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Abstract

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 naïve 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.

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Abstract

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.

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Abstract

Escherichia coli K12 (EcK12) is commonly used for gene technology purposes and regarded as a security strain due to its inability to adhere to epithelial cells. The conventional intestinal microbiota composition is critical for physiological colonization resistance against most bacterial species including pathogens. We were therefore interested whether intestinal colonization by a genetically modified EcK12 (W3110) strain carrying a chloramphenicol resistance cassette was facilitated following broad-spectrum antibiotic treatment eradicating the intestinal microbiota or induction of small intestinal inflammation accompanied by distinct microbiota shifts. Whereas conventional C57BL/6 and BALB/c mice had virtually expelled the EcK12 (W3110) strain within the first 3 days upon peroral infection, EcK12 (W3110) could establish within the small and large intestines of gnotobiotic mice generated by quintuple antibiotic treatment. Gnotobiotic mice perorally infected with EcK12 (W3110) plus fecal transplant from conventional donors harbored lower intestinal EcK12 (W3110) loads compared to animals challenged with EcK12 (W3110) alone. Furthermore, EcK12 (W3110) infection of conventional mice after but not before induction of ileitis resulted in stable colonization of ileum and colon by EcK12 (W3110). Taken together, broad-spectrum antibiotic treatment and intestinal inflammation compromise colonization resistance and thus facilitate colonization of the intestinal tract with genetically modified EcK12 security strains.

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European Journal of Microbiology and Immunology
Authors: Marie E. Alutis, Ursula Grundmann, Ulrike Hagen, André Fischer, Anja A. Kühl, Ulf B. Göbel, Stefan Bereswill, and Markus M. Heimesaat

Increased levels of the matrix metalloproteinases (MMPs)-2 and -9 (also referred to gelatinase-A and -B, respectively) can be detected in the inflamed gut. We have recently shown that synthetic gelatinase blockage reduces colonic apoptosis and pro-inflammatory immune responses following murine Campylobacter (C.) jejuni infection. In order to dissect whether MMP-2 and/or MMP-9 is involved in mediating C. jejuni-induced immune responses, infant MMP-2-/-, MMP-9-/-, and wildtype (WT) mice were perorally infected with the C. jejuni strain B2 immediately after weaning. Whereas, at day 2 postinfection (p.i.), fecal C. jejuni B2 loads were comparable in mice of either genotype, mice expelled the pathogen from the intestinal tract until day 4 p.i. Six days p.i., colonic MMP-2 but not MMP-9 mRNA was upregulated in WT mice. Remarkably, infected MMP-2-/- mice exhibited less frequent abundance of blood in feces, less distinct colonic histopathology and apoptosis, lower numbers of effector as well as innate and adaptive immune cells within the colonic mucosa, and higher colonic IL-22 mRNA levels as compared to infected WT mice. In conclusion, these results point towards an important role of MMP-2 in mediating C. jejuni-induced intestinal immunopathogenesis.

Open access
European Journal of Microbiology and Immunology
Authors: Sascha Cording, Diana Fleissner, Markus M. Heimesaat, Stefan Bereswill, Christoph Loddenkemper, Satoshi Uematsu, Shizuo Akira, Alf Hamann, and Jochen Huehn

Abstract

Compelling evidence demonstrates that intestinal commensal microbiota modulate conventional and regulatory T cell (Treg) responses that are required for effective host defence against pathogens and avoidance of autoimmunity and other immunopathologic conditions. Here, we investigated the contribution of the commensal microbiota and Toll-like receptor (TLR) signaling to homeostasis of Foxp3 conventional CD4+ T cells and Foxp3+ Tregs. Upon long-term antibiotics treatment, we observed a significant reduction of conventional CD4+ T cell proliferation in a systemic manner, whereas Foxp3+ Treg proliferation was locally impaired in gut-draining mesenteric lymph nodes and Peyer's patches. The proliferative response to microbial components was not mediated by TLRs as MyD88- and various TLR-deficient mice displayed normal or even increased conventional T cell and Foxp3+ Treg proliferation. Thus, commensal microbiota-derived stimuli support cycling of both conventional CD4+ T cells and Foxp3+ Tregs with TLR-mediated recognition of bacterial components not being the major mechanism controlling microbiota-driven T cell homeostasis.

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European Journal of Microbiology and Immunology
Authors: Markus M. Heimesaat, Lea-Maxie Haag, André Fischer, Bettina Otto, Anja A. Kühl, Ulf B. Göbel, and Stefan Bereswill

Abstract

Campylobacter jejuni is among the most frequently reported bacterial pathogens causing diarrhea in humans worldwide. We recently reported a murine infection model mimicking key features of human campylobacteriosis. Six days following oral C. jejuni infection immediately after weaning, infant mice developed acute enterocolitis resolving within 2 weeks. Thereafter, C. jejuni could still be isolated from the intestines of asymptomatic mice at low levels accompanied by distinct immune responses, both at intestinal and extra-intestinal locations. We here show that, at day 103 post infection (p.i.), long-term C. jejuni-infected mice exhibited higher numbers of T lymphocytes in liver, lung, kindneys, and cardiac muscle as compared to uninfected controls. In addition, B lymphocytes were slightly higher, but macrophage numbers were significantly lower in liver and lung of C. jejuni-infected versus naive mice. As compared to uninfected control animals, proliferating cells were significantly lower in liver, lung, kidneys, cardiac muscle, and spleen at day 103 p.i., whereas more apoptotic cells were abundant in the spleen with predominance in the red pulp. This study underlines that post-infectious, immunological sequelae at extra-intestinal locations are of importance even in asymptomatic long-term C. jejuni carriers and need to be further studied in order to unravel the underlying molecular mechanisms.

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European Journal of Microbiology and Immunology
Authors: Markus M. Heimesaat, Gernot Reifenberger, Viktoria Vicena, Anita Illes, Gabriella Horvath, Andrea Tamas, Balazs D. Fulop, Stefan Bereswill, and Dora Reglodi

Pituitary adenylate cyclase activating polypetide (PACAP) constitutes a neuropeptide that is widely distributed in the host exerting essential cytoprotective properties, whereas PACAP−/− mice display increased susceptibility to distinct immunopathological conditions. The orchestrated interplay between the gut microbiota and the host is pivotal in immune homeostasis and resistance to disease. Potential pertubations of the intestinal microbiota in PACAP−/− mice, however, have not been addressed so far. For the first time, we performed a comprehensive survey of the intestinal microbiota composition in PACAP−/− and wildtype (WT) mice starting 2 weeks postpartum until 18 months of age applying quantitative culture-independent techniques. Fecal enterobacteria and enterococci were lower in PACAP−/− than WT mice aged 1 month and ≥6 months, respectively. Whereas Mouse Intestinal Bacteroides were slightly higher in PACAP−/− versus WT mice aged 1 and 6 months, this later in life held true for Bacteroides/Prevotella spp. (≥12 months) and lactobacilli (>15 months of age). Strikingly, health-beneficial bifidobacteria were virtually absent in the intestines of PACAP−/− mice, even when still breastfed. In conclusion, PACAP deficiency is accompanied by distinct changes in fecal microbiota composition with virtually absent bifidobacteria as a major hallmark that might be linked to increased susceptibility to disease.

Open access
European Journal of Microbiology and Immunology
Authors: Manja Boehm, Daniel Simson, Ulrike Escher, Anna-Maria Schmidt, Stefan Bereswill, Nicole Tegtmeyer, Steffen Backert, and Markus M. Heimesaat

Campylobacter jejuni is a major food-borne zoonotic pathogen, responsible for a large proportion of bacterial gastroenteritis cases, as well as Guillian-Barré and Miller-Fisher syndromes. During infection, tissue damage is mainly caused by bacteria invading epithelial cells and traversing the intestinal barrier. C. jejuni is able to enter the lamina propria and the bloodstream and may move into other organs, such as spleen, liver, or mesenteric lymph nodes. However, the involved molecular mechanisms are not fully understood. C. jejuni can transmigrate effectively across polarized intestinal epithelial cells mainly by the paracellular route using the serine protease high-temperature requirement A (HtrA). However, it appears that HtrA has a dual function, as it also acts as a chaperone, interacting with denatured or misfolded periplasmic proteins under stress conditions. Here, we review recent progress on the role of HtrA in C. jejuni pathogenesis. HtrA can be transported into the extracellular space and cleaves cell-to-cell junction factors, such as E-cadherin and probably others, disrupting the epithelial barrier and enabling paracellular transmigration of the bacteria. The secretion of HtrA is a newly discovered strategy also utilized by other pathogens. Thus, secreted HtrA proteases represent highly attractive targets for anti-bacterial treatment and may provide a suitable candidate for vaccine development.

Open access

Abstract

Introduction

Surrogate endpoints are widely used in clinical trials, especially in situations where the endpoint of interest is not directly observable or to avoid long trial periods. A typical example for this case is frequently found in clinical trials in oncology, where overall survival (OS) as endpoint of interest and progression free survival (PFS) as surrogate endpoint are discriminated.

Methods

Based on the perspective of case definitions on surrogate endpoints, we provide a formal definition of such endpoints followed by a description of the structure of surrogate endpoints.

Results

Surrogate endpoints can be considered as case definitions for the endpoint of interest. Therefore, the performance of surrogate endpoints can be described using the classical terminology of diagnostic tests including sensitivity and specificity. Since such endpoints always focus on sensitivity with necessarily reduced specificity, efficacy estimates based on such endpoints are in general biased.

Conclusion

The abovementioned has to be taken into account while interpreting the results of clinical trials and should not be ignored while planning or conducting a study.

Open access
European Journal of Microbiology and Immunology
Authors: Marie E. Alutis, Ursula Grundmann, André Fischer, Ulrike Hagen, Anja A. Kühl, Ulf B. Göbel, Stefan Bereswill, and Markus M. Heimesaat

Matrix metalloproteinases (MMP)-2 and -9 (also referred to gelatinases-A and -B, respectively) are upregulated in the inflamed gut of mice and men. We recently demonstrated that synthetic gelatinase blockage reduced large intestinal pro-inflammatory immune responses and apoptosis following murine Campylobacter (C.) jejuni infection. In order to address which gelatinase mediates C. jejuni-induced immune responses, gnotobiotic MMP-2−/−, MMP-9−/−, and wildtype (WT) mice were generated by broadspectrum antibiotic treatment and perorally infected with C. jejuni strain 81-176. The pathogen stably colonized the murine intestinal tract irrespective of the genotype but did not translocate to extra-intestinal compartments. At days 8 and 14 postinfection (p.i.), less pronounced colonic histopathological changes were observed in infected MMP-2−/− mice, less distinct epithelial apoptosis, but more epithelial proliferation in both MMP-2−/− and MMP-9−/− mice, as compared to WT controls. Reduced immune responses in gelatinase- deficient mice were characterized by lower numbers of effector as well as innate and adaptive immune cells within the colonic mucosa and lamina propria. The expression of IL-22, IL-18, IL-17A, and IL-1β mRNA was higher in the colon of MMP-2−/− as compared to WT mice. In conclusion, both MMP-2 and MMP-9 are differentially involved in mediating C. jejuni-induced intestinal immunopathology.

Open access