Klaus Fellermann

Publications of Klaus Fellermann

• Association between azithromycin therapy and duration of bacterial shedding among patients with Shiga toxin-producing enteroaggregative Escherichia coli O104:H4.

  Authors: Martin Nitschke, Friedhelm Sayk, Christoph Härtel, Rahel Tabea Roseland, Susanne Hauswaldt, Jürgen Steinhoff, Klaus Fellermann, Inge Derad, Peter Wellhöner, Jürgen Büning, Bettina Tiemer, Alexander Katalinic, Jan Rupp, Hendrik Lehnert, Werner Solbach, Johannes K-M Knobloch

  JAMA : the journal of the American Medical Association. 03/2012; 307(10):1046-52.

  An outbreak of Shiga toxin-producing enteroaggregative Escherichia coli (STEC O104:H4) infection with a high incidence of hemolytic uremic syndrome (HUS) occurred in Germany in May 2011. AntibioticAn outbreak of Shiga toxin-producing enteroaggregative Escherichia coli (STEC O104:H4) infection with a high incidence of hemolytic uremic syndrome (HUS) occurred in Germany in May 2011. Antibiotic treatment of STEC infection is discouraged because it might increase the risk of HUS development. However, antibiotic therapy is widely used to treat enteroaggregative E coli infection. In the German outbreak, a substantial number of patients received prophylactic azithromycin treatment as part of a therapeutic regimen with the C5 antibody eculizumab. To analyze the duration of bacterial shedding in patients with STEC infection who did and did not receive oral azithromycin therapy. At a single center in Lübeck, Germany, 65 patients with STEC infection, including patients with HUS as well as STEC-infected outpatients without manifestation of HUS, were investigated between May 15 and July 26, 2011, and were monitored for a mean of 39.3 days after onset of clinical symptoms. Carriage of STEC after azithromycin therapy. Twenty-two patients received oral azithromycin and 43 patients did not receive antibiotic treatment. Among antibiotic-treated patients, long-term STEC carriage (>28 days) was observed in 1 of 22 patients (4.5%; 95% CI, 0%-13.3%) compared with 35 of 43 patients (81.4%; 95% CI, 69.8%-93.0%) who were not treated with antibiotics (P < .001). All 22 patients receiving azithromycin treatment had at least 3 STEC-negative stool specimens after the completion of treatment, and no recurrence of STEC was observed in these patients. As proof of principle, 15 patients who initially were not treated with antibiotics and were long-term STEC carriers were treated with oral azithromycin given for 3 days and subsequently had negative stool specimens. Treatment with azithromycin was associated with a lower frequency of long-term STEC O104:H4 carriage.

• Cyclophosphamide therapy in Sweet's syndrome complicating refractory Crohn's disease − Efficacy and mechanism of action.

  Authors: Christian Meinhardt, Jürgen Büning, Klaus Fellermann, Hendrik Lehnert, Klaus J Schmidt

  Journal of Crohn's & colitis. 12/2011; 5(6):633-7.

  Sweet's syndrome is a rare extraintestinal manifestation of Crohn's disease that is usually treated by corticosteroids. Cyclophosphamide therapy has been shown to be effective in steroid-refractorySweet's syndrome is a rare extraintestinal manifestation of Crohn's disease that is usually treated by corticosteroids. Cyclophosphamide therapy has been shown to be effective in steroid-refractory Crohn's disease with extraintestinal manifestations. The mechanism of action remains obscure. Here, we report about a case of steroid-refractory Sweet's syndrome accompanying Crohn's colitis treated by cyclophosphamide. At baseline and two weeks after initiating cyclophosphamide pulse therapy, clinical symptoms were evaluated and apoptosis in mononuclear cells of the colon mucosa was quantified via immunofluorescence TUNEL-labeling. Ongoing clinical follow-up lasts for more than three years. Cyclophosphamide pulse therapy resulted in complete resolution of luminal activity and extraintestinal manifestations. TUNEL-marked CD4(+), CD8(+) and CD68(+) cells in intestinal biopsies showed a 338% increase as compared to baseline. Cyclophosphamide therapy was highly effective in steroid-refractory Crohn's colitis accompanied by Sweet's syndrome for induction of remission. Furthermore, apoptosis of mononuclear cells in the colon mucosa, including CD68(+) macrophages as well as CD4(+) and CD8(+) cells, appears to be a component of the anti-inflammatory effect of cyclophosphamide in Crohn's disease.

• The CD40-CD40L pathway contributes to the proinflammatory function of intestinal epithelial cells in inflammatory bowel disease.

  Authors: Frauke Borcherding, Martin Nitschke, Gheorghe Hundorfean, Jan Rupp, Dorthe von Smolinski, Katja Bieber, Cees van Kooten, Hendrik Lehnert, Klaus Fellermann, Jürgen Büning

  The American journal of pathology. 04/2010; 176(4):1816-27.

  In inflammatory bowel diseases (IBD), intestinal epithelial cells (IECs) are involved in the outbalanced immune responses toward luminal antigens. However, the signals responsible for thisIn inflammatory bowel diseases (IBD), intestinal epithelial cells (IECs) are involved in the outbalanced immune responses toward luminal antigens. However, the signals responsible for this proinflammatory capacity of IECs in IBD remain unclear. The CD40/CD40L interaction activates various pathways in immune and nonimmune cells related to inflammation and was shown to be critical for the development of IBD. Here we demonstrate CD40 expression within IECs during active IBD. Endoscopically obtained biopsies taken from Crohn's disease (n = 112) and ulcerative colitis patients (n = 67) consistently showed immunofluorescence staining for CD40 in IECs of inflamed ileal or colonic mucosa. In noninvolved mucosa during active disease, tissue obtained during Crohn's disease or ulcerative colitis in remission and biopsies from healthy controls (n = 38) IECs almost entirely lacked CD40 staining. Flow cytometry and RT-PCR analysis using different intestinal epithelial cell lines (HT29, SW480, and T84) showed IFN-gamma to effectively induce CD40 in IECs. Cells were virtually unresponsive to LPS or whole E. coli regarding CD40 expression. In addition, a moderate induction of CD40 was found in response to TNF-alpha, which exerted synergistical effects with IFN-gamma. CD40 ligation by CD40L-transfected murine fibroblasts or soluble CD40L increased the secretion of IL-8 in IFN-gamma pretreated HT29 cells. Our findings provide evidence for the epithelial expression and modulation of CD40 in IBD-affected mucosa and indicate its involvement in the proinflammatory function of IECs.

• Crohn's targeted therapy: myth or real goal?

  Authors: Klaus J Schmidt, Jürgen Büning, Carsten Jankowiak, Hendrik Lehnert, Klaus Fellermann

  Current drug discovery technologies. 12/2009; 6(4):290-8.

  The broad spectrum of chronic inflammatory bowel diseases encompasses the two main entities Crohn's disease (CD) and ulcerative colitis (UC). Rapid action and long-term duration is the superordinateThe broad spectrum of chronic inflammatory bowel diseases encompasses the two main entities Crohn's disease (CD) and ulcerative colitis (UC). Rapid action and long-term duration is the superordinate goal in most therapeutic approaches facing chronic inflammation including inflammatory bowel diseases (IBD) for induction and maintenance of remission. The availability of antibodies targeting TNF-alpha or alpha 4 integrin has recently offered new opportunities apart from classical remedies for the treatment of CD, a major challenge for future therapeutic concepts. Classical way of treating CD is an escalation scheme ("step-up") whereas the novel and still controverse approach ("top-down") favors a biological as initial drug. Today, four biologicals have proven efficacy and safety in CD treatment strategies and have received approval by FDA and, with the exception of natalizumab and certolizumab, by EMEA. Infliximab was the first TNF-alpha blocker and extended the care in CD. Adalimumab and certolizumab pegol followed as humanized second generation TNF-alpha blockers. Another targeted therapy option is natalizumab, an alpha 4 integrin monoclonal antibody, which blocks the migration of leukocytes into inflamed gut tissue. Nevertheless, a considerable number of patients remain refractory, lose response or render intolerant to these biologicals. An overall long lasting remission of less than 30% with scheduled administration of TNF-alpha blockers in patients with steroid dependent or refractory CD seems to be lifelike and alternative therapeutic options are warranted. Broad acting antimetabolites come into focus again and recent data provide substantial evidence for the efficacy and safety of cyclophosphamide pulse therapy in ileocolonic, refractory CD. This therapeutic option should be kept in mind as a reasonable agent to target the inflammatory process in severely disabled patients. Hence, targeted therapy in CD seems to be still a myth at present owing to the complex nature of disease.

• 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.

• 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.

• How effective is natalizumab induction therapy in patients with Crohn's disease?

  Authors: Klaus Fellermann, Eduard F Stange

  Nature clinical practice. Gastroenterology & hepatology. 03/2008; 5(2):78-9.

• The Paneth cell alpha-defensin deficiency of ileal Crohn's disease is linked to Wnt/Tcf-4.

  Authors: Jan Wehkamp, Guoxing Wang, Irmgard Kübler, Sabine Nuding, Alex Gregorieff, Anke Schnabel, Robert J Kays, Klaus Fellermann, Oliver Burk, Matthias Schwab, Hans Clevers, Charles L Bevins, Eduard F Stange

  Journal of immunology (Baltimore, Md. : 1950). 10/2007; 179(5):3109-18.

  Ileal Crohn's disease (CD), a chronic mucosal inflammation, is characterized by two pertinent features: a specific decrease of Paneth cell-produced antimicrobial alpha-defensins and the presence ofIleal Crohn's disease (CD), a chronic mucosal inflammation, is characterized by two pertinent features: a specific decrease of Paneth cell-produced antimicrobial alpha-defensins and the presence of mucosal-adherent bacteria. A mutation in NOD2, the muramyl dipeptide recognition receptor, is found in some patients, which leads to an even more pronounced alpha-defensin decrease. However, the underlying mechanism remains unclear for the majority of patients. In this study, we report a reduced expression in ileal CD of the Wnt-signaling pathway transcription factor Tcf-4, a known regulator of Paneth cell differentiation and alpha-defensin expression. Within specimens, the levels of Tcf-4 mRNA showed a high degree of correlation with both HD5 and HD6 mRNA. The levels of Tcf-4 mRNA were decreased in patients with ileal disease irrespective of degree of inflammation, but were not decreased in colonic CD or ulcerative colitis. As a functional indicator of Tcf-4 protein, quantitative binding analysis with nuclear extracts from small intestine biopsies to a Tcf-4 high-affinity binding site in the HD-5 and HD-6 promoters showed significantly reduced activity in ileal CD. Furthermore, a causal link was shown in a murine Tcf-4 knockout model, where the comparably reduced expression of Tcf-4 in heterozygous (+/-) mice was sufficient to cause a significant decrease of both Paneth cell alpha-defensin levels and bacterial killing activity. Finally, the association between Paneth cell alpha-defensins and Tcf-4 was found to be independent of the NOD2 genotype. This new link established between a human inflammatory bowel disease and the Wnt pathway/Tcf-4 provides a novel mechanism for pathogenesis in patients with ileal CD.

• Reduced mucosal antimicrobial activity in Crohn's disease of the colon.

  Authors: Sabine Nuding, Klaus Fellermann, Jan Wehkamp, Eduard F Stange

  Gut. 10/2007; 56(9):1240-7.

  OBJECTIVES: In order to maintain the mucosal barrier against luminal microorganisms the intestinal epithelial cells synthesise various broad-spectrum antimicrobial peptides including defensins andOBJECTIVES: In order to maintain the mucosal barrier against luminal microorganisms the intestinal epithelial cells synthesise various broad-spectrum antimicrobial peptides including defensins and cathelicidins. Recent studies indicate that both may be deficient in Crohn's disease. To elucidate the possible functional consequences of this deficiency antimicrobial activity in colonic mucosa from patients with inflammatory bowel disease and healthy controls was investigated. METHODS: A flow cytometric assay was established to quantitate bacterial killing and test the antibacterial activity of cationic peptide extracts from colonic biopsies taken from patients with active or inactive ileocolonic or colonic Crohn's disease (n = 22), ulcerative colitis (n = 29) and controls (n = 13) against clinical isolates of Bacteroides vulgatus and Enterococcus faecalis or the reference strains Escherichia coli American Type Culture Collection (ATCC) 25922 and Staphylococcus aureus ATCC 25923. RESULTS: Compared with controls and ulcerative colitis there was a reduced antimicrobial effect in Crohn's disease extracts that was most evident against B. vulgatus. The antimicrobial effect against E. coli and E. faecalis was significantly lower in Crohn's disease compared with ulcerative colitis. Activity against S. aureus disclosed a similar pattern, but was less pronounced. The differences were independent of the inflammation status or concurrent steroid treatment. Bacteria incubated with biopsy extracts from ulcerative colitis patients frequently showed a characteristic change in cell size and granularity, compatible with more extensive membrane disintegration, compared with bacteria incubated with extracts from controls or Crohn's disease. CONCLUSION: Crohn's disease of the colon is characterized by a diminished functional antimicrobial activity that is consistent with the reported low antibacterial peptide expression.

• Herpes simplex virus colitis complicating ulcerative colitis: A case report and brief review on superinfections.

  Authors: Marco Oliver Schunter, Thorsten Walles, Peter Fritz, Uta Meyding-Lamadé, Klaus-Peter Thon, Klaus Fellermann, Eduard Friedrich Stange, Wolfram Lamadé

  Journal of Crohn's & colitis. 09/2007; 1(1):41-6.

  In patients with inflammatory bowel disease herpes simplex virus infection has been described as a major cause of morbidity and mortality, especially in immunocompromised individuals. Here we presentIn patients with inflammatory bowel disease herpes simplex virus infection has been described as a major cause of morbidity and mortality, especially in immunocompromised individuals. Here we present the case of a 35-year old woman with an exacerbation of ulcerative colitis caused by herlpes simplex virus infection (HSV-2). The diagnosis was confirmed histologically following subtotal colectomy. After intravenous treatment with aciclovir for 2 weeks postoperative hematochezia stopped. Herpes simplex virus colitis is a rare but potentially fatal complication of immunosuppressive treatment in patients with inflammatory bowel disease. Prompt diagnosis and efficient antiviral therapy are mandatory to improve prognosis.

• Induction of human beta-defensin 2 by the probiotic Escherichia coli Nissle 1917 is mediated through flagellin.

  Authors: Miriam Schlee, Jan Wehkamp, Artur Altenhoefer, Tobias A Oelschlaeger, Eduard F Stange, Klaus Fellermann

  Infection and immunity. 06/2007; 75(5):2399-407.

  Human beta-defensin 2 (hBD-2) is an inducible antimicrobial peptide synthesized by the epithelium to counteract bacterial adherence and invasion. Proinflammatory cytokines, as well as certainHuman beta-defensin 2 (hBD-2) is an inducible antimicrobial peptide synthesized by the epithelium to counteract bacterial adherence and invasion. Proinflammatory cytokines, as well as certain bacterial strains, have been identified as potent endogenous inducers. Recently, we have found that hBD-2 induction by probiotic Escherichia coli Nissle 1917 was mediated through NF-kappaB- and AP-1-dependent pathways. The aim of the present study was to identify the responsible bacterial factor. E. coli Nissle 1917 culture supernatant was found to be more potent than the pellet, indicating a soluble or shed factor. Chemical analysis demonstrated the factor to be heat resistant and proteinase digestible. Several E. coli Nissle 1917 deletion mutants were constructed and tested for their ability to induce hBD-2 expression in Caco-2 cells. Deletion mutants for flagellin specifically exhibited an impaired immunostimulatory capacity. Reinsertion of the flagellin gene restored the induction capacity to normal levels. Isolated flagellin from E. coli Nissle 1917 and from Salmonella enterica serovar Enteritidis induced hBD-2 mRNA significantly in contrast to the flagellin of the apathogenic E. coli strain ATCC 25922. H1 flagellin antiserum abrogated hBD-2 expression induced by flagellin as well as E. coli Nissle 1917 supernatant, confirming that flagellin is the major stimulatory factor of E. coli Nissle 1917.

• Attenuated induction of epithelial and leukocyte serine antiproteases elafin and secretory leukocyte protease inhibitor in Crohn's disease.

  Authors: Michael Schmid, Klaus Fellermann, Peter Fritz, Oliver Wiedow, Eduard F Stange, Jan Wehkamp

  Journal of leukocyte biology. 05/2007; 81(4):907-15.

  Elafin (or skin-derived antileukoprotease) and secretory leukocyte protease inhibitor (SLPI) are serine antiproteases antagonizing human neutrophil elastase (HNE), thereby preventing tissue injuryElafin (or skin-derived antileukoprotease) and secretory leukocyte protease inhibitor (SLPI) are serine antiproteases antagonizing human neutrophil elastase (HNE), thereby preventing tissue injury from excessive release of proteolytic enzymes by inflammatory cells. Furthermore, elafin and SLPI are "defensin-like" molecules with broad antimicrobial activity. The balance between proteases and antagonists may critically determine inflammatory processes in Crohn's disease (CD) and ulcerative colitis (UC). Real-time PCR was performed to quantitate colonic, proinflammatory cytokine IL-8, protease (HNE), and antiprotease mRNA (elafin and SLPI) in a total of 340 biopsies from 117 patients (47 CD, 45 UC, 25 controls). Histological inflammation was scored, and HNE, elafin, and SLPI were localized and semiquantified by immunostaining in 51 colonic paraffin sections (23 CD, 11 UC, 17 controls). Proinflammatory IL-8, degree of histological inflammation, and granulocyte content were similar in UC and CD. Elafin stained predominantly in the epithelium and SLPI in mucosal inflammatory cells. HNE mRNA levels and immunostaining were increased equally in both forms of inflammatory bowel disease. Levels of mRNA and immunostaining of the antiproteases elafin and SLPI were enhanced strongly in inflamed versus noninflamed UC. It is surprising that comparing inflamed versus noninflamed CD, this increase was significantly less pronounced for elafin and even lacking for SLPI. Despite comparable degrees of inflammation and protease levels, the induction of both antiproteases was attenuated in CD. This could contribute to the transmural depth of tissue destruction in CD. Elafin and SLPI may be added to the list of defensin-like peptides with diminished induction in CD versus UC.

• A chromosome 8 gene-cluster polymorphism with low human beta-defensin 2 gene copy number predisposes to Crohn disease of the colon.

  Authors: Klaus Fellermann, Daniel E Stange, Elke Schaeffeler, Hartmut Schmalzl, Jan Wehkamp, Charles L Bevins, Walter Reinisch, Alexander Teml, Matthias Schwab, Peter Lichter, Bernhard Radlwimmer, Eduard F Stange

  American journal of human genetics. 10/2006; 79(3):439-48.

  Defensins are endogenous antimicrobial peptides that protect the intestinal mucosa against bacterial invasion. It has been suggested that deficient defensin expression may underlie the chronicDefensins are endogenous antimicrobial peptides that protect the intestinal mucosa against bacterial invasion. It has been suggested that deficient defensin expression may underlie the chronic inflammation of Crohn disease (CD). The DNA copy number of the beta-defensin gene cluster on chromosome 8p23.1 is highly polymorphic within the healthy population, which suggests that the defective beta-defensin induction in colonic CD could be due to low beta-defensin-gene copy number. Here, we tested this hypothesis, using genomewide DNA copy number profiling by array-based comparative genomic hybridization and quantitative polymerase-chain-reaction analysis of the human beta-defensin 2 (HBD-2) gene. We showed that healthy individuals, as well as patients with ulcerative colitis, have a median of 4 (range 2-10) HBD-2 gene copies per genome. In a surgical cohort with ileal or colonic CD and in a second large cohort with inflammatory bowel diseases, those with ileal resections/disease exhibited a normal median HBD-2 copy number of 4, whereas those with colonic CD had a median of only 3 copies per genome (P=.008 for the surgical cohort; P=.032 for the second cohort). Overall, the copy number distribution in colonic CD was shifted to lower numbers compared with controls (P=.002 for both the surgical cohort and the cohort with inflammatory bowel diseases). Individuals with < or = 3 copies have a significantly higher risk of developing colonic CD than did individuals with > or = 4 copies (odds ratio 3.06; 95% confidence interval 1.46-6.45). An HBD-2 gene copy number of < 4 was associated with diminished mucosal HBD-2 mRNA expression (P=.033). In conclusion, a lower HBD-2 gene copy number in the beta-defensin locus predisposes to colonic CD, most likely through diminished beta-defensin expression.

  Download

• Heterogeneous expression of human cathelicidin hCAP18/LL-37 in inflammatory bowel diseases.

  Authors: Jürgen Schauber, Daniel Rieger, Frank Weiler, Jan Wehkamp, Matthias Eck, Klaus Fellermann, Wolfgang Scheppach, Richard L Gallo, Eduard F Stange

  European journal of gastroenterology & hepatology. 07/2006; 18(6):615-21.

  BACKGROUND: Inflammatory bowel diseases (IBDs) are characterized by a breakdown of colon epithelial barrier function. Antimicrobial peptides like cathelicidins are molecules of the innate immuneBACKGROUND: Inflammatory bowel diseases (IBDs) are characterized by a breakdown of colon epithelial barrier function. Antimicrobial peptides like cathelicidins are molecules of the innate immune system located at epithelial surfaces. Cathelicidins influence microbial growth and inflammation and may play a role in IBD. In this study, the expression of human cathelicidin hCAP18/LL-37 was investigated in the intestinal mucosa from patients suffering from ulcerative colitis or Crohn's disease. METHODS: Biopsy material from colon and ileal mucosa of a total of 89 patients (34 with Crohn's disease, 27 with ulcerative colitis, 28 control patients) was evaluated for cathelicidin expression by real-time reverse-transcriptase polymerase chain reaction and immunohistochemistry. Colon epithelial cells were stimulated in vitro with various cytokines to evaluate mechanisms that influence cathelicidin production. RESULTS: Cathelicidin expression was significantly increased in inflamed and non-inflamed colon mucosa from ulcerative colitis patients compared to non-inflamed control mucosa. In patients with Crohn's disease cathelicidin expression was not changed in inflamed or non-inflamed colon or ileal mucosa independent of NOD2 status. Biopsies evaluated by immunohistochemistry showed epithelial cathelicidin expression in the upper crypt that was diffuse in controls and only basal in IBD patients. Inflammation mediators, alone or in combination with the known cathelicidin inducer butyrate, had no effect on cathelicidin expression in cultured colon cells. CONCLUSIONS: In IBD the colonic expression of human cathelicidin is altered: cathelicidin expression is increased in inflamed and non-inflamed mucosa in patients suffering from ulcerative colitis but not in Crohn's disease. This deficiency may further compromise the antimicrobial barrier in Crohn's disease.

• A flow cytometric assay to monitor antimicrobial activity of defensins and cationic tissue extracts.

  Authors: Sabine Nuding, Klaus Fellermann, Jan Wehkamp, Holger A G Mueller, Eduard F Stange

  Journal of microbiological methods. 05/2006; 65(2):335-45.

  To determine the antibacterial activity of defensins and other antimicrobial peptides in biopsy extracts, we evaluated a flow cytometric method with the membrane potential sensitive dyeTo determine the antibacterial activity of defensins and other antimicrobial peptides in biopsy extracts, we evaluated a flow cytometric method with the membrane potential sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. This assay enables us to discriminate intact non-fluorescent and depolarized fluorescent bacteria after exposure to antimicrobial peptides by measurement at the direct target, the cytoplasmic membrane and the membrane potential. The feasibility of the flow cytometric assay was evaluated with recombinant human beta-defensin 3 (HBD-3) against 25 bacterial strains representing 12 species. HBD-3 showed a broad-spectrum dose dependent activity and the minimal dose to cause depolarization ranged from 1.25 to >15 microg/ml HBD-3, depending on the species tested. The antibacterial effect was diminished with sodium chloride or dithiothreitol and could be abrogated with a HBD-3 antibody. Additionally, isolated cationic extracts from human intestinal biopsies showed a strong bactericidal effect against Escherichia coli K12, E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923, which was diminished towards E. coli at 150 mM NaCl, whereas the activity towards S. aureus ATCC 25923 remained unaffected at physiological salt concentrations. DTT blocked the bactericidal effect of biopsy extracts completely.

• Reduced Paneth cell alpha-defensins in ileal Crohn's disease.

  Authors: Jan Wehkamp, Nita H Salzman, Edith Porter, Sabine Nuding, Michael Weichenthal, Robert E. Petras, Bo Shen, Elke Schaeffeler, Matthias Schwab, Rose Linzmeier, Ryan W Feathers, Hiutung Chu, Heriberto Lima, Klaus Fellermann, Tomas Ganz, Eduard F Stange, Charles L Bevins

  Proceedings of the National Academy of Sciences of the United States of America. 01/2006; 102(50):18129-34.

  The pathogenesis of Crohn's disease (CD), an idiopathic inflammatory bowel disease, is attributed, in part, to intestinal bacteria that may initiate and perpetuate mucosal inflammation in geneticallyThe pathogenesis of Crohn's disease (CD), an idiopathic inflammatory bowel disease, is attributed, in part, to intestinal bacteria that may initiate and perpetuate mucosal inflammation in genetically susceptible individuals. Paneth cells (PC) are the major source of antimicrobial peptides in the small intestine, including human alpha-defensins HD5 and HD6. We tested the hypothesis that reduced expression of PC alpha-defensins compromises mucosal host defenses and predisposes patients to CD of the ileum. We report that patients with CD of the ileum have reduced antibacterial activity in their intestinal mucosal extracts. These specimens also showed decreased expression of PC alpha-defensins, whereas the expression of eight other PC products either remained unchanged or increased when compared with controls. The specific decrease of alpha-defensins was independent of the degree of inflammation in the specimens and was not observed in either CD of the colon, ulcerative colitis, or pouchitis. The functional consequence of alpha-defensin expression levels was examined by using a transgenic mouse model, where we found changes in HD5 expression levels, comparable to those observed in CD, had a pronounced impact on the luminal microbiota. Thus, the specific deficiency of PC defensins that characterizes ileal CD may compromise innate immune defenses of the ileal mucosa and initiate and/or perpetuate this disease.

• Mechanisms of disease: defensins in gastrointestinal diseases.

  Authors: Jan Wehkamp, Klaus Fellermann, Klaus R Herrlinger, Charles L Bevins, Eduard F Stange

  Nature clinical practice. Gastroenterology & hepatology. 10/2005; 2(9):406-15.

  Defensins are endogenous antibiotics with microbicidal activity against Gram-negative and Gram-positive bacteria, fungi, viruses and protozoa. In the gastrointestinal tract, defensins help regulateDefensins are endogenous antibiotics with microbicidal activity against Gram-negative and Gram-positive bacteria, fungi, viruses and protozoa. In the gastrointestinal tract, defensins help regulate the composition and number of colonizing microbes, and protect the host from food-borne and water-borne pathogens. In health, the normal host relationship with the commensal luminal microbiota is beneficial, but the same commensal bacteria could have a pathogenic role in inflammatory diseases. A disturbance in antimicrobial defense, as provided by Paneth cells of the small intestine, seems to be a critical factor in the pathogenesis of ileal Crohn's disease, an inflammatory disease of the intestinal tract. The disruption of the critical balance between antimicrobial peptides and luminal bacteria might also explain other gastrointestinal infections and diseases. Elucidating the underlying mechanisms involved in the regulation and biology of defensins could open up new therapeutic avenues.

• Defensin deficiency, intestinal microbes, and the clinical phenotypes of Crohn's disease.

  Authors: Jan Wehkamp, Michael Schmid, Klaus Fellermann, Eduard F Stange

  Journal of leukocyte biology. 05/2005; 77(4):460-5.

  Crohn's disease is a chronic, inflammatory disease of the intestinal mucosa. Although intestinal bacteria are implicated in disease pathogenesis, the etiology is still unclear. The main location ofCrohn's disease is a chronic, inflammatory disease of the intestinal mucosa. Although intestinal bacteria are implicated in disease pathogenesis, the etiology is still unclear. The main location of disease is the small intestine (ileum) and the colon. Ileal disease has been linked to a mutation in the NOD2 gene. Defensins are antimicrobial peptides and in the ileum, are mainly expressed in Paneth cells, epithelial cells that also express NOD2. In the colon, defensins are expressed by enterocytes or metaplastic Paneth cells. Crohn's disease patients with ileal involvement, compared with controls or Crohn's patients without ileal involvement, have diminished expression of ileal Paneth cell defensins. This decrease is even more pronounced in Crohn's patients displaying a NOD2 mutation. In contrast, Crohn's disease of the colon is characterized by an impaired induction of beta-defensins in enterocytes. The colonic expression of the constitutive beta-defensin 1 is also decreased in the inflamed colonic mucosa, but this decrease is less specific to Crohn's disease, as it can also be found in ulcerative colitis patients. In conclusion, the regional localizations of Crohn's disease, ileal or colonic disease, can be linked to different defensin profiles. Crohn's disease of the ileum is associated with diminished defensin expression in Paneth cells. Crohn's disease of the colon is associated with diminished beta-defensin expression in enterocytes. Thus, it can be speculated that decreased defensin levels lead to a weakened intestinal barrier function to intestinal microbes and might be crucial in the pathophysiology of Crohn's disease.