Resveratrol Prevents Campylobacter jejuni-Induced Leaky gut by Restoring Occludin and Claudin-5 in the Paracellular Leak Pathway.

Campylobacter jejuni is a bacterial human pathogen causing gastroenteritis and sequelae like irritable bowel syndrome. Epidemiologists count the human campylobacteriosis by C. jejuni as the most common foodborne zoonosis and bacterial diarrheal disease worldwide. Based on bioinformatics predictions for potential protective compounds in campylobacteriosis, the question was raised whether the plant-based polyphenol resveratrol is sufficient to attenuate intestinal epithelial damage induced by C. jejuni. We investigated this by performing experimental infection studies in an epithelial cell culture and the secondary abiotic IL-10-/- mouse model. In C. jejuni-infected human colonic HT-29/B6 cell monolayers, transepithelial electrical resistance (TER) was decreased and the paracellular marker flux of fluorescein (332 Da) increased. Concomitantly, the tight junction (TJ) proteins occludin and claudin-5 were re-distributed off the tight junction domain. This was accompanied by an increased induction of epithelial apoptosis, both changes contributing to compromised barrier function and the opening of the leak pathway induced by C. jejuni. In parallel, the recovery experiments with the application of resveratrol revealed a functional improvement of the disturbed epithelial barrier in both models in vitro and in vivo. During treatment with resveratrol, TJ localization of occludin and claudin-5 was fully restored in the paracellular domain of HT-29/B6 cells. Moreover, resveratrol decreased the rate of epithelial apoptosis. These resveratrol-induced molecular and cellular effects would therefore be expected to improve epithelial barrier function, thereby minimizing the so-called leaky gut phenomenon. In conclusion, the induction of the leak pathway by C. jejuni and the restoration of barrier function by resveratrol demonstrates its effectiveness as a potential preventive or therapeutic method of mitigating the leaky gut associated with campylobacteriosis.

Evidence for a role of RUNX1 as recombinase cofactor for TCRß rearrangements and pathological deletions in ETV6-RUNX1 ALL.

T-cell receptor gene beta (TCRß) gene rearrangement represents a complex, tightly regulated molecular mechanism involving excision, deletion and recombination of DNA during T-cell development. RUNX1, a well-known transcription factor for T-cell differentiation, has recently been described to act in addition as a recombinase cofactor for TCRδ gene rearrangements. In this work we employed a RUNX1 knock-out mouse model and demonstrate by deep TCRß sequencing, immunostaining and chromatin immunoprecipitation that RUNX1 binds to the initiation site of TCRß rearrangement and its homozygous inactivation induces severe structural changes of the rearranged TCRß gene, whereas heterozygous inactivation has almost no impact. To compare the mouse model results to the situation in Acute Lymphoblastic Leukemia (ALL) we analyzed TCRß gene rearrangements in T-ALL samples harboring heterozygous Runx1 mutations. Comparable to the Runx1+/- mouse model, heterozygous Runx1 mutations in T-ALL patients displayed no detectable impact on TCRß rearrangements. Furthermore, we reanalyzed published sequence data from recurrent deletion borders of ALL patients carrying an ETV6-RUNX1 translocation. RUNX1 motifs were significantly overrepresented at the deletion ends arguing for a role of RUNX1 in the deletion mechanism. Collectively, our data imply a role of RUNX1 as recombinase cofactor for both physiological and aberrant deletions.

Preventive Anti-CD2 Treatment does not Impair Parasite Control in a Murine Toxoplasmosis Model.

Targeting human CD2 with the monoclonal antibody (mAb) CB.219 reduces intestinal inflammation in a colitis model where T cells carry human CD2. Here, we asked whether this mAb has adverse effects on infection control. Mice expressing human CD2 on T cells (huCD2tg) were orally infected with Toxoplasma (T.) gondii and treated with the human CD2-specific mAb CB.219 in a preventive setting. The intestinal T. gondii loads in CB.219 treated mice did not differ from the control group. Histologically, huCD2tg mice showed moderate ileal inflammation that did not change with CB.219 treatment. In the ileum, CB.219 treatment reduced the protein levels of interferon-γ, transforming growth factor ß and interleukin-6, whereas interleukin-18 mRNA was slightly increased. The infiltration of neutrophils, macrophages, and T cells into the ileum was unaffected by CB.219 treatment. However, CB.219 treatment decreased the numbers of forkhead box P3(+) regulatory T cells (Treg) in ileum and liver of huCD2tg mice. This was confirmed in vitro using human peripheral blood mononuclear cells. Taken together, targeting CD2(+) T cells by the human CD2 mAb CB.219 does not prevent beneficial immune reactions necessary for pathogen control. Further experiments will address gut specificity, underlying mechanisms, and general applicability of CB.219 treatment.

Selective gelatinase inhibition reduces apoptosis and pro-inflammatory immune cell responses in Campylobacter jejuni-infected gnotobiotic IL-10 deficient mice.

Increased levels of the matrix metalloproteinases-2 and -9 (also referred to gelatinase-A and -B, respectively) can be detected in intestinal inflammation. We have recently shown that selective gelatinase blockage by the synthetic compound RO28-2653 ameliorates acute murine ileitis and colitis. We here investigated whether RO28-2653 exerts anti-inflammatory effects in acute Campylobacter jejuni-induced enterocolitis of gnotobiotic IL-10(-/-) mice generated following antibiotic treatment. Mice were perorally infected with C. jejuni (day 0) and either treated with RO28-2653 (75 mg/kg body weight/day) or placebo from day 1 until day 6 post infection (p.i.) by gavage. Irrespective of the treatment, infected mice displayed comparable pathogen loads within the gastrointestinal tract. Following RO28-2653 administration, however, infected mice exhibited less severe symptoms such as bloody diarrhea as compared to placebo controls. Furthermore, less distinct apoptosis but higher numbers of proliferating cells could be detected in the colon of RO28-2653-treated as compared to placebo-treated mice at day 7 p.i. Remarkably, gelatinase blockage resulted in lower numbers of T- and B-lymphocytes as well as macrophages and monocytes in the colonic mucosa of C. jejuni-infected gnotobiotic IL-10(-/-) mice. Taken together, synthetic gelatinase inhibition exerts anti-inflammatory effects in experimental campylobacteriosis.

The intestinal micro-environment imprints stromal cells to promote efficient Treg induction in gut-draining lymph nodes.

De novo induction of Foxp3⁺ regulatory T cells (Tregs) is particularly efficient in gut-draining mesenteric and celiac lymph nodes (mLN and celLN). Here we used LN transplantations to dissect the contribution of stromal cells and environmental factors to the high Treg-inducing capacity of these LN. After transplantation into the popliteal fossa, mLN and celLN retained their high Treg-inducing capacity, whereas transplantation of skin-draining LN into the gut mesenteries did not enable efficient Treg induction. However, de novo Treg induction was abolished in the absence of dendritic cells (DC), indicating that this process depends on synergistic contributions of stromal and DC. Stromal cells themselves were influenced by environmental signals as mLN grafts taken from germ-free donors and celLN grafts taken from vitamin A-deficient donors did not show any superior Treg-inducing capacity. Collectively, our observations reveal a hitherto unrecognized role of LN stromal cells for the de novo induction of Foxp3⁺ Tregs.

Can microbiota transplantation abrogate murine colonization resistance against Campylobacter jejuni?

Enterocolitis caused by Campylobacter jejuni represents an important socioeconomic burden worldwide. The host-specific intestinal microbiota is essential for maintaining colonization resistance (CR) against C. jejuni in conventional mice. Notably, CR is abrogated by shifts of the intestinal microbiota towards overgrowth with commensal E. coli during acute ileitis. Thus, we investigated whether oral transplantation (TX) of ileal microbiota derived from C. jejuni susceptible mice with acute ileitis overcomes CR of healthy conventional animals. Four days following ileitis microbiota TX or ileitis induction and right before C. jejuni infection, mice displayed comparable loads of main intestinal bacterial groups as shown by culture. Eight days following ileitis induction, but not ileal microbiota TX, however, C. jejuni could readily colonize the gastrointestinal tract of conventional mice and also translocate to extra-intestinal tissue sites such as mesenteric lymph nodes, spleen, liver, and blood within 4 days following oral infection. Of note, C. jejuni did not further deteriorate histopathology following ileitis induction. Lack of C. jejuni colonization in TX mice was accompanied by a decrease of commensal E. coli loads in the feces 4 days following C. jejuni infection. In summary, oral ileal microbiota TX from susceptible donors is not sufficient to abrogate murine CR against C. jejuni.

Pro-inflammatory potential of Escherichia coli strains K12 and Nissle 1917 in a murine model of acute ileitis.

Non-pathogenic Escherichia coli (Ec) strains K12 (EcK12) and Nissle 1917 (EcN) are used for gene technology and probiotic treatment of intestinal inflammation, respectively. We investigated intestinal colonization and potential pro-inflammatory properties of EcK12, EcN, and commensal E. coli (EcCo) strains in Toxoplasma (T.) gondii-induced acute ileitis. Whereas gnotobiotic animals generated by quintuple antibiotic treatment were protected from ileitis, mice replenished with conventional microbiota suffered from small intestinal necrosis 7 days post-T. gondii infection (p.i.). Irrespective of the Ec strain, recolonized mice revealed mild to moderate histopathological changes in their ileal mucosa. Upon stable recolonization with EcK12, EcN, or EcCo, development of inflammation was accompanied by pro-inflammatory responses at day 7 p.i., including increased ileal T lymphocyte and apoptotic cell numbers compared to T. gondii-infected gnotobiotic controls. Strikingly, either Ec strain was capable to translocate to extra-intestinal locations, such as MLN, spleen, and liver. Taken together, Ec strains used in gene technology and probiotic treatment are able to exert inflammatory responses in a murine model of small intestinal inflammation. In conclusion, the therapeutic use of Ec strains in patients with broad-spectrum antibiotic treatment and/or intestinal inflammation should be considered with caution.

Tim-3 is differently expressed in genetically susceptible C57BL/6 and resistant BALB/c mice during oral infection with Toxoplasma gondii.

Tim-3 has opposing roles in innate and adaptive immunities. It not only dampens CD4+ and CD8+ T cells responses but also enhances the ability of macrophages to eliminate intracellular pathogens. After peroral infection with 100 cysts of Toxoplasma gondii genetically susceptible C57BL/6 mice develop an unchecked Th1 response associated with the development of small intestinal immunopathology. Here we report that upon infection with T. gondii, both susceptible C57BL/6 and resistant BALB/c mice exhibit increased frequencies of Tim-3+ cells in spleens and mesenteric lymph nodes. The number of Tim-3+ cells was significantly higher in C57BL/6 than in BALB/c mice. Tim-3 was expressed by macrophages, dendritic, natural killer, as well as CD4+ and CD8+ T cells. Highest frequencies of Tim-3+ cells were observed at the peak of Th1 responses (day 7 post infection) concurrent with the development of ileal immunopathology. Infected Tim-3-deficient BALB/c mice did not develop ileal immunopathology nor did their parasite loads differ from those in wildtype BALB/c mice. Thus, although Tim-3 is markedly upregulated upon infection and differentially regulated in susceptible and resistant mice upon infection with T. gondii, the absence of Tim-3 is not sufficient to overcome the genetic resistance of BALB/c mice to the development of Th1-driven small intestinal immunopathology.

Mesenteric fat - control site for bacterial translocation in colitis?

In Crohn's disease bacteria could be detected in the adjacent mesenteric fat characterized by hypertrophy of unknown function. This study aimed to define effector responses of this compartment induced by bacterial translocation during intestinal inflammation. Dextran sulfate sodium-induced colitis served as a model of intestinal inflammation. Translocation of peptides and bacteria into mesenteric fat was evaluated. Innate functions of mesenteric fat and epithelium were characterized at whole tissue, cellular, and effector molecule levels. Orally applied peptides translocated in healthy wild-type (WT) mice. Bacterial translocation was not detected in healthy and acute but increased in chronic colitis. Mesenteric fat from colitic mice released elevated levels of cytokines and was infiltrated by immune cells. In MyD88(-/-) mice bacterial translocation occurred in health and increased in colitis. The exaggerated cytokine production in mesenteric fat accompanying colonic inflammation in WT mice was less distinct in MyD88(-/-) mice. In vitro studies revealed that fat not only increases cytokine production following contact with bacterial products, but also that preadipocytes are potent phagocytes. Colonic inflammation is accompanied by massive cytokine production and immune cell infiltration in adjacent adipose tissue. These effects can be considered as protective mechanisms of the mesenteric fat in the defense of bacterial translocation.

Campylobacter jejuni infection of infant mice: acute enterocolitis is followed by asymptomatic intestinal and extra-intestinal immune responses.

Campylobacter (C.) jejuni is among the leading bacterial agents causing enterocolitis worldwide. Despite the high prevalence of C. jejuni infections and its significant medical and economical consequences, intestinal pathogenesis is poorly understood. This is mainly due to the lack of appropriate animal models. In the age of 3 months, adult mice display strong colonization resistance (CR) against C. jejuni. Previous studies underlined the substantial role of the murine intestinal microbiota in maintaining CR. Due to the fact that the host-specific gut flora establishes after weaning, we investigated CR against C. jejuni in 3-week-old mice and studied intestinal and extra-intestinal immunopathogenesis as well as age dependent differences of the murine colon microbiota. In infant animals infected orally immediately after weaning C. jejuni strain B2 could stably colonize the gastrointestinal tract for more than 100 days. Within six days following infection, infant mice developed acute enterocolitis as indicated by bloody diarrhea, colonic shortening, and increased apoptotic cell numbers in the colon mucosa. Similar to human campylobacteriosis clinical disease manifestations were self-limited and disappeared within two weeks. Interestingly, long-term C. jejuni infection was accompanied by distinct intestinal immune and inflammatory responses as indicated by increased numbers of T- and B-lymphocytes, regulatory T-cells, neutrophils, as well as apoptotic cells in the colon mucosa. Strikingly, C. jejuni infection also induced a pronounced influx of immune cells into extra-intestinal sites such as liver, lung, and kidney. Furthermore, C. jejuni susceptible weaned mice harbored a different microbiota as compared to resistant adult animals. These results support the essential role of the microflora composition in CR against C. jejuni and demonstrate that infant mouse models resemble C. jejuni mediated immunopathogenesis including the characteristic self-limited enterocolitis in human campylobacteriosis. Furthermore, potential clinical and immunological sequelae of chronic C. jejuni carriers in humans can be further elucidated by investigation of long-term infected infant mice. The observed extraintestinal disease manifestations might help to unravel the mechanisms causing complications such as reactive arthritis or Guillain-Barré syndrome.

The synthetic hydroxyproline-containing collagen analogue (Gly-Pro-Hyp)10 promotes enzymatic activity of matrixmetalloproteinase-2 in vitro.

Diseases such as liver fibrosis and intestinal inflammation are characterized by accumulated components of the extracellular matrix (ECM). Given that fibrillar collagen structures were shown to serve as storage site for inactive proforms of matrixmetalloproteinases (MMPs), modulating this MMP-collagen interaction might offer a rational interventional (therapeutic) approach to enhance degradation of accumulated ECM. The synthetic triple helical collagen analogue (Gly-Pro-Hyp)10 - (GPO)10 - was shown to trigger release and enzymatic activation of collagen sequestered proMMP-2. In the presented study, we, for the first time, investigated how MMP-(GPO)10 interaction impacts cellular responses in vitro. We found that recombinant proMMP-2 induced proliferation of hepatic stellate cells (HSC), which was enhanced after addition of (GPO)10 reaching comparable levels following incubation with fully activated MMP-2. In addition, (GPO)10 induced HSC migration similar to the platelet-derived growth factor subunit-B. Further, the MMP-2-dependent invasion of HT1080 fibrosarcoma cells through an ECM membrane was enhanced after addition of (GPO)10. Since cellular proliferation and migration concomitant with matrix degradation is stimulated, we conclude that the MMP-(GPO)10 interaction also functions in a physiological environment. Thus, a potential therapeutic effect of (GPO)10 should be further tested in animal models for MMP-associated diseases such as colitis or fibrosis.

The synthetic hydroxyproline-containing collagen analogue (Gly-Pro-Hyp)10 ameliorates acute DSS colitis.

In experimental models of and humans with intestinal inflammation, increased levels of the matrix-degrading gelatinases MMP-2 and -9 in inflamed tissues can be detected. The synthetic collagen analogue (Gly-Pro-Hyp)10, (GPO)10, has been identified as a relevant binding structure for proMMP-2/-9 and promotes enzymatic activity of proMMP-2. Since targeted MMP strategies might offer promising anti-inflammatory treatment options, we for the first time studied in vivo actions exerted by (GPO)10 applying an acute dextrane sulfate sodium (DSS) induced colitis model. Seven-day intraperitoneal (GPO)10 treatment ameliorated clinical symptoms and histopathological colonic changes as compared to placebo controls with severe colitis. (GPO)10-treated mice displayed a diminished influx of neutrophils, and T- and B-lymphocytes into their colonic mucosa whereas numbers of regulatory T-cells and regenerative cells were higher as compared to placebo controls. Furthermore, IL-6 secretion was down-regulated in ex vivo colonic biopsies derived from (GPO)10-treated mice whereas higher concentrations of the anti-inflammatory cytokine IL-10 in extra-intestinal compartments such as MLN and spleen could be detected. Strikingly, influx of inflammatory cells into lungs was abolished following (GPO)10 application. We therefore propose (GPO)10 as a promising effective and safe treatment option of intestinal and extra-intestinal inflammatory conditions in humans.

Campylobacter jejuni induces extra-intestinal immune responses via Toll-like-receptor-4 signaling in conventional IL-10 deficient mice with chronic colitis.

Campylobacter jejuni is one of the predominant causes for foodborne bacterial infections worldwide. We investigated whether signaling of C. jejuni-lipoproteins and -lipooligosaccharide via Toll-like-receptor (TLR) -2 and -4, respectively, is inducing intestinal and extra-intestinal immune responses following infection of conventional IL-10(-/-) mice with chronic colitis. At day 3 following oral infection, IL-10(-/-) mice lacking TLR-2 or TLR-4 harbored comparable C. jejuni strain ATCC 43431 loads in their colon. Interestingly, infected TLR-4(-/-) IL-10(-/-) mice displayed less compromized epithelial barrier function as indicated by lower translocation rates of live gut commensals into mesenteric lymphnodes (MLNs), and exhibited less distinct B lymphocyte responses in their colonic mucosa as compared to naїve IL-10(-/-) controls. Furthermore, in extra-intestinal compartments such as MLNs and spleens, abundance of myeloid cells was less distinct whereas relative percentages of activated T helper cells and cytotoxic T cells were higher in spleens and dendritic cells more abundant in MLNs of infected IL-10(-/-) animals lacking TLR-4 as compared to IL-10(-/-) controls. Taken together, in conventionally colonized IL-10(-/-) mice, TLR-4, but not TLR-2, is involved in mediating extra-intestinal pro-inflammatory immune responses following C. jejuni infection. Thus, conventional IL-10(-/-) mice are well suited to further dissect mechanisms underlying Campylobacter infections in vivo.

Treatment with interleukin-18 binding protein ameliorates Toxoplasma gondii-induced small intestinal pathology that is induced by bone marrow cell-derived interleukin-18.

Peroral infection with Toxoplasma gondii results in a Th1-type immunopathology characterized by small intestinal necrosis and is dependent on IL-18. In the present study, we investigated whether treatment with IL-18 binding protein (IL-18bp) prevents ileal pathology. We observed increased expression of IL-18bp in intestinal biopsies of mice following infection. Whereas small intestines of control mice showed severe necrosis with complete destruction of the small intestinal architecture, mice treated with IL-18bp daily displayed only mild inflammatory changes including flattening of villi and edema in the space between the epithelium and lamina propria. Small intestinal parasite loads and concentrations of pro-inflammatory cytokines did not differ in control and IL-18bp-treated mice. Binding of IL-18 to immobilized IL-18bp revealed a remarkably slow dissociation rate, indicating high affinity. Using chimeric mice we observed that bone marrow-derived rather than stromal cells were the primary source of IL-18 that resulted in small intestinal pathology following peroral infection with T. gondii. In conclusion, the results presented here suggest that IL-18bp may be an effective and safe treatment for small intestinal inflammation. Antigen-presenting rather than epithelial cells appear to be the main source of IL-18 in T. gondii-induced small intestinal inflammation.

[Campylobacteriosis of man : livestock as reservoir for Campylobacter species]. / Die Campylobacteriose des Menschen : Nutztiere als Reservoir für Campylobacter-Arten.

Over the last few years, infections with Campylobacter have significantly increased in Europe and Germany and these bacteria have even surpassed Salmonella as the most prevalent bacteria, causing gastroenteritis. Especially contamination during the handling and consumption of meat products seems to be the most important risk factor which plays a prominent role for transmission to man. In addition, contact with pets and other animals, drinking raw or improperly pasteurized milk, and the tenacity of Campylobacter in different environments, especially water, have also to be considered for an adequate risk assessment. Besides gastroenteritis, arthralgia, and Guillain-Barré syndrome are important clinical complications of Campylobacter infections in man. At the same time, it is mostly unclear why the course of infection in man and in reservoir animals differs significantly, especially as only a few classical bacterial virulence factors have been identified so far. For these reasons, the development of efficient prevention strategies is of utmost importance in order to control campylobacteriosis.

Selective gelatinase blockage ameliorates acute DSS colitis.

In the experimental models of intestinal inflammation and humans with inflammatory bowel diseases (IBD), increased levels of the matrix metalloproteinases (MMPs), MMP-2 and -9 (also referred to as gelatinase A and B, respectively), in inflamed tissue sites can be detected. In the presented study, we investigated potential beneficial effects exerted by doxycycline nonselectively blocking MMPs and the selective gelatinase inhibitor RO28-2653 in acute DSS colitis. Treatment with either compound for 8 days ameliorated clinical colitis pathology with a superior outcome in RO28-2653-treated animals. As compared to placebo controls, histopathological changes in the colon were less distinct following MMP blockage and IL-6 secretion in ex vivo biopsies was downregulated, paralleled by a diminished influx of pro-inflammatory immune cells and lack of overgrowth of the colonic lumen by potentially pro-inflammatory Escherichia coli of the commensal colon flora. We conclude that selective gelatinase inhibition not only exerts beneficial effects by disrupting the vicious cycle of positive feedback between immune cell stimulation and MMP induction but also prevents overgrowth of the colonic lumen by pro-inflammatory E. coli despite a lack of direct anti-bacterial properties, thus unaffecting the commensal gut microbiota. These findings put RO28-2653 into a center stage for development of intervention strategies in human IBD.

What you eat is what you get: Novel Campylobacter models in the quadrangle relationship between nutrition, obesity, microbiota and susceptibility to infection.

Enterocolitis caused by Campylobacter jejuni-infections represents an important socioeconomic burden worldwide. Recent results from novel murine infection models reveal that the intestinal microbiota is essential for maintaining colonization resistance against C. jejuni. We extended these studies to investigate the role of nutrition and obesity in susceptibility to C. jejuni-infection. Gnotobiotic (GB) mice generated by antibiotic treatment, which were fed with a human cafeteria diet (CAF), as well as obese (ob/ob) mice with a conventional microbiota harbored higher Escherichia coli loads in their colon as compared to respective controls. Following oral infection, C. jejuni 43431 ATCC readily colonized the intestines of CAF and ob/ob mice, whereas GB mice fed with a standard chow (MUD) eradicated the pathogen within days. Furthermore, live C. jejuni translocated into mesenteric lymph nodes of CAF, but not MUD mice. Strikingly, stably infected animals developed enterocolitis as indicated by increased numbers of immune and apoptotic cells in the colon in situ. We conclude that a specific human diet and obesity render mice susceptible to C. jejuni infection. The corresponding murine models are excellently suited for the study of C. jejuni pathogenesis and will help to get further insights into interplays between C. jejuni, microbiota, diet, obesity and immunity.

The distinct roles of MMP-2 and MMP-9 in acute DSS colitis.

Expression of gelatinases A and B, also referred to matrixmetalloproteinases (MMP)-2 and -9, respectively, is increased in inflamed tissues of experimental intestinal inflammation and humans with inflammatory bowel disease (IBDs). Given that we recently reported that treatment with the selective gelatinase inhibitor RO28-2653 ameliorates acute dextrane sulfate sodium (DSS) colitis, we asked whether gelatinase A or B expression is pivotal in mediating large intestinal inflammation. Results from our study reveal that symptoms of acute DSS colitis as well as histopathological colonic changes were ameliorated in MMP-2-, but not MMP-9-deficient mice, and were paralleled by a diminished influx of immune cells. In MMP-2-deficient mice, we observed lower expression of pro-inflammatory cytokines including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IL-6 in colonic biopsies and less overgrowth of the colonic lumen by potentially pro-inflammatory enterobacteria from the commensal gut microbiota. We conclude that rather MMP-2 than MMP-9 is causative for the establishment of DSS colitis in mice. The discrepancy of these data to prior reports might be due to substantial differences in the intestinal microbiota composition of the mice bred at different animal facilities impacting susceptibility to inflammatory stimuli. Consequently, a detailed survey of the gut microbiota should be implemented in immunological/inflammatory studies in the future in order to allow comparison of data from different facilities.

Exacerbation of murine ileitis by Toll-like receptor 4 mediated sensing of lipopolysaccharide from commensal Escherichia coli.

BACKGROUND: In the course of inflammatory bowel diseases (IBD) and acute murine ileitis following peroral Toxoplasma gondii infection, commensal Escherichia coli accumulate at inflamed mucosal sites and aggravate small intestinal immunopathology. AIM: To unravel the molecular mechanisms by which commensal E coli exacerbate ileitis. METHODS: Ileitis was investigated in mice that lack Toll-like receptors (TLR) 2 or 4, specific for bacterial lipoproteins (LP) or lipopolysaccharide (LPS), respectively. Gnotobiotic mice, in which any cultivable gut bacteria were eradicated by antibiotic treatment, were used to study the role of LPS in ileitis. RESULTS: Microbiological analyses revealed that E coli increase in the inflamed ileum. TLR4(-/-), but not TLR2(-/-), mice displayed reduced mortality and small intestinal immunopathology. Decreased interferon (IFN)-gamma and nitric oxide (NO) levels in the inflamed terminal ileum of TLR4(-/-) mice indicated that TLR4 signalling aggravates ileitis via local mediator release from immune cells. E coli strains isolated from the inflamed ileum activated cultured mouse macrophages and induced TLR4-dependent nuclear factor kappaB activation and NO production in human embryonic kidney 293 cells and in peritoneal macrophages, respectively. Most strikingly, in contrast with wild-type mice, gnotobiotic TLR4(-/-) mice were protected from induction of ileitis by treatment with purified E coli lipid A or colonisation with live E coli. Finally, prophylactic treatment with the LPS scavenger polymyxin B ameliorated T gondii-induced ileitis. CONCLUSION: These findings highlight the innate immune system as a key player in T gondii-induced ileal immunopathology. Treatment with LPS or TLR4 antagonists may represent a novel strategy for prophylaxis and/or therapy of small intestinal inflammation in IBD.

Prevalence of Clostridium difficile toxins A and B and Clostridium perfringens enterotoxin A in stool samples of patients with antibiotic-associated diarrhea.

BACKGROUND: Antibiotic-associated diarrhea (AAD) is a major nosocomial as well as a community health problem. Clostridium difficile toxins (CDT) can be detected in only 10-25% of patients with AAD. The role of Clostridium perfringens enterotoxin A (CPEnt) as a cause of AAD remains to be elucidated. We, therefore, prospectively investigated the prevalences of both CPEnt and CDT in stool samples of patients with AAD. MATERIALS AND METHODS: A total of 693 stool samples consecutively submitted to our department from patients with AAD were screened for CDT and CPEnt using commercially available enzyme-linked immunoassays (ELISA). C. difficile and C. perfringens were detected by standard culture techniques. In addition, samples being CPEnt positive and/ or harboring C. perfringens were screened for the CPEnt gene by duplex PCR. RESULTS: CDT was detected in 79 (11.4%) of 693 stool samples. Of these, 49 (62.0%) harbored C. difficile. In one (0.14%) of 693 samples, CPEnt could be detected by ELISA. This respective CPEnt-positive stool sample also harbored C. perfringens. 147 (21.2%) of all stool samples were culture positive for C. perfringens. We did not detect samples positive for both CPEnt and CDT. In five (3.4%) of 147 C. perfringens isolates, the CPEnt gene could be detected by duplex PCR. PCR was positive in two (40%) of the five stool samples harboring CPEnt gene-positive C. perfringens isolates. CONCLUSION: The present prospective study revealed a prevalence of CDT of 11.4%, whereas the prevalence of CPEnt was less than 1%. Routine screening of stool samples for CPEnt does not appear to be justified in patients with AAD.