SPF Conditions Research Articles

Housing RCS rats under specific pathogen-free conditions mildly ameliorates retinal degeneration and alters intestine microbiota

Retinitis pigmentosa (RP) is a genetic blinding disease with over 80 causative genes. Disease progression varies between patients with similar genetic backgrounds. We assessed the association between environment, gut microbiota, and retinal degeneration in the RP rat model Royal College of Surgeons (RCS). The rats were born and raised for two generations under specific pathogen-free (SPF, n = 69) or non-SPF conditions (n = 48). At the age of four weeks, SPF rats had significantly shorter dark-adapted a-wave and dark and light-adapted b-wave implicit times by electroretinogram (p = 0.014, p = 9.5*10−6, p = 0.009, respectively). The SPF rats had significantly less photoreceptor apoptosis at ages four, eight, and twelve weeks (all p < 0.022), significantly thicker debris zone at age 14 weeks, and smaller hypofluorescent lesions in SPF rats at ages 10–16 weeks, especially in the inferior retina. The non-SPF rats had significantly higher microbiota alpha diversity (p = 0.037) and failed to present the age-related maturation of Proteobacteria, Spirochaetes, Actinobacteria, and Bacteroidetes seen in SPF conditions. Specific microbial amplicon sequence variants were reduced in rats with more severe retinal degeneration. Our data suggest an environmental effect on retinal deterioration in RCS rats. These findings may lead to the development of novel microbiome-related interventions for retinal degeneration.

Vasoactive Intestinal Polypeptide Plays a Key Role in the Microbial-Neuroimmune Control of Intestinal Motility

Background and AimsAlthough chronic diarrhea and constipation are common, the treatment is symptomatic as their pathophysiology is poorly understood. Accumulating evidence suggests that the microbiota modulates gut function but the underlying mechanisms are unknown. We therefore investigated the pathways by which microbiota modulates gastrointestinal motility in different sections of the alimentary tract. MethodsGastric emptying, intestinal transit, muscle contractility, acetylcholine release, gene expression and vasoactive intestinal polypeptide (VIP) immunoreactivity were assessed in wild-type and Myd88-/-Trif-/- mice in germ-free, gnotobiotic and SPF conditions. Effects of transient colonization and antimicrobials, as well as immune cell blockade were investigated. VIP levels were assessed in human full thickness biopsies by Western blot. ResultsGerm-free mice had similar gastric emptying but slower intestinal transit compared with SPF mice, or mice monocolonized with Lactobacillus rhamnosus or Escherichia coli, the latter having stronger effects. While muscle contractility was unaffected, its neural control was modulated by microbiota by upregulating jejunal VIP, which co-localized with and controlled cholinergic nerve function. This process was responsive to changes in the microbial composition and load, and mediated through TLR signaling, with enteric glia cells playing a key role. Jejunal VIP was lower in patients with chronic intestinal pseudo-obstruction compared with control subjects. ConclusionMicrobial control of gastrointestinal motility is both region- and bacteria-specific, it reacts to environmental changes, and is mediated by innate immunity-neural system interactions. Small intestinal VIP, by regulating cholinergic nerves, plays a key role in this process, thus providing a new therapeutic target for patients with motility disorders.

Free lipoproteins from Bifidobacterium longum alleviate osteoarthritis through modulation of the gut microbiome.

Aim: The "gut-joint" axis is suspected to be involved in the pathophysiology of osteoarthritis (OA). The present study aims at investigating the potential of lipoproteins (Lpps) secreted by Bifidobacterium longum to alleviate OA progression in the rat. Methods: Experimental OA was induced in rats harbouring Schaedler Flora maintained in SPF conditions. Two weeks post-injection, 20 rats were randomized to water (n = 10) or 0.3 mg/L Lpps solution (n = 10). Weight and food intake were monitored for 6 weeks. At sacrifice, joints were scored using macroscopic and histological criteria. Serum LPS, Schaedler flora as well as selected intestinal bacteria were analyzed. Results: Lpps intake prevents OA progression. The protected rats showed a significant increase in lactobacilli along the intestine as well as in Mucispirillum schaedleri in the colon and a significant decrease in Parabacteroides goldsteini and Akkermansia in caecum and colon, respectively. There was no significant difference in serum lipopolysaccharide or bacteria translocating in Peyer's patches. Labelled Lpps were not detected in bone marrow of the OA joint. The principal component analysis points out that OA prevention is primarily associated with bacteria involved in the tryptophane degradation pathway and SCFA formation. Conclusion: In rats deprived of bifidobacteria, intake of B.longum Lpps prevented OA development and modulated the intestinal microbiome with a possible impact on the bacterial end-products. The link between Lpps and the gut microbial metabolome warrants further investigation.

Oral dextran sulfate sodium administration induces peripheral spondyloarthritis features in SKG mice accompanied by intestinal bacterial translocation and systemic Th1 and Th17 cell activation

BackgroundSpondyloarthritis (SpA) is an autoimmune and autoinflammatory musculoskeletal disease characterised by systemic enthesitis. Recent research has focused on subclinical inflammatory bowel disease (IBD) in SpA pathogenesis. SKG mice, harbouring the Zap70 W163C mutation, increase autoreactive Th17 cells intrinsically, and in a conventional environment, they exhibit spontaneous arthritis with fungal factors. Under SPF conditions, they show SpA features, including enteritis, after peritoneal injection of β-1,3-glucan. This study aimed to clarify whether oral dextran sulfate sodium (DSS) administration, utilised in IBD model mice, can provoke SpA features in SKG mice under SPF conditions, focusing on the relationship between gut microorganisms and SpA pathogenesis.MethodsBALB/c and SKG mice were administered oral DSS, and their body weights, arthritis, and enthesitis scores were recorded. In another cohort, antibiotics (meropenem and vancomycin) or an anti-fungal agent (amphotericin B) was administered orally before DSS administration. The splenic Th1 and Th17 cell populations were examined before and after DSS administration using flow cytometry. Furthermore, the amount of circulating bacterial DNA in whole blood was measured by absolute quantitative polymerase chain reaction (qPCR), and the number and characteristics of bacterial species corresponding to these circulating DNA were analysed by next-generation sequencing (NGS).ResultsAnkle enthesitis as a peripheral SpA feature was elicited in half of DSS-administered SKG mice, and none of the BALB/c mice. Pre-administration of antibiotics suppressed enthesitis, whilst an anti-fungal agent could not. Th1 and Th17 cell levels in the spleen increased after DSS administration, and this was suppressed by pre-administration of antibiotics. SKG mice have a larger amount of bacterial DNA in whole blood than BALB/c mice before and 1 day after the initiation of DSS administration. The number of bacterial species in whole blood increased after DSS administration in BALB/c and SKG mice. Some genera and species significantly specific to the DSS-treated SKG mouse group were also detected.ConclusionOral DSS administration alone elicited peripheral enthesitis in SKG mice with bacterial translocation accompanied by increased splenic Th1 and Th17 cell levels. Pre-administration of antibiotics ameliorated these DSS-induced SpA features. These findings suggest that intestinal bacterial leakage plays a pivotal role in SpA pathogenesis.

A Murine Point Mutation of Sgpl1 Skin Is Enriched With Vγ6 IL17-Producing Cell and Revealed With Hyperpigmentation After Imiquimod Treatment.

Sphingosine-1-phosphate lyase is encoded by the Sgpl1 gene, degrades S1P, and is crucial for S1P homeostasis in animal models and humans. S1P lyase deficient patients suffer from adrenal insufficiency, severe lymphopenia, and skin disorders. In this study, we used random mutagenesis screening to identify a mouse line carrying a missense mutation of Sgpl1 (M467K). This mutation caused similar pathologies as Sgpl1 knock-out mice in multiple organs, but greatly preserved its lifespan, which M467K mutation mice look normal under SPF conditions for over 40 weeks, in contrast, the knock-out mice live no more than 6 weeks. When treated with Imiquimod, Sgpl1M467K mice experienced exacerbated skin inflammation, as revealed by aggravated acanthosis and orthokeratotic hyperkeratosis. We also demonstrated that the IL17a producing Vγ6+ cell was enriched in Sgpl1M467K skin and caused severe pathology after imiquimod treatment. Interestingly, hyperchromic plaque occurred in the mutant mice one month after Imiquimod treatment but not in the controls, which resembled the skin disorder found in Sgpl1 deficient patients. Therefore, our results demonstrate that Sgpl1M467K point mutation mice successfully modeled a human disease after being treated with Imiquimod. We also revealed a major subset of γδT cells in the skin, IL17 secreting Vγ6 T cells were augmented by Sgpl1 deficiency and led to skin pathology. Therefore, we have, for the first time, linked the IL17a and γδT cells to SPL insufficiency.

Breeding and Maintenance of Immunodeficient Mouse Lines under SPF Conditions-A Call for Individualized Severity Analyses and Approval Procedures.

Simple SummaryIn the EU, the breeding of genetically modified laboratory animals is defined as an animal experiment, if the animals may experience pain, suffering or harm due to the genetic modification. In most cases, modifications of the immune system do not lead to pain, suffering, or harm given the animals are kept under specific pathogen-free housing conditions. Nevertheless, the EU working group on genetically altered animals defined SPF housing conditions as “Refinement”. According to this working group, applied refinement strategies for breeding genetically modified animals do not exclude them from project licensing. Thus, theoretical suffering, pain or harm is assumed without applying methods of actual burden assessment. Furthermore, the definition of SPF as a refinement strategy rather than a standard husbandry procedure leads to incomprehension among the scientific community in the EU. Here, we discuss the ethical basis of animal experiments as well as the current legal situation for immunodeficient animals. Furthermore, we discuss potential pain, suffering, or harm of immunodeficient animals from an immunological perspective. Finally, we briefly outline an animal welfare-oriented approach to severity assessment of immunodeficient mice.In the EU, the breeding of genetically modified laboratory animals is, by definition, an animal experiment if the offspring may experience pain, suffering, or harm. In order to determine the actual burden of genetically modified mice, established methods are available. However, the breeding of immunodeficient mice is considered an experiment requiring a permit, even if no pain, suffering or harm is observed under scientifically required defined hygienic housing conditions, as determined by established methods of severity assessment. This seems contradictory and leads to uncertainty among scientists. With this commentary, we would like to shed light on this topic from different perspectives and propose a solution in terms of individualized severity assessment and approval procedures.

Lack of bone marrow stromal cell antigen-1 (BST-1)/CD157 ameliorated colitis induced by dextran sodium sulfate (DSS)

Abstract Bone marrow stromal cell antigen-1(BST-1)/CD157 is GPI-anchored ectoenzyme having adenosine diphosphate(ADP)-ribosyl cyclase and cyclic ADP-ribose hydrolase activities. BST-1 also functions as a receptor for signal transduction. Recently, various groups reported novel functions of BST-1, such as regulation of intestinal or mesenchymal stem cells. Re-analyses of BST-1 deficient mice (Bst1KO) in C57BL/6 background revealed roles in defense against tuberculosis, and prevention of depressive or anxious behaviors. Thus, BST-1 is now appreciated as an entero-neuro-immune regulator. Since expression of BST-1 in the intestines is common in human and mouse, we examined the roles for BST-1 in DSS-induced colitis using Bst1KO. Drinking water containing 3% DSS was supplied ad libitum to Bst1KO and C57BL/6 (WT) in SPF condition for 5 days, and then water without DSS for 2 days. Decreases of body weights in Bst1KO were similar to WT until day 5, but since then they became significantly slower than WT. Clinical scores based on bleeding and diarrhea were similar in both genotypes. Histological analyses with HE staining revealed the ratio of destroyed epitheliums in the total colon of Bst1KO was 15% whereas that of WT was 50%. Flow cytometrical analyses revealed no difference in the cell numbers of neutrophils and macrophages increased in the colon at day 7. Quantitative real-time PCR revealed that increases of Bst1 expression in the colons of WT started on day 1 and reached to the level more than thousand times at day 5. In the colons of Bst1KO, increases of Il6 expression at day 5 were markedly decreased compared with those of WT. These data indicate that BST-1 has pro-inflammatory roles in DSS-induced colitis through positive regulation of IL-6 production.

High-dimensional single cell mass cytometry analysis of the murine hematopoietic system reveals signatures induced by ageing and physiological pathogen challenges

BackgroundImmune ageing is a result of repetitive microbial challenges along with cell intrinsic or systemic changes occurring during ageing. Mice under ‘specific-pathogen-free’ (SPF) conditions are frequently used to assess immune ageing in long-term experiments. However, physiological pathogenic challenges are reduced in SPF mice. The question arises to what extent murine experiments performed under SPF conditions are suited to analyze immune ageing in mice and serve as models for human immune ageing. Our previous comparisons of same aged mice with different microbial exposures, unambiguously identified distinct clusters of immune cells characteristic for numerous previous pathogen encounters in particular in pet shop mice.ResultsWe here performed single cell mass cytometry assessing splenic as secondary and bone marrow as primary lymphoid organ-derived leukocytes isolated from young versus aged SPF mice in order to delineate alterations of the murine hematopoietic system induced during ageing. We then compared immune clusters from young and aged SPF mice to pet shop mice in order to delineate alterations of the murine hematopoietic system induced by physiological pathogenic challenges and those caused by cell intrinsic or systemic changes during ageing. Notably, distinct immune signatures were similarly altered in both pet shop and aged SPF mice in comparison to young SPF mice, including increased frequencies of memory T lymphocytes, effector-cytokine producing T cells, plasma cells and mature NK cells. However, elevated frequencies of CD4+ T cells, total NK cells, granulocytes, pDCs, cDCs and decreased frequencies of naïve B cells were specifically identified only in pet shop mice. In aged SPF mice specifically the frequencies of splenic IgM+ plasma cells, CD8+ T cells and CD4+ CD25+ Treg were increased as compared to pet shop mice and young mice.ConclusionsOur study dissects firstly how ageing impacts both innate and adaptive immune cells in primary and secondary lymphoid organs. Secondly, it partly distinguishes murine intrinsic immune ageing alterations from those induced by physiological pathogen challenges highlighting the importance of designing mouse models for their use in preclinical research including vaccines and immunotherapies.

The microbiome does not influence cartilage degradation following joint destabilisation in mice when performed under stringently controlled conditions

Purpose: The influence of gut bacteria in the modulation of disease severity of OA is poorly understood. Reports to date on the role of the microbiome in germ free (GF) models of OA are conflicting and haven’t always been stringently controlled. In the present study, surgical induction of OA by destabilisation of the medial meniscus (DMM) was used to examine the potential influence of the microbiome on cartilage damage severity. Methods: Male C57BL/6J mice used in the present study were bred and housed in the gnotobiotic facility at the Kennedy Institute. Microbiome analysis by 16S rRNA amplicon sequencing was performed on stool samples from mice prior to DMM surgery. At 10 weeks of age, mice were randomly selected for sham (n=6-8) or DMM (n=17-20) surgery, which was performed in sterile Class II cabinets. Knees from operated mice were harvested at 8 or 12wks post DMM. Faecal samples were collected post surgery and at the end of the experiment to test GF status. Joints were sectioned and severity of cartilage degradation was assessed in a blinded method according to OARSI guidelines. No mice were excluded from data analysis. Differences were analysed by ANOVA with Tukey post test correction and P values less than 0.05 were considered statistically significant. Control (specific pathogen free, SPF) male C57BL/6J mice (originally derived from the GF founders) were bred under SPF conditions but housed in an environment to mimic the GF conditions in terms of bedding, food (irradiated) and water, and underwent sham (n=4-6) or DMM (n=11-13) surgery at 10 weeks for analysis after 8 and 12wks. Results: All mice were confirmed GF at the time of surgery but 50% tested positive for microbes at the end of the surgical procedure suggesting contamination at the time of surgery (indicated in red dots). Proteoglycan loss and cartilage erosion were evident in SPF and GF mice 12 wks after surgery. Cartilage damage following DMM was significantly higher than sham in both groups, but was not different between GF and SPF mice. Cartilage damage seen at 8wks was modest and only reached significance over sham-operated in GF mice. No significant differences in scores between the GF and SPF DMM groups were demonstrated. No apparent relationship between those animals that became contaminated at the time of surgery and disease scores was seen. Conclusions: The present study shows that significant cartilage degradation occurs in GF mice undergoing DMM at both timepoints. The disease severity was not altered by the absence of a microbiome, nor apparently when mice lost their GF status during the experimental course. When SPF mice were housed under tightly matched cage conditions to the GF mice, disease appeared indistinguishable. The absence of a gut microbiome does not appear to protect the cartilage from erosion after joint destabilisation.

Contribution of the Microbiome to Maintenance of the CD4:CD8 Ratio in Inbred and Congenic Mouse Strains

Abstract The gut microbiome is known to play an integral role in the regulation of the immune system. Previous studies indicate that species diversity and overall abundance of commensal bacteria in the gut influences immune system function by altering total T cell counts and the ratio of CD4+ to CD8+ T cells in peripheral lymphoid organs and blood. In normal individuals, the CD4:CD8 ratio is greater than one. However, the congenic B10.A mouse strain in our conventional facility consistently demonstrates an inverted CD4:CD8 ratio in a sex- and age-dependent manner, in contrast to both the parental C57Bl/10 strain and B10.A kept under SPF conditions. To determine whether this phenomenon is due to the composition and diversity of the gut microbiome affecting the homeostasis of the CD4:CD8 ratio, we altered the microbiome by supplementing the diet with the prebiotic inulin and looked for changes in the number of Peyer’s patches and changes in the CD4:CD8 ratio in gut lymphoid organs and blood in both B10.A and C57Bl/10 strains. In addition, we followed the changes in the microbiome upon treatment. We find that the mice in our facility have lower microbiome diversity and very different composition from mice kept at SPF conditions. The inulin treatment significantly increased the CD4:CD8 ratio in the blood of the C57Bl/10 mice, but not the B10.A mice. In addition, males of both strains had a significant increase in Peyer’s patch number, while the females had a significant increase in the CD4:CD8 ratio in the Peyer’s patches.

Adoption of Exhaust Air Dust Testing in SPF Rodent Facilities.

Reliable detection of unwanted microbial agents is essential for meaningful health monitoring in laboratory animal facilities. Most rodents at our institution are housed in IVC rack systems to minimize aerogenic transmission of infectious agents between cages. The most commonly used rodent health monitoring systems expose live sentinel rodents to soiled bedding collected from other rodent cages on IVC racks and subsequently test these soiled-bedding sentinels for evidence of infection with excluded agents. However, infectious agents might go undetected when using this health surveillance method, due to inefficient organism shedding or transmission failure. In 2016, our institution switched the health monitoring methodology for the majority of our SPF rodent colonies to real-time PCR testing of environmental samples collected from the exhaust plenums of IVC racks. Here we describe our rationale for this conversion, describe some interesting health monitoring cases that arose soon after the conversion, and discuss a potential problem with the conversion-residual nucleic acids. We compared cost and implementation effort associated with 2 sampling methods, sticky swabs and in-line collection media. We also compared the ability of these 2 sampling methods to detect 2 prevalent microbes in our facilities, Helicobacter and murine norovirus. Our institution-wide switch to health monitoring by real-time PCR assay of exhaust air dust samples thus far has provided a sensitive, simple, and reliable approach for maintenance of SPF conditions in laboratory rodents and has dramatically reduced the use of live sentinel animals.

Degradation of the anticancer drug flutamide by solar photo-Fenton treatment at near-neutral pH: Identification of transformation products and in silico (Q)SAR risk assessment

Flutamide (FLUT) is a non-steroidal drug mainly used in the treatment of prostate cancer and has been detected in the aquatic environment at ng L−1 levels. The environmental fate and effects of FLUT have not yet been studied. Conventional treatment technologies fail to completely remove pharmaceuticals, so the solar photo-Fenton process (SPF) has been proposed as an alternative. In this study, the degradation of FLUT, at two different initial concentrations in ultra-pure water, was carried out by SPF. The initial SPF conditions were pH0 5, [Fe2+]0 = 5 mg L−1, and [H2O2]0 = 50 mg L−1. Preliminary elimination rates of 53.4% and 73.4%. The kinetics of FLUT degradation could be fitted by a pseudo-first order model and the kobs were 6.57 × 10−3 and 9.13 × 10−3 min−1 t30W and the half-life times were 95.62 and 73.10 min t30W were achieved for [FLUT]0 of 5 mg L−1 and 500 μg L−1, respectively. Analysis using LC-QTOF MS identified thirteen transformation products (TPs) during the FLUT degradation process. The main degradation pathways proposed were hydroxylation, hydrogen abstraction, demethylation, NO2 elimination, cleavage, and aromatic ring opening. Different in silico (quantitative) structure-activity relationship ((Q)SAR) freeware models were used to predict the toxicities and environmental fates of FLUT and the TPs. The in silico predictions indicated that these substances were not biodegradable, while some TPs were classified near the threshold point to be considered as PBT compounds. The in silico (Q)SAR predictions gave positive alerts concerning the mutagenicity and carcinogenicity endpoints. Additionally, the (Q)SAR toolbox software provided structural alerts corresponding to the positive alerts obtained with the different mutagenicity and carcinogenicity models, supporting the positive alerts with more proactive information.

2017-P: Gut Microbes after Bariatric Surgery in Humans Improve Glucose Control in Mice without Fat Loss

Bariatric surgery (BS) is the most efficacious treatment against severe obesity and T2D. Among bariatric procedures, biliopancreatic diversion with duodenal switch (BPD-DS) can produce superior metabolic benefits compared with other surgical options. The mechanisms by which BPD-DS improve metabolic health are not well-understood, but there is evidence implicating the gut microbiota (GM) as contributor to changes in adiposity and consequently blood glucose control. We aimed to determine how the GM before and after BPD-DS alter host metabolism. Fecal samples from female patients subjected to BPD-DS were collected before (pre-surgery) and 12 months after surgery (post-surgery) and used to colonize female C57BL6NTac mice housed either in germ-free (GF) or SPF conditions. Oral glucose tolerance (OGT) was assessed in mice 3 and 7 weeks post-colonization. Three weeks after fecal microbiota transplantation (FMT), glucose tolerance was similar in GF mice receiving pre- and post-surgery fecal slurries. However, GF mice had lower glucose during OGT test 7 weeks after FMT derived from BPD-DS post-surgery compared to GF mice receiving pre-surgery fecal slurry. No changes in fat mass accretion were seen between pre- and post-surgery mice. SPF mice colonized with the fecal microbiota pre- and post-surgery showed no difference in glucose tolerance after 3 and 7 weeks of FMT. These data suggest that the GM after BPD-DS is an independent and transmissible contributor to improved host glucose control. Furthermore, while our data show that the mouse model (e.g., SPF vs. GF) dictates the glycemic response to FMT, exposure time (e.g., at least 7 weeks) is a key factor in microbe transmissible changes in glycemia. Disclosure F. Forato Anhê: None. K.P. Foley: None. N.G. Barra: None. B.M. Duggan: None. J.F. Cavallari: None. B.D. Henriksbo: None. P.D. Cani: Board Member; Self; A-Mansia Biotech. Consultant; Self; Biocodex, Pilèje, Valbiotis. Research Support; Self; Tate &amp; Lyle. Stock/Shareholder; Self; A-Mansia Biotech, Enterosys. L. Biertho: None. A. Tchernof: Research Support; Self; Johnson &amp; Johnson Medical Devices Companies, Medtronic, Pfizer Inc. A. Marette: Advisory Panel; Self; Plexus, Valbiotis. Consultant; Self; Danone Nutricia Research. J.D. Schertzer: None. Funding Canadian Institutes of Health Research; Diabetes Canada

Genotoxic Escherichia coli Strains Encoding Colibactin, Cytolethal Distending Toxin, and Cytotoxic Necrotizing Factor in Laboratory Rats.

Although many Escherichia coli strains are considered commensals in mammals, strains encoding the cyclomodulin genotoxins are associated with clinical and subclinical disease in the urogenital and gastrointestinal tracts, meningitis, and inflammatory disorders. These genotoxins include the polyketide synthase (pks) pathogenicity island, cytolethal distending toxin (cdt), and hemolysin-associated cytotoxic necrotizing factor (cnf). E. coli strains are not excluded from rodents housed under SPF conditions in academic or vendor facilities. This study isolated and characterized genotoxin-encoding E. coli from laboratory rats obtained from 4 academic institutions and 3 vendors. A total of 69 distinct E. coli isolates were cultured from feces, rectal swab, nares, or vaginal swab of 52 rats and characterized biochemically. PCR analysis for cyclomodulin genes and phylogroup was performed on all 69 isolates. Of the 69 isolates, 45 (65%) were positive for pks, 20/69 (29%) were positive for cdt, and 4 (6%) were positive for cnf. Colibactin was the sole genotoxin identified in 21 of 45 pks+ isolates (47%), whereas cdt or cnf was also present in the remaining 24 isolates (53%); cdt and cnf were never present together or without pks. All genotoxin-associated strains were members of pathogen-associated phylogroup B2. Fisher exact and χ² tests demonstrated significant differences in genotoxin prevalence and API code distribution with regard to vendor. Select E. coli isolates were characterized by HeLa cell in vitro cytotoxicity assays, serotyped, and whole-genome sequenced. All isolates encoding cyclomodulins induced megalocytosis. Serotypes corresponded with vendor origin and cyclomodulin composition, with the cnf+ serotype representing a known human uropathogen. Whole-genome sequencing confirmed the presence of complete pks, cdt, and hemolysin-cnf pathogenicity islands. These findings indicate that genotoxin-encoding E. coli colonize laboratory rats from multiple commercial vendors and academic institutions and suggest the potential to contribute to clinical disease and introduce confounding variables into experimental rat models.

Mechanical behaviour modelling and finite element simulation of simple part of Ti-6Al-4V sheet under hot/warm stamping conditions

In order to reduce production costs and environmental impact, the cycle time reductions associated with a decrease in temperature levels are relevant. This study focuses on the behaviour modelling of Ti-6Al-4V alloy at temperatures between 400 °C and 500 °C in order to obtain greater formability than at room temperature, whilst remaining below SPF conditions to reduce tool, workshop and energy costs. Mechanical tests are conducted to identify elasto-viscoplastic model parameters. They use displacement field measurements obtained by Digital Image Correlation (DIC) and based on innovative surface preparation patterns adapted to high temperature exposures. Different material parameters are identified to define a model that is able to predict the mechanical behaviour of Ti-6Al-4V alloy under hot/warm stamping conditions. Then, an omega shape forming test is developed to validate the behaviour model. Finally, the experimental results are compared with numerical simulations which require the implementation of the behaviour model formulation into an FE code.

Receptor tyrosine kinase MerTK suppresses an allogenic type I IFN response to promote transplant tolerance.

Recipient infusion of donor apoptotic cells is an emerging strategy for inducing robust transplantation tolerance. Daily clearance of billions of self-apoptotic cells relies on homeostatic engagement of phagocytic receptors, in particular, receptors of the tyrosine kinase family TAM (Tyro3, Axl, and MerTK), to maintain self-tolerance. However, an outstanding question is if allogeneic apoptotic cells trigger the same receptor system for inducing allogeneic tolerance. Here, we employed allogeneic apoptotic splenocytes and discovered that the efferocytic receptor MerTK on recipient phagocytes is a critical mediator for transplantation tolerance induced by this strategy. Our findings indicate that the tolerogenic properties of allogeneic apoptotic splenocytes require MerTK transmission of intracellular signaling to suppress the production of inflammatory cytokine interferon α (IFN-α). We further demonstrate that MerTK is crucial for subsequent expansion of myeloid-derived suppressor cells and for promoting their immunomodulatory function, including maintaining graft-infiltrating CD4+ CD25+ Foxp3+ regulatory T cells. Consequently, recipient MerTK deficiency resulted in failure of tolerance by donor apoptotic cells, and this failure could be effectively rescued by IFN-α receptor blockade. These findings underscore the importance of the efferocytic receptor MerTK in mediating transplantation tolerance by donor apoptotic cells and implicate MerTK agonism as a promising target for promoting transplantation tolerance.

Superplasticity in Fine Grain Ti-6Al-4V Alloy: Mechanical Behavior and Microstructural Evolution

Titanium Ti-6Al-4V alloys are known to exhibit interesting superplastic properties for different conditions of temperature and strain rate, depending on the initial grain size. Even if superplasticity is generally explained in terms of grain boundary sliding (GBS) accompanied by several accommodation mechanisms, it appears that the micromechanisms of superplasticity are still controversial especially at the grain scale and even more at lower scale. These micromechanisms, involving microstructural evolution, depend also on the SPF conditions (temperature, strain rate and initial microstructure). In this study, the flow stress in the Ti-6Al-4V alloy is investigated for different strain rate and for temperature in the range of the α/β transformation. The preferred orientation evolution of alpha phase grains for different percentage of deformation is studied for a non-optimal SPF regime (920°C-10-4 s-1) in order to highlight the microstructural evolution and so the deformation mechanisms involved. For that, mechanical interrupted test combined with Scanning Electron Microscopy (SEM) and Electron Back Scatter Diffraction (EBSD) are used.

Increased NCR negative ILC 3 may be indicative for the early development of spontaneous colitis in the IL 10 knockout mouse

Abstract The IL10 knockout (KO) mouse is a model for environmentally and genetically triggered spontaneous colitis. The response of the adaptive immune system to these changes has been studied extensively in this model. However, the response of the innate immune system, specifically the response of innate lymphoid cell (ILCs) populations to the genetic background of the mouse and the environmental stimulus of introducing microbiota has not been well studied. Herein, we evaluated the response of ILCs and Lin marker positive lymphocytes to changes in genotype (wild type Mice B6 in SPF to IL10 KO B6 mice in SPF) and also compared IL10KO mice who were either germ free or SPF exposed. To accomplish this, IL10 knockout mice were exposed to SPF conditions for three weeks. LPMCs were isolated from the colon and small bowel. Innate lymphoid cells were identified as Lin− CD90.2+CD127+cells, and within total ILC, ILC2s were ST2+ cells, ILC1s were ST2−CD117− cells, and ILC3s were ST2−CD117+NKp46+/−cells. As expected, we saw a less developed adaptive immune response in GF IL10 KO mice, with up regulation in this compartment following SPF exposure (Lin+ population increase 1.87 fold). Upon comparison of WT SPF versus IL10 KO SPF, we observed a 5 fold increase in total ILCs and an 18% increase in NCR-ILC3 in the SPF. Comparison of IL10 KO GF versus SPF revealed a 1.8 fold increase in total ILC and a 3% increase in NCR-ILC3s. In conclusion, we show that both genetic and environmental factors play a role in the innate immune changes that occur prior to the development of spontaneous colitis in the IL10 KO mouse and that the shift towards NCR-ILC 3 maybe an early marker for the development of spontaneous colitis.

Prolonged transfer of feces from the lean mice modulates gut microbiota in obese mice

BackgroundTransplanting a fecal sample from lean, healthy donors to obese recipients has been shown to improve metabolic syndrome symptoms. We therefore examined the gut microbiota in mice after administering a long-term, high-fat diet (HFD) supplemented with feces from lean mice through the fecal-oral route.MethodsC57BL6/W mice were allowed to adapt to a non-specific pathogen free (SFP) environment for 2 weeks before being divided into three groups of 16 animals. Animals were fed for 28 weeks with a normal diet (ND), HFD or HFD supplemented with feces from ND-fed mice (HFDS). The composition of colonizing bacteria was evaluated in droppings collected under SPF conditions at the beginning of the study and at 12 and 28 weeks using an 16S Metagenomics Kit on Ion PGM sequencer.ResultsHFD and HFDS-fed mice attained (p < 0.05) greater body weights by weeks 6 and 5, respectively. HFDS-fed mice gained more weight than HFD-fed mice by week 25. Both species diversity and richness indices increased with time in HFDS mice only.ConclusionsProlonged HFD-fed mice supplementation with feces from lean mice altered bacteria species diversity and richness, accelerated the onset of obesity, and caused increased weight gain in the later weeks of the HFD regimen.Electronic supplementary materialThe online version of this article (doi:10.1186/s12986-016-0116-8) contains supplementary material, which is available to authorized users.

Conducting and automating Morris water maze test under SPF conditions

The Morris water maze is the basic test to study spatial learning and memory in laboratory rodents. It is part of the set of tests necessary for behavioral phenotyping of mutant and transgenic mice. However, the SPF conditions impose very strict regulations on pathogen control, which hinders performance of this test. Another problem is a low contrast of a white animal on the whitened water surface, interfering with automated animal tracking. A unique installation based on EthoStudio has been designed at the Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences and Institute of Automation and Electrometry, Siberian Branch, Russian Academy of Sciences. This tool automates the tracking of mice of any coat color under SPF conditions and comprises a plastic water tank (110 × 40 cm), a digital camera, and a light source. The water for the tank is sterilized using a Van Erp Blue Lagoon UV-C Tech 15000 ultraviolet decontaminator. The image of an animal is processed in a frame-by-frame manner using the EthoStudio software; the following parameters are calculated: escape latency, path length, cumulative distance to the platform, and the time spent in the sectors of tank. This installation has allowed us to study the spatial learning and memory in C57BL/6 and C57BL/6/Kaiso mice; in the latter strain, the gene encoding the methyl-DNA binding Kaiso protein is knocked out. It has been shown that the mice of these strains are capable of learning to find the platform in the Morris water maze and remember the location of the platform for at least the next four days.