[Show abstract][Hide abstract] ABSTRACT: A dysfunction of the Na(+)/H(+) exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine. We previously reported a strong decrease in the NHERF family member PDZK1 (NHERF3), which binds to NHE3 and regulates its function in a mouse model of colitis. The present study investigates whether a causal relationship exists between the decreased PDZK1 expression and the NHE3 dysfunction in human and murine intestinal inflammation. Biopsies from the colon of patients with ulcerative colitis, murine inflamed ileal and colonic mucosa, NHE3-transfected Caco-2BBe colonic cells with short hairpin RNA (shRNA) knockdown of PDZK1, and Pdzk1-gene-deleted mice were studied. PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not. Inflamed human and murine intestinal tissues displayed correct brush border localization of NHE3 but reduced acid-activated NHE3 transport activity. A similar NHE3 transport defect was observed when PDZK1 protein content was decreased by shRNA knockdown in Caco-2BBe cells or when enterocyte PDZK1 protein content was decreased to similar levels as found in inflamed mucosa by heterozygote breeding of Pdzk1-gene-deleted and WT mice. We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes. We therefore hypothesize that inflammation-induced loss of PDZK1 expression may contribute to the NHE3 dysfunction observed in the inflamed intestine.
Pflugers Archiv : European journal of physiology. 10/2014;
[Show abstract][Hide abstract] ABSTRACT: Necroptosis has emerged as an important pathway of programmed cell death in embryonic development, tissue homeostasis, immunity and inflammation. RIPK1 is implicated in inflammatory and cell death signalling and its kinase activity is believed to drive RIPK3-mediated necroptosis. Here we show that kinase-independent scaffolding RIPK1 functions regulate homeostasis and prevent inflammation in barrier tissues by inhibiting epithelial cell apoptosis and necroptosis. Intestinal epithelial cell (IEC)-specific RIPK1 knockout caused IEC apoptosis, villus atrophy, loss of goblet and Paneth cells and premature death in mice. This pathology developed independently of the microbiota and of MyD88 signalling but was partly rescued by TNFR1 (also known as TNFRSF1A) deficiency. Epithelial FADD ablation inhibited IEC apoptosis and prevented the premature death of mice with IEC-specific RIPK1 knockout. However, mice lacking both RIPK1 and FADD in IECs displayed RIPK3-dependent IEC necroptosis, Paneth cell loss and focal erosive inflammatory lesions in the colon. Moreover, a RIPK1 kinase inactive knock-in delayed but did not prevent inflammation caused by FADD deficiency in IECs or keratinocytes, showing that RIPK3-dependent necroptosis of FADD-deficient epithelial cells only partly requires RIPK1 kinase activity. Epidermis-specific RIPK1 knockout triggered keratinocyte apoptosis and necroptosis and caused severe skin inflammation that was prevented by RIPK3 but not FADD deficiency. These findings revealed that RIPK1 inhibits RIPK3-mediated necroptosis in keratinocytes in vivo and identified necroptosis as a more potent trigger of inflammation compared with apoptosis. Therefore, RIPK1 is a master regulator of epithelial cell survival, homeostasis and inflammation in the intestine and the skin.
[Show abstract][Hide abstract] ABSTRACT: Intestinal inflammation is often associated with an increased level of serotonin (5-HT), an important gastrointestinal signaling molecule involved in gut homeostasis through stimulation of specific receptors. In this study, we investigated the role of 5-HT7 receptor (5-HT7R) in the induction and development of intestinal inflammation using a mouse model of acute and chronic colitis and human patients with Crohn's disease (CD).
[Show abstract][Hide abstract] ABSTRACT: Intestinal epithelial cells (IEC) express toll-like receptors (TLR) that facilitate microbial recognition. Stimulation of TLR ligands induces a transient increase in epithelial cell shedding, a mechanism that serves the antibacterial and antiviral host defence of the epithelium and promotes elimination of intracellular pathogens. Although activation of the extrinsic apoptosis pathway has been described during inflammatory shedding, its functional involvement is currently unclear.
[Show abstract][Hide abstract] ABSTRACT: Infection may trigger clinically overt mucosal inflammation in patients with predisposition for inflammatory bowel disease. However, the impact of particular enteropathogenic microorganisms is ill-defined. In this study, the influence of murine norovirus (MNV) infection on clinical, histopathological, and immunological features of mucosal inflammation in the IL10-deficient (Il10) mouse model of inflammatory bowel disease was examined.
C57BL/6J and C3H/HeJBir wild-type and Il10 mice kept under special pathogen-free conditions and devoid of clinical and histopathological signs of mucosal inflammation were monitored after MNV infection for structural and functional intestinal barrier changes by in situ MNV reverse transcription PCR, transgene reporter gene technology, histology, flux measurements, quantitative real-time PCR, immunohistology, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. In addition, the influence of the enteric microbiota was analyzed in MNV-infected germfree Il10 mice.
Although MNV-infected wild-type mice remained asymptomatic, mucosal inflammation was noted in previously healthy Il10 mice 2 to 4 weeks after infection. MNV-induced changes in Il10 mice included increased paracellular permeability indicated by increased mucosal mannitol flux, reduced gene expression of tight junction molecules, and an enhanced rate of epithelial apoptosis. MNV-induced reduction of tight junction protein expression and inflammatory lesions were absent in germfree Il10 mice, whereas epithelial apoptosis was still observed.
Despite its subclinical course in wild-type animals, MNV causes epithelial barrier disruption in Il10 animals representing a potent colitogenic stimulus that largely depends on the presence of the enteric microbiota. MNV might thus trigger overt clinical disease in individuals with a nonsymptomatic predisposition for inflammatory bowel disease by impairment of the intestinal mucosa.
[Show abstract][Hide abstract] ABSTRACT: The rodents Pasteurellaceae have to be excluded from the specified pathogen free experimental animal facilities. Despite the biological and economic importance of Pasteurellaceae in relation to experimental animals just a few molecular based methods are available for their detection and identification. The aim of the present investigation was to develop a multiplex PCR assay allowing detection of all rodent Pasteurellaceae and identification of [Pasteurella] pneumotropica biotype Jawetz, [Pasteurella] pneumotropica biotype Heyl and [Actinobacillus] muris, as the most prevalent members of the group. For this, a Pasteurellaceae common forward primer located on the 16S rRNA gene was used in conjunction with four different reverse primers specific for [Pasteurella] pneumotropica biotype Jawetz, [Pasteurella] pneumotropica biotype Heyl, [Actinobacillus] muris and a common reverse primer for all rodent Pasteurellaceae, all targeting the 16S-23S rRNA internal transcribed spacer sequences. The performance characteristics of the assay were tested against 125 Pasteurellaceae isolates belonging to eleven different species and including 34 strains of [Pasteurella] pneumotropica biotype Jawetz, 44 strains of [Pasteurella] pneumotropica biotype Heyl and 37 strains of [Actinobacillus] muris. Additionally, eight other mouse associated bacterial species which could pose a diagnostic problem were included. The assay showed 100% sensitivity and specificity. Identification of the clinical isolates was validated by ITS profiling and when necessary by 16S rRNA gene sequencing. This multiplex PCR represents the first molecular tool able to detect and differentiate in a single assay among the Pasteurellaceae found in laboratory mouse and may become a reliable alternative to the present diagnostic methods.
Journal of microbiological methods 09/2013; · 2.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inflammatory bowel disease (IBD) summarises a group of chronic intestinal disorders with Crohn's disease and ulcerative colitis being most prominent. Though much effort is put into identification of causative factors, etiology is still not understood. Risk factors for disease development include genetic predisposition and environmental trigger. Crucial for identification and analysis of relevant factors are mouse models. Experimental IBD in mice occurs spontaneously or is induced by chemicals, cell transfer, pathogens, or genetic mutation. These models were utilized for analysing genetic contribution to disease and genotype-environmental interactions. In these studies, a variety of modifier loci were identified thereby demonstrating the complexity of disease. A major contribution of distal chromosome 3 was independently replicated in several studies. The first colitogenic QTL in this region was detected using the IL-10 deficient mouse model and called cytokine deficiency induced colitis susceptibility (Cdcs)1. This QTL contains at least three subintervals with independent genetic factors. This locus or defined subintervals were replicated in at least seven studies, using models based on dysregulation of innate or adaptive immunity or pathogen control. In this review we illustrate the various models used for genetic mapping of susceptibility to experimental IBD and display Cdcs1-related loci as well as the mechanism of their contribution identified so far.
[Show abstract][Hide abstract] ABSTRACT: Helicobacter pylori maintains colonization in its human host using a limited set of taxis sensors. TlpD is a proposed energy taxis sensor of H. pylori and dominant under environmental conditions of low bacterial energy yield. We studied the impact of H. pylori TlpD on colonization in vivo using a gerbil infection model, which closely mimics the gastric physiology of humans. A gerbil-adapted H. pylori strain, HP87 P7, showed energy-dependent behavior, while its isogenic tlpD mutant had lost it. A TlpD-complemented strain regained the wild type phenotype. Gerbil infections including the complemented strain demonstrated that TlpD was important for persistent infection in antrum and corpus, and suggested a role of TlpD in horizontal navigation and persistent corpus colonization. As a part of the full characterization of the model and to gain insight into the genetic basis of H. pylori adapation to the gerbil, we determined the complete genome sequences of the gerbil-adapted strain HP87 P7, two HP87 P7 tlpD mutants before and after gerbil passage, and the original human isolate HP87. The integrity of the genome including a functional cagPAI was maintained after gerbil adaptation. Genetic and phenotypical differences between the strains were observed. Major differences emerged between the gerbil-adapted strain and the human isolate, including evidence of recent recombination. Passage of the tlpD mutant through the gerbil selected for gain-of-function variation in a fucosyltransferase gene, futC/HP0093. In conclusion, a gerbil-adapted H. pylori strain with a stable genome has helped to establish important TlpD functions for persistent colonization in the stomach.
Infection and immunity 07/2013; · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using interleukin 10-deficient (IL-10(-/-) ) and wild-type mice monoassociated with either the adherent-invasive Escherichia coli UNC or the probiotic E. coli Nissle, the effect of a mild intestinal inflammation on the bacterial proteome was studied. Within 8 weeks, IL-10(-/-) mice monoassociated with E. coli UNC exhibited an increased expression of several proinflammatory markers in caecal mucosa. Escherichia coli Nissle-associated IL-10(-/-) mice did not do so. As observed previously for E. coli from mice with acute colitis, glycolytic enzymes were downregulated in intestinal E. coli UNC from IL-10(-/-) mice. In addition, the inhibitor of vertebrate C-type lysozyme, Ivy, was upregulated on messenger RNA (mRNA) and protein level in E. coli Nissle from IL-10(-/-) mice compared with E. coli UNC from these mice. Higher expression of Ivy in E. coli Nissle correlated with an improved growth of this probiotic strain in the presence of lysozyme-ethylenediaminetetraacetic acid (EDTA). By overexpressing Ivy, we demonstrated that Ivy contributes to a higher lysozyme resistance of E. coli, supporting the role of Ivy as a potential fitness factor. However, deletion of Ivy did not alter the growth phenotype of E. coli Nissle in the presence of lysozyme-EDTA, suggesting the existence of additional lysozyme inhibitors that can take over the function of Ivy.
[Show abstract][Hide abstract] ABSTRACT: HYPOTHESIS: The present study was performed to examine the impact of the release rate of ciprofloxacin from prostheses coated with nanoporous silica layers on the outcome of an acute bacterial infection of the middle ear of rabbits. BACKGROUND: Middle ear prostheses are often implanted in an infectious environment because of chronic otitis media and cholesteatoma. Bacterial colonization leads to healing disorders after surgery and may lead to the extrusion of the implants. Nanoporous silica layers appear promising as a drug delivery system for antibiotics placed on implants. Before clinical applications can be envisioned, it is necessary to find an optimal release rate. METHODS: White New Zealand rabbits were provided unilaterally with either a "slow release" or a "burst release" ciprofloxacin-containing middle ear Bioverit II prosthesis. After implantation, the middle ears were infected with a solution of Pseudomonas aeruginosa. Afterwards, animals were monitored clinically and, after 3 months, sacrificed to perform necropsy and microbiologic examinations. RESULTS: In the "slow release" group, 7 of 12 animals had to be euthanized preterm because of their poor clinical condition compared with 2 of 12 animals of the "burst release" group (p < 0.05). Clinical and microbiologic examination also showed a better outcome for animals of the burst release group. CONCLUSION: A burst release of ciprofloxacin from middle ear implants is important to combat a perioperative infection with Ps. aeruginosa in the middle ear model of the rabbit.
Otology & neurotology: official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology 04/2013; · 1.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inflammatory bowel diseases are a critical public health issue, and as treatment options remain limited, there is a need to unravel the underlying pathomechanisms in order to identify new therapeutic targets. Complement activation was found in patients suffering from inflammatory bowel disease, and the complement anaphylatoxin C5a and its receptor C5aR have been implicated in disease pathogenesis in animal models of bowel inflammation. To further characterize complement-related pathomechanisms in inflammatory bowel disease, we have investigated the role of the anaphylatoxin C3a receptor in acute dextran sulfate sodium-induced colitis in mice. For this, colitis was induced in C3a receptor-deficient BALB/c and C57BL/6 mice, and disease severity was evaluated by clinical and histological examination, and by measuring the mRNA expression or protein levels of inflammatory mediators in the tissue. C3a receptor deficiency was partially protective in BALB/c mice, which had significantly reduced weight loss, clinical and histological scores, colon shortening, and CXCL-1/KC mRNA, myeloperoxidase and interleukin-6 tissue levels compared to the corresponding wild type mice. In C57BL/6 mice the differences between wild type and C3a receptor-deficient animals were much smaller and reached no significance. Our data demonstrate that the contribution of C3a receptor to disease pathogenesis and severity of dextran sulfate sodium-induced colitis in mice depends on the genetic background. Further studies will be required to clarify whether targeting of C3a receptor, possibly in combination with C5a receptor, might be considered as a therapeutic strategy for inflammatory bowel disease.
PLoS ONE 01/2013; 8(4):e62257. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mouse pathobiont Helicobacter hepaticus can induce typhlocolitis in interleukin-10-deficient mice, and H. hepaticus infection of immunodeficient mice is widely used as a model to study the role of pathogens and commensal bacteria in the pathogenesis of inflammatory bowel disease. C57BL/6J Il10(-/-) mice kept under specific pathogen-free conditions in two different facilities (MHH and MIT), displayed strong differences with respect to their susceptibilities to H. hepaticus-induced intestinal pathology. Mice at MIT developed robust typhlocolitis after infection with H. hepaticus, while mice at MHH developed no significant pathology after infection with the same H. hepaticus strain. We hypothesized that the intestinal microbiota might be responsible for these differences and therefore performed high resolution analysis of the intestinal microbiota composition in uninfected mice from the two facilities by deep sequencing of partial 16S rRNA amplicons. The microbiota composition differed markedly between mice from both facilities. Significant differences were also detected between two groups of MHH mice born in different years. Of the 119 operational taxonomic units (OTUs) that occurred in at least half the cecum or colon samples of at least one mouse group, 24 were only found in MIT mice, and another 13 OTUs could only be found in MHH samples. While most of the MHH-specific OTUs could only be identified to class or family level, the MIT-specific set contained OTUs identified to genus or species level, including the opportunistic pathogen, Bilophila wadsworthia. The susceptibility to H. hepaticus-induced colitis differed considerably between Il10(-/-) mice originating from the two institutions. This was associated with significant differences in microbiota composition, highlighting the importance of characterizing the intestinal microbiome when studying murine models of IBD.
PLoS ONE 01/2013; 8(8):e70783. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The anti-inflammatory cytokine interleukin-10 and its viral homologs were chosen as model proteins for the development of drug delivery systems based on probiotic carriers like E. coli Nissle 1917, E. coli G3/10, and Saccharomyces boulardii. Exterior cytokine secretion was achieved by a modified E. coli hemolysin transporter. Release of interleukin-10 transported to the periplasm via the OmpF signal peptide was enabled by a T4 phage lysis system under control of the araC PBAD activator-promoter. The yield of interleukin-10 delivered by the phage lysis system was too low for functional analysis whereas the fusion protein secreted by the hemolysin transporter proved to be biologically inactive. Moreover, partial processing of the fusion protein by the E. coli membrane protease OmpT had no effect on the protein's functionality. Using the α-mating factor signal sequence, the yeast S. boulardii proved to be suitable for secretory expression of biologically active viral interleukin-10.
[Show abstract][Hide abstract] ABSTRACT: Chronic otitis media is a common disease often accompanied by recurrent bacterial infections. These may lead to the destruction of the middle ear bones such that prostheses have to be implanted to restore sound transmission. Surface coatings with layered double hydroxides (LDHs) are evaluated here as a possibility for drug delivery systems with convenient advantages such as low cytotoxicity and easy synthesis. Male New Zealand White rabbits were implanted with Bioverit(®) II middle ear prostheses coated with the LDH Mg(4)Al(2)(OH)(12)(SO(4))(2)·6H(2)O impregnated with ciprofloxacin. 12 (group 1) were directly infected with Pseudomonas aeruginosa and another 12 (group 2) 1 week after the implantation. Clinical outcome, blood counts, histological analyses and microbiological examination showed an excellent antimicrobial activity for group 1, whereas this effect was attenuated in animals where infection was performed 1 week after implantation. This is the first study to demonstrate an efficient drug delivery system with an LDH coating on prostheses in the middle ear.
Journal of Materials Science Materials in Medicine 10/2012; · 2.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nanoporous silica layers are able to host molecules and release them over a certain period of time. These local drug delivery systems for antibiotics could be a new approach in the treatment of chronic otitis media. The aim of this study was to examine the efficacy of nanoporous silica coatings on middle ear prostheses as a delivery system for antibiotics in vivo. Pseudomonas aeruginosa was inoculated into the middle ear of rabbits to induce an otitis media. The control group received coated Bioverit®II implants without antibiotics. Coated prostheses with loaded ciprofloxacin were implanted into the middle ears of the study group. After 1week, the rabbits were sacrificed. The clinical examination as well as the microbiological and histological examinations of organs and middle ear irrigation revealed clear differences between the two groups. P. aeruginosa was detected in every middle ear of the control group and was almost completely eliminated in the study group. Organ examinations revealed the presence of P. aeruginosa in the control group and a prevention of a bacterial spread in the study group. The nanoporous silica layer as antibiotic delivery system showed convincing efficacy in induced pseudomonal otitis media in the rabbit.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Although magnetic resonance imaging (MRI) is an increasingly used diagnostic tool in the assessment of inflammatory bowel disease (IBD) in humans, diagnosis and quantitation of intestinal inflammation in animal models of IBD still depends on ex vivo techniques. The aim of this study was to evaluate whether high-field MRI is suitable for the quantitative phenotyping of gut inflammation in a dextran sulfate sodium (DSS)-triggered interleukin (IL)10-deficient (IL-10(-/-) ) mouse model of IBD, especially in longitudinal studies. METHODS: Using colitis-susceptible and -resistant backgrounds, MRI and ex vivo analyses were applied to characterize this specific model, differentiating disease severity and time-dependent alterations. Colon wall thickness, cecum wall tissue intensity, spleen, and mesenteric lymph node (MLN) volumes were evaluated 1, 2, 4, and 12 weeks after disease onset by T2-weighted MRI. Ex vivo parameters included histology, spleen, and MLN weight and analysis of cytokine expression. RESULTS: MRI and ex vivo determined parameters correlated well, revealing a mouse strain-specific colitis development over time with characteristics typical for the DSS model in the initial and for the IL-10(-/-) model in the chronic phase. To evaluate the use of high-field MRI for monitoring therapeutic studies, mice with a profound colitis were treated with IL-10-producing Saccharomyces boulardii and monitored by MRI. CONCLUSIONS: MRI can be utilized to quantify colitis development in the IL-10(-/-) model of IBD. Therefore, this noninvasive technique might be highly advantageous for an individual follow-up of colitis development in chronic models of IBD, facilitating the reduction of animal numbers in this kind of research. (Inflamm Bowel Dis 2012).
[Show abstract][Hide abstract] ABSTRACT: Rotavirus is a major cause of diarrhea worldwide and exhibits a pronounced small intestinal epithelial cell (IEC) tropism. Both human infants and neonatal mice are highly susceptible, whereas adult individuals remain asymptomatic and shed only low numbers of viral particles. Here we investigated age-dependent mechanisms of the intestinal epithelial innate immune response to rotavirus infection in an oral mouse infection model. Expression of the innate immune receptor for viral dsRNA, Toll-like receptor (Tlr) 3 was low in the epithelium of suckling mice but strongly increased during the postnatal period inversely correlating with rotavirus susceptibility, viral shedding and histological damage. Adult mice deficient in Tlr3 (Tlr3(-/-)) or the adaptor molecule Trif (Trif(Lps2/Lps2)) exerted significantly higher viral shedding and decreased epithelial expression of proinflammatory and antiviral genes as compared to wild-type animals. In contrast, neonatal mice deficient in Tlr3 or Trif did not display impaired cell stimulation or enhanced rotavirus susceptibility. Using chimeric mice, a major contribution of the non-hematopoietic cell compartment in the Trif-mediated antiviral host response was detected in adult animals. Finally, a significant age-dependent increase of TLR3 expression was also detected in human small intestinal biopsies. Thus, upregulation of epithelial TLR3 expression during infancy might contribute to the age-dependent susceptibility to rotavirus infection.
[Show abstract][Hide abstract] ABSTRACT: The structure of the human gut microbial community is determined by host genetics and environmental factors, where alterations in its structure have been associated with the onset of different diseases. Establishing a defined human gut microbial community within inbred rodent models provides a means to study microbial-related pathologies, however, an in-depth comparison of the established human gut microbiota in the different models is lacking. We compared the efficiency of establishing the bacterial component of a defined human microbial community within germ-free (GF) rats, GF mice, and antibiotic-treated specific pathogen-free mice. Remarkable differences were observed between the different rodent models. While the majority of abundant human-donor bacterial phylotypes were established in the GF rats, only a subset was present in the GF mice. Despite the fact that members of the phylum Bacteriodetes were well established in all rodent models, mice enriched for phylotypes related to species of Bacteroides. In contrary to the efficiency of Clostridiales to populate the GF rat in relative proportions to that of the human-donor, members of Clostridia cluster IV only poorly colonize the mouse gut. Thus, the genetic background of the different recipient rodent systems (that is, rats and mice) strongly influences the nature of the populating human gut microbiota, determining each model's biological suitability.
[Show abstract][Hide abstract] ABSTRACT: The gut microbiota (GM) composition and its impact on animal experiments has become currently dramatically relevant in our days: (1) recent progress in metagenomic technologies, (2) the availability of large scale quantitative analyses to characterize even subtle phenotypes, (3) the limited diversity of laboratory rodent GM due to strict barriers at laboratory animal vendors, and (4) the availability of up to 300.000 different transgenic mouse strains from different sources displaying a huge variety in their GM composition. In this review the GM is described as a variable in animal experiments which need to be reduced for scientific as well as ethical reasons, and strategies how to implement this in routine diagnostic procedures are proposed. We conclude that we have both enough information available to state that the GM has an essential impact on animal models, as well as the methods available to start dealing with these impacts.