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Search Results: 1 - 10 of 555885 matches for " Anja A. Kühl "
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Combined Pulse Electroporation – A Novel Strategy for Highly Efficient Transfection of Human and Mouse Cells
Thorsten Stroh,Ulrike Erben,Anja A. Kühl,Martin Zeitz,Britta Siegmund
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0009488
Abstract: The type of a nucleic acid and the type of the cell to be transfected generally affect the efficiency of electroporation, the versatile method of choice for gene regulation studies or for recombinant protein expression. We here present a combined square pulse electroporation strategy to reproducibly and efficiently transfect eukaryotic cells. Cells suspended in a universal buffer system received an initial high voltage pulse that was continuously combined with a subsequent low voltage pulse with independently defined electric parameters of the effective field and the duration of each pulse. At comparable viable cell recoveries and transfection efficiencies of up to 95% of all cells, a wide variety of cells especially profited from this combined pulse strategy by high protein expression levels of individual cells after transfection. Long-term silencing of gene expression by transfected small interfering RNA was most likely due to the uptake of large nucleic acid amounts as shown by direct detection of fluorochromated small interfering RNA. The highly efficient combined pulse electroporation strategy enables for external regulation of the number of naked nucleic acid molecules taken up and can be easily adapted for cells considered difficult to transfect.
Small Intestinal Nematode Infection of Mice Is Associated with Increased Enterobacterial Loads alongside the Intestinal Tract
Sebastian Rausch, Josephin Held, André Fischer, Markus M. Heimesaat, Anja A. Kühl, Stefan Bereswill, Susanne Hartmann
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0074026
Abstract: Parasitic nematodes are potent modulators of immune reactivity in mice and men. Intestinal nematodes live in close contact with commensal gut bacteria, provoke biased Th2 immune responses upon infection, and subsequently lead to changes in gut physiology. We hypothesized that murine nematode infection is associated with distinct changes of the intestinal bacterial microbiota composition. We here studied intestinal inflammatory and immune responses in mice following infection with the hookworm Heligmosomoides polygyrus bakeri and applied cultural and molecular techniques to quantitatively assess intestinal microbiota changes in the ileum, cecum and colon. At day 14 post nematode infection, mice harbored significantly higher numbers of γ-Proteobacteria/Enterobacteriaceae and members of the Bacteroides/Prevotella group in their cecum as compared to uninfected controls. Abundance of Gram-positive species such as Lactobacilli, Clostridia as well as the total bacterial load was not affected by worm infection. The altered microbiota composition was independent of the IL-4/-13 – STAT6 signaling axis, as infected IL-4Rα-/- mice showed a similar increase in enterobacterial loads. In conclusion, infection with an enteric nematode is accompanied by distinct intestinal microbiota changes towards higher abundance of gram-negative commensal species at the small intestinal site of infection (and inflammation), but also in the parasite-free large intestinal tract. Further studies should unravel the impact of nematode-induced microbiota changes in inflammatory bowel disease to allow for a better understanding of how theses parasites interfere with intestinal inflammation and bacterial communities in men.
Characterization of Chromosomal Instability in Murine Colitis-Associated Colorectal Cancer
Marco Gerling,Rainer Glauben,Jens K. Habermann,Anja A. Kühl,Christoph Loddenkemper,Hans-Anton Lehr,Martin Zeitz,Britta Siegmund
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0022114
Abstract: Patients suffering from ulcerative colitis (UC) bear an increased risk for colorectal cancer. Due to the sparsity of colitis-associated cancer (CAC) and the long duration between UC initiation and overt carcinoma, elucidating mechanisms of inflammation-associated carcinogenesis in the gut is particularly challenging. Adequate murine models are thus highly desirable. For human CACs a high frequency of chromosomal instability (CIN) reflected by aneuploidy could be shown, exceeding that of sporadic carcinomas. The aim of this study was to analyze mouse models of CAC with regard to CIN. Additionally, protein expression of p53, beta-catenin and Ki67 was measured to further characterize murine tumor development in comparison to UC-associated carcinogenesis in men.
Impact of Campylobacter jejuni cj0268c Knockout Mutation on Intestinal Colonization, Translocation, and Induction of Immunopathology in Gnotobiotic IL-10 Deficient Mice
Markus M. Heimesaat, Raimond Lugert, André Fischer, Marie Alutis, Anja A. Kühl, Andreas E. Zautner, A. Malik Tareen, Ulf B. G?bel, Stefan Bereswill
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0090148
Abstract: Background Although Campylobacter jejuni infections have a high prevalence worldwide and represent a significant socioeconomic burden, the underlying molecular mechanisms of induced intestinal immunopathology are still not well understood. We have recently generated a C. jejuni mutant strain NCTC11168::cj0268c, which has been shown to be involved in cellular adhesion and invasion. The immunopathological impact of this gene, however, has not been investigated in vivo so far. Methodology/Principal Findings Gnotobiotic IL-10 deficient mice were generated by quintuple antibiotic treatment and perorally infected with C. jejuni mutant strain NCTC11168::cj0268c, its complemented version (NCTC11168::cj0268c-comp-cj0268c), or the parental strain NCTC11168. Kinetic analyses of fecal pathogen loads until day 6 post infection (p.i.) revealed that knockout of cj0268c did not compromise intestinal C. jejuni colonization capacities. Whereas animals irrespective of the analysed C. jejuni strain developed similar clinical symptoms of campylobacteriosis (i.e. enteritis), mice infected with the NCTC11168::cj0268c mutant strain displayed significant longer small as well as large intestinal lengths indicative for less distinct C. jejuni induced pathology when compared to infected control groups at day 6 p.i. This was further supported by significantly lower apoptotic and T cell numbers in the colonic mucosa and lamina propria, which were paralleled by lower intestinal IFN-γ and IL-6 concentrations at day 6 following knockout mutant NCTC11168::cj0268c as compared to parental strain infection. Remarkably, less intestinal immunopathology was accompanied by lower IFN-γ secretion in ex vivo biopsies taken from mesenteric lymphnodes of NCTC11168::cj0268c infected mice versus controls. Conclusion/Significance We here for the first time show that the cj0268c gene is involved in mediating C. jejuni induced immunopathogenesis in vivo. Future studies will provide further deep insights into the immunological and molecular interplays between C. jejuni and innate immunity in human campylobacteriosis.
Comprehensive Postmortem Analyses of Intestinal Microbiota Changes and Bacterial Translocation in Human Flora Associated Mice
Markus M. Heimesaat, Silvia Boelke, André Fischer, Lea-Maxie Haag, Christoph Loddenkemper, Anja A. Kühl, Ulf B. G?bel, Stefan Bereswill
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0040758
Abstract: Background Postmortem microbiological examinations are performed in forensic and medical pathology for defining uncertain causes of deaths and for screening of deceased tissue donors. Interpretation of bacteriological data, however, is hampered by false-positive results due to agonal spread of microorganisms, postmortem bacterial translocation, and environmental contamination. Methodology/Principal Findings We performed a kinetic survey of naturally occurring postmortem gut flora changes in the small and large intestines of conventional and gnotobiotic mice associated with a human microbiota (hfa) applying cultural and molecular methods. Sacrificed mice were kept under ambient conditions for up to 72 hours postmortem. Intestinal microbiota changes were most pronounced in the ileal lumen where enterobacteria and enterococci increased by 3–5 orders of magnitude in conventional and hfa mice. Interestingly, comparable intestinal overgrowth was shown in acute and chronic intestinal inflammation in mice and men. In hfa mice, ileal overgrowth with enterococci and enterobacteria started 3 and 24 hours postmortem, respectively. Strikingly, intestinal bacteria translocated to extra-intestinal compartments such as mesenteric lymphnodes, spleen, liver, kidney, and cardiac blood as early as 5 min after death. Furthermore, intestinal tissue destruction was characterized by increased numbers of apoptotic cells and neutrophils within 3 hours postmortem, whereas counts of proliferative cells as well as T- and B-lymphocytes and regulatory T-cells decreased between 3 and 12 hours postmortem. Conclusions/Significance We conclude that kinetics of ileal overgrowth with enterobacteria and enterococci in hfa mice can be used as an indicator for compromized intestinal functionality and for more precisely defining the time point of death under defined ambient conditions. The rapid translocation of intestinal bacteria starting within a few minutes after death will help to distinguish between relevant bacteria and secondary contaminants thus providing important informations for routine applications and future studies in applied microbiology, forensic pathology, and criminal medicine.
Anti-Inflammatory Effects of Resveratrol, Curcumin and Simvastatin in Acute Small Intestinal Inflammation
Stefan Bereswill,Melba Mu?oz,André Fischer,Rita Plickert,Lea-Maxie Haag,Bettina Otto,Anja A. Kühl,Christoph Loddenkemper,Ulf B. G?bel,Markus M. Heimesaat
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015099
Abstract: The health beneficial effects of Resveratrol, Curcumin and Simvastatin have been demonstrated in various experimental models of inflammation. We investigated the potential anti-inflammatory and immunomodulatory mechanisms of the above mentioned compounds in a murine model of hyper-acute Th1-type ileitis following peroral infection with Toxoplasma gondii.
Campylobacter jejuni Induces Acute Enterocolitis in Gnotobiotic IL-10?/? Mice via Toll-Like-Receptor-2 and -4 Signaling
Lea-Maxie Haag, André Fischer, Bettina Otto, Rita Plickert, Anja A. Kühl, Ulf B. G?bel, Stefan Bereswill, Markus M. Heimesaat
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0040761
Abstract: Background Campylobacter jejuni is a leading cause of foodborne bacterial enterocolitis worldwide. Investigation of immunopathology is hampered by a lack of suitable vertebrate models. We have recently shown that gnotobiotic mice as well as conventional IL-10?/? animals are susceptible to C. jejuni infection and develop intestinal immune responses. However, clinical symptoms of C. jejuni infection were rather subtle and did not reflect acute bloody diarrhea seen in human campylobacteriosis. Methodology/Principal Findings In order to overcome these limitations we generated gnotobiotic IL-10?/? mice by quintuple antibiotic treatment starting right after weaning. The early treatment was essential to prevent these animals from chronic colitis. Following oral infection C. jejuni colonized the gastrointestinal tract at high levels and induced acute enterocolitis within 7 days as indicated by bloody diarrhea and pronounced histopathological changes of the colonic mucosa. Immunopathology was further characterized by increased numbers of apoptotic cells, regulatory T-cells, T- and B-lymphocytes as well as elevated TNF-α, IFN-γ, and MCP-1 concentrations in the inflamed colon. The induction of enterocolitis was specific for C. jejuni given that control animals infected with a commensal E. coli strain did not display any signs of disease. Most strikingly, intestinal immunopathology was ameliorated in mice lacking Toll-like-receptors-2 or -4 indicating that C. jejuni lipoproteins and lipooligosaccharide are essential for induction and progression of immunopathology. Conclusion/Significance Gnotobiotic IL-10?/? mice develop acute enterocolitis following C. jejuni infection mimicking severe episodes of human campylobacteriosis and are thus well suited to further dissect mechanisms underlying Campylobacter infections in vivo.
Intestinal Microbiota Shifts towards Elevated Commensal Escherichia coli Loads Abrogate Colonization Resistance against Campylobacter jejuni in Mice
Lea-Maxie Haag, André Fischer, Bettina Otto, Rita Plickert, Anja A. Kühl, Ulf B. G?bel, Stefan Bereswill, Markus M. Heimesaat
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0035988
Abstract: Background The zoonotic pathogen Campylobacter jejuni is a leading cause of bacterial foodborne enterocolitis in humans worldwide. The understanding of immunopathology underlying human campylobacteriosis is hampered by the fact that mice display strong colonization resistance against the pathogen due to their host specific gut microbiota composition. Methodology/Principal Findings Since the microbiota composition changes significantly during intestinal inflammation we dissected factors contributing to colonization resistance against C. jejuni in murine ileitis, colitis and in infant mice. In contrast to healthy animals C. jejuni could stably colonize mice suffering from intestinal inflammation. Strikingly, in mice with Toxoplasma gondii-induced acute ileitis, C. jejuni disseminated to mesenteric lymphnodes, spleen, liver, kidney, and blood. In infant mice C. jejuni infection induced enterocolitis. Mice suffering from intestinal inflammation and C. jejuni susceptible infant mice displayed characteristical microbiota shifts dominated by increased numbers of commensal Escherichia coli. To further dissect the pivotal role of those distinct microbiota shifts in abrogating colonization resistance, we investigated C. jejuni infection in healthy adult mice in which the microbiota was artificially modified by feeding live commensal E. coli. Strikingly, in animals harboring supra-physiological intestinal E. coli loads, colonization resistance was significantly diminished and C. jejuni infection induced enterocolitis mimicking key features of human campylobacteriosis. Conclusion/Significance Murine colonization resistance against C. jejuni is abrogated by changes in the microbiota composition towards elevated E. coli loads during intestinal inflammation as well as in infant mice. Intestinal inflammation and microbiota shifts thus represent potential risk factors for C. jejuni infection. Corresponding interplays between C. jejuni and microbiota might occur in human campylobacteriosis. Murine models introduced here mimick key features of human campylobacteriosis and allow for further analysis of immunological and molecular mechanisms of C. jejuni – host interactions.
Stable T-bet+GATA-3+ Th1/Th2 Hybrid Cells Arise In Vivo, Can Develop Directly from Naive Precursors, and Limit Immunopathologic Inflammation
Michael Peine,Sebastian Rausch equal contributor,Caroline Helmstetter equal contributor,Anja Fr?hlich,Ahmed N. Hegazy,Anja A. Kühl,Christoph G. Grevelding,Thomas H?fer,Susanne Hartmann,Max L?hning
PLOS Biology , 2013, DOI: 10.1371/journal.pbio.1001633
Abstract: Differentiated T helper (Th) cell lineages are thought to emerge from alternative cell fate decisions. However, recent studies indicated that differentiated Th cells can adopt mixed phenotypes during secondary immunological challenges. Here we show that natural primary immune responses against parasites generate bifunctional Th1 and Th2 hybrid cells that co-express the lineage-specifying transcription factors T-bet and GATA-3 and co-produce Th1 and Th2 cytokines. The integration of Th1-promoting interferon (IFN)-γ and interleukin (IL)-12 signals together with Th2-favoring IL-4 signals commits naive Th cells directly and homogeneously to the hybrid Th1/2 phenotype. Specifically, IFN-γ signals are essential for T-bet+GATA-3+ cells to develop in vitro and in vivo by breaking the dominance of IL-4 over IL-12 signals. The hybrid Th1/2 phenotype is stably maintained in memory cells in vivo for months. It resists reprogramming into classic Th1 or Th2 cells by Th1- or Th2-promoting stimuli, which rather induce quantitative modulations of the combined Th1 and Th2 programs without abolishing either. The hybrid phenotype is associated with intermediate manifestations of both Th1 and Th2 cell properties. Consistently, hybrid Th1/2 cells support inflammatory type-1 and type-2 immune responses but cause less immunopathology than Th1 and Th2 cells, respectively. Thus, we propose the self-limitation of effector T cells based on the stable cell-intrinsic balance of two opposing differentiation programs as a novel concept of how the immune system can prevent excessive inflammation.
Novel Murine Infection Models Provide Deep Insights into the “Ménage à Trois” of Campylobacter jejuni, Microbiota and Host Innate Immunity
Stefan Bereswill,André Fischer,Rita Plickert,Lea-Maxie Haag,Bettina Otto,Anja A. Kühl,Javid I. Dashti,Andreas E. Zautner,Melba Mu?oz,Christoph Loddenkemper,Uwe Gro?,Ulf B. G?bel,Markus M. Heimesaat
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0020953
Abstract: Although Campylobacter jejuni-infections have a high prevalence worldwide and represent a significant socioeconomic burden, it is still not well understood how C. jejuni causes intestinal inflammation. Detailed investigation of C. jejuni-mediated intestinal immunopathology is hampered by the lack of appropriate vertebrate models. In particular, mice display colonization resistance against this pathogen.
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