasin were also predominantly expressed in the oesophagus (P<0.005). In contrast, LL-37 and bactericidal/permeability-increasing protein were only marginally expressed. Cationic tissue extracts from both the oesophagus as well as the stomach showed potent antibacterial activity against E. coli. Consistent with susceptibility to Candida infection, the esophageal extracts exhibited a weaker activity against C. albicans (P=0.026). CONCLUSION: Despite dominant expression of antimicrobial host peptides, oesophageal tissue shows a weakened potency to kill C. albicans. These data suggest an important role of yet unknown antimicrobial molecules.

• Diminished Expression of Apical Sodium-Dependent Bile Acid Transporter in Gallstone Disease Is Independent of Ileal Inflammation.

  Authors: Arthur Holzer, Simone Harsch, Olga Renner, André Strohmeyer, Silke Schimmel, Jan Wehkamp, Peter Fritz, Eduard F Stange

  Digestion. 11/2008; 78(1):52-59.

  Background: Non-obese gallstone patients exhibit a diminished expression of apical sodium-dependent bile acid transporter (ASBT) in terminal ileum. Crohn's ileitis demonstrates a significantBackground: Non-obese gallstone patients exhibit a diminished expression of apical sodium-dependent bile acid transporter (ASBT) in terminal ileum. Crohn's ileitis demonstrates a significant downregulation of this transporter. Aim: To test whether subclinical ileal inflammation contributes to gallstone disease. Methods: Biopsies from terminal ileum of female subjects with gallstone disease (n = 7), active Crohn's disease (n = 17) and controls (n = 22) were investigated. mRNA expression of ASBT, tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-8, c-jun and c-fos was measured. c-jun and c-fos protein levels were determined and hematoxylin and eosin staining was applied for ileal histology. Results: ASBT expression was comparably low both in gallstone (47% of controls, p = 0.0093) and Crohn's disease (42% of controls, p = 0.0008). In gallstone disease there was a non-significant trend towards elevated TNF-alpha and IL-1beta, but all cytokines were increased in active Crohn's disease. c-jun and c-fos were slightly diminished in patients with gallstones. Neither cytokines nor transcription factors correlated significantly with ASBT. The gallstone-associated ileal biopsies exhibited no histological inflammation. Conclusion: Although the expression of ASBT was similarly diminished in both gallstone and Crohn's disease, subclinical ileal inflammation does not appear to be relevant in gallstone patients. The mechanisms of transcriptional repression of ASBT in both diseases are apparently different.

• Crohn's disease-Defect in innate defence.

  Authors: Michael Gersemann, Jan Wehkamp, Klaus Fellermann, Eduard Friedrich Stange

  World journal of gastroenterology : WJG. 10/2008; 14(36):5499-503.

  Crohn's disease may prinicipally involve the whole gastrointestinal tract. Most commonly, the inflammation occurs in the small intestine and/or in the colon with stable disease location over theCrohn's disease may prinicipally involve the whole gastrointestinal tract. Most commonly, the inflammation occurs in the small intestine and/or in the colon with stable disease location over the years. The pathogenesis of both disease phenotypes is complex, the likely primary defect lies in the innate rather than adaptive immunity, particularly in the chemical antimicrobial barrier of the mucosa. Crohn's ileitis is associated with a reduced expression of the Wnt signalling pathway transcription factor T-cell factor 4 (TCF4), which is regulating Paneth cell differentiation. As a result, the alpha-defensins and principal Paneth cell products HD5 and HD6 are deficiently expressed in ileal disease, independent of current inflammation. In contrast, Crohn's colitis is typically associated with an impaired induction of the beta-defensins HBD2 and HBD3 caused by fewer gene copy numbers in the gene locus of the beta-defensins on chromosome 8. This ileal and colonic defect in innate defence mediated by a deficiency of the protective alpha- and beta-defensins may enable the luminal microbes to invade the mucosa and trigger the inflammation. A better understanding of the exact molecular mechanisms behind ileal and colonic Crohn's disease may give rise to new therapeutic strategies based on a stimulation of the protective innate immune system.