Specific Pathogen-free Conditions Research Articles

Measurement of the Intestinal pH in Mice under Various Conditions Reveals Alkalization Induced by Antibiotics.

The intestinal pH can greatly influence the stability and absorption of oral drugs. Therefore, knowledge of intestinal pH is necessary to understand the conditions for drug delivery. This has previously been measured in humans and rats. However, information on intestinal pH in mice is insufficient despite these animals being used often in preclinical testing. In this study, 72 female ICR mice housed in SPF (specific pathogen-free) conditions were separated into nine groups to determine the intestinal pH under conditions that might cause pH fluctuations, including high-protein diet, ageing, proton pump inhibitor (PPI) treatment, several antibiotic treatment regimens and germ-free mice. pH was measured in samples collected from the ileum, cecum and colon, and compared to control animals. An electrode, 3 mm in diameter, enabled accurate pH measurements with a small amount of gastrointestinal content. Consequently, the pH values in the cecum and colon were increased by high-protein diet, and the pH in the ileum was decreased by PPI. Drastic alkalization was induced by antibiotics, especially in the cecum and colon. The alkalized pH values in germ-free mice suggested that the reduction in the intestinal bacteria caused by antibiotics led to alkalization. Alkalization of the intestinal pH caused by antibiotic treatment was verified in mice. We need further investigations in clinical settings to check whether the same phenomena occur in patients.

Trophoblast Stem-Cell-Derived Exosomes Improve Doxorubicin-Induced Dilated Cardiomyopathy by Modulating the let-7i/YAP Pathway

Trophoblast stem cells (TSCs) have been confirmed to play a cardioprotective role in heart failure. However, whether TSC-derived exosomes (TSC-exos) can protect against cardiac injury remains unclear. In the present study, TSC-exos were isolated from the supernatant of TSCs using the ultracentrifugation method and characterized by transmission electron microscopy and western blotting. Utilizing the public Gene Expression Omnibus (GEO) database, we found that let-7i and Yes-associated protein 1 (YAP) could participate in the development of heart failure. In vitro, AC16 cardiomyocytes subjected to doxorubicin (DOX) were treated with TSC-exos or let-7i mimic. Flow cytometry showed that TSC-exos and let-7i both decreased cardiomyocyte apoptosis. In vivo, mice that were intraperitoneally injected into DOX received either PBS, TSC-exos, or AAV9-let7iup for let-7i overexpression. Mice receiving TSC-exos and AAV9-let7iup showed improved cardiac function and decreased inflammatory responses, accompanied by downregulated YAP signaling. Mechanistically, TSC-exos could transfer let-7i to cardiomyocytes and silence the YAP signaling pathway. In conclusion, TSC-exos could alleviate DOX-induced cardiac injury via the let-7i/YAP pathway, which sheds new light on the application of TSC-exos as a potential therapeutic tool for heart failure.

Stress hormones or general well-being are not altered in immune-deficient mice lacking either T- and B- lymphocytes or Interferon gamma signaling if kept under specific pathogen free housing conditions.

It is controversially discussed whether immune-deficient mice experience severity in the absence of infection. Because a comprehensive analysis of the well-being of immune-deficient mice under specific pathogen free conditions is missing, we used a multi-parametric test analyzing, corticosterone, weight, nest building and facial expression over a period of 9 month to determine the well-being of two immune-deficient mouse lines (recombination activating gene 2- and interferon gamma receptor-deficient mice). We do not find evidence for severity when comparing immune-deficient mice to their heterozygous immune-competent littermates. Our data challenge the assumption that immune-deficiency per se regardless of housing conditions causes severity. Based on our study we propose to use objective non-invasive parameters determined by laboratory animal science for decisions concerning severity of immune-deficient mice.

Immune cell composition in normal human kidneys

An understanding of immunological mechanisms in kidney diseases has advanced using mouse kidneys. However, the profiling of immune cell subsets in human kidneys remains undetermined, particularly compared with mouse kidneys. Normal human kidneys were obtained from radically nephrectomised patients with urogenital malignancy (n = 15). Subsequently, human kidney immune cell subsets were analysed using multicolor flow cytometry and compared with subsets from C57BL/6 or BALB/c mice under specific pathogen-free conditions. Twenty kidney sections from healthy kidney donors or subjects without specific renal lesions were additionally analysed by immunohistochemistry. In human kidneys, 47% ± 12% (maximum 63%) of immune cells were CD3+ T cells. Kidney CD4+ and CD8+ T cells comprised 44% and 56% of total T cells. Of these, 47% ± 15% of T cells displayed an effector memory phenotype (CCR7− CD45RA− CD69−), and 48% ± 19% were kidney-resident cells (CCR7− CD45RA− CD69+). However, the proportions of human CD14+ and CD16+ myeloid cells were approximately 10% of total immune cells. A predominance of CD3+ T cells and a low proportion of CD14+ or CD68+ myeloid cells were also identified in healthy human kidney sections. In mouse kidneys, kidney-resident macrophages (CD11blow F4/80high) were the most predominant subset (up to 50%) but the proportion of CD3+ T cells was less than 20%. These results will be of use in studies in which mouse results are translated into human cases under homeostatic conditions or with disease.

Ascorbic acid deficiency induces hepatic and intestinal expression of inflammation-related genes irrespective of the presence or absence of gut microbiota in ODS rats

We have previously demonstrated that ascorbic acid (AsA) deficiency causes inflammatory changes in the liver and intestine in Osteogenic Disorder Shionogi (ODS) rats, which are unable to synthesize AsA. We have suggested that AsA deficiency increased intestinal interleukine (IL)-6 production, stimulating hepatic acute phase proteins (APPs) expression via the portal vein. In this study, we determined whether these hepatic and intestinal inflammatory changes by AsA deficiency are induced in germ-free (GF) ODS rats. For 18 days, male specific pathogen-free (SPF) ODS rats were fed the basal diet containing 600 mg AsA/kg (control group) or the AsA-free diet (AsA-deficient group) in SPF conditions, while male GF ODS rats were fed the basal diet (control group) or the AsA-free diet (AsA-deficient group) in GF conditions. Firstly, AsA deficiency significantly elevated the hepatic expression of APPs in both SPF and GF rats. In hepatic mRNA levels of some APPs, significant interaction between GF and AsA-deficiency effects was observed. Secondly, AsA deficiency elevated intestinal IL-6 and IL-1β mRNA levels in both SPF and GF rats, and significant interaction between GF and AsA-deficiency effects was observed in these mRNA levels of jejunum and cecum. In SPF and GF rats, AsA deficiency elevated portal IL-6 concentration. These results show that AsA deficiency caused hepatic and intestinal inflammatory changes in both the GF and SPF ODS rats and indicate that AsA deficiency could directly induce intestinal inflammatory changes without the involvement of gut microbiota.

Effect of Porphyromonas gingivalis infection on atherosclerosis in apolipoprotein-E knockout mice

Studies have indicated that periodontal pathogen Porphyromonas gingivalis (P. gingivalis) infection may contributed to accelerate the development of atherosclerosis. The aim of this study was to investigate the effect of inflammation, oxidative stress and the mechanism on atherosclerosis in apolipoprotein-E knockout (ApoE-/-) mice with P. gingivalis infection. Eight-week-old male ApoE-/- mice (C57BL/6) were maintained under specific pathogen-free conditions and fed regular chow and sterile water after 1 weeks of housing. The animals were randomly divided into two groups: (a) ApoE-/- + PBS (n=8); (b) ApoE-/- + P.gingivalis strain FDC381 (n=8). Both of the groups received intravenous injections 3 times per week for 4 weeks since 8 weeks of age. The sham control group received injections with phosphate buffered saline only, while the P. gingivalis-challenged group with P.gingivalis strain FDC381at the same time. After 4 weeks, oxidative stress mediators and inflammation cytokines were analyzed by oil red O in heart, Enzyme linked immunosorbent assay (ELISA) in serum, quantitative real-time PCR and Western blot in aorta. In our study, we found accelerated development of atherosclerosis and plaque formation in aorta with oil red O staining, increased oxidative stress markers [8-hydroxy-2-deoxyguanosine (8-OHdG), NADPH oxidase (NOX)-2 and NOX-4], as well as increased inflammation cytokines [interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α)] in the serum and aorta of the P. gingivalis-infected ApoE-/- mice. Compared with the control group, there was a significant increase protein level of nuclear factor-kappa B (NF-κB) in aorta after P. gingivalis infection. Our results suggest that chronic intravenous infection of P. gingivalis in ApoE-/- mice could accelerate the development of atherosclerosis by disturbing the lipid profile and inducing oxidative stress and inflammation. The NF-κB signaling pathway might play a potential role in the P. gingivalis-accelerated atherogenesis.

Mutation in Sodium-Glucose Cotransporter 2 Results in Down-Regulation of Amyloid Beta (A4) Precursor-Like Protein 1 in Young Age, Which May Lead to Poor Memory Retention in Old Age.

Senescence-accelerated mouse prone 10 (SAMP10) exhibits cerebral atrophy and depression-like behavior. A line of SAMP10 with spontaneous mutation in the Slc5a2 gene encoding the sodium-glucose cotransporter (SGLT) 2 was named SAMP10/TaSlc-Slc5a2slc (SAMP10-ΔSglt2) and was identified as a renal diabetes model. In contrast, a line of SAMP10 with no mutation in SGLT2 (SAMP10/TaIdrSlc, SAMP10(+)) was recently established under a specific pathogen-free condition. Here, we examined the mutation effect in SGLT2 on brain function and longevity. No differences were found in the survival curve, depression-like behavior, and age-related brain atrophy between SAMP10-ΔSglt2 and SAMP10(+). However, memory retention was lower in SAMP10-ΔSglt2 mice than SAMP10(+). Amyloid beta (A4) precursor-like protein 1 (Aplp1) expression was significantly lower in the hippocampus of SAMP10-ΔSGLT2 than in SAMP10(+) at 2 months of age, but was similar at 12 months of age. CaM kinase-like vesicle association (Camkv) expression was remarkably lower in SAMP10(+). These genes have been reported to be involved in dendrite function. Amyloid precursor proteins have been reported to involve in maintaining homeostasis of glucose and insulin. These results suggest that mutation in SGLT2 results in down-regulation of Aplp1 in young age, which can lead to poor memory retention in old age.

Severe Calorie Restriction Induces Gut Microbiota-Dependent Intestinal Stem Cell Dysfunction

Abstract Objectives Anorexia nervosa (AN) is a life-threatening illness, primarily characterized by extreme calorie restriction (CR). Given the substantial undernourishment in patients with AN, it is essential to understand the effects of severe CR on the small intestinal epithelium which is the primary tissue responsible for nutrient absorption and is therefore critical for the refeeding process. While we have previously reported that patients with AN harbor an altered intestinal microbiota, it remains unknown whether an AN-associated gut microbial community, induced by a nutrient-deprived environment, impacts intestinal epithelial physiology. Methods Conventionally-raised (CONVR) and germ-free (GF) mice were individually housed and placed on a 60% CR (60CR) diet for 9 days. CONVR mice were housed in standard specific-pathogen free conditions while GF mice were bred and housed in sterile isolators to occlude exposure to microorganisms. Mice were injected intraperitoneally with 4 mg/kg 5-ethynyl-2’deoxyuridine (EdU) 90 minutes before necropsy to mark proliferating cells. Intestinal tissue was collected for histology and intestinal crypts were isolated for ex vivo organoid culture. Results CONVR and GF mice on a 60CR diet lost significant body weight (28%±1 and 24%±1, respectively). 60CR CONVR and GF mice both displayed a significant increase in ISCs (+1.27 ± 0.02-fold and + 1.15 ± 0.05-fold, respectively) yet a significant decrease in proliferating cells (1.7 ± 0.2-fold and 1.7 ± 0.1-fold, respectively) compared to ad libitum controls. Interestingly, ex vivo organoids derived from crypts of CONVR 60CR mice were significantly smaller compared to organoids yielded from control crypts, but, strikingly, no differences in organoid size were observed between GF crypts from either diet group. Conclusions While ISC from 60CR CONVR and GF mice exhibited similar features, 60CR GF ISC function was maintained as indicated by the similar organoid growth relative to GF ad libitum controls. These data suggest that severe CR induces ISC dysfunction resulting in impaired de novo generation of digestive epithelial mass, and that these changes are microbiota-dependent. Funding Sources National Institutes of Health, North Carolina Translational and Clinical Sciences Institute, NCSU College of Veterinary Medicine, UNC Center for Gastrointestinal Biology and Disease.

Crypt residing bacteria and proximal colonic carcinogenesis in a mouse model of Lynch syndrome.

Colorectal cancer is a multifactorial disease involving inherited DNA mutations, environmental factors, gut inflammation and intestinal microbiota. Certain germline mutations within the DNA mismatch repair system are associated with Lynch syndrome tumors including right‐sided colorectal cancer with mucinous phenotype and presence of an inflammatory infiltrate. Such tumors are more often associated with bacterial biofilms, which may contribute to disease onset and progression. Inflammatory bowel diseases are also associated with colorectal cancer and intestinal dysbiosis. Herein we addressed the question, whether inflammation can aggravate colorectal cancer development under mismatch repair deficiency. MSH2loxP/loxP Vill‐cre mice were crossed into the IL‐10−/− background to study the importance of inflammation and mucosal bacteria as a driver of tumorigenesis in a Lynch syndrome mouse model. An increase in large bowel tumorigenesis was found in double knockout mice both under conventional housing and under specific pathogen‐free conditions. This increase was mostly due to the development of proximal tumors, a hotspot for tumorigenesis in Lynch syndrome, and was associated with a higher degree of inflammation. Additionally, bacterial invasion into the mucus of tumor crypts was observed in the proximal tumors. Inflammation shifted fecal and mucosal microbiota composition and was associated with enrichment in Escherichia‐Shigella as well as Akkermansia, Bacteroides and Parabacteroides genera in fecal samples. Tumor‐bearing double knockout mice showed a similar enrichment for Escherichia‐Shigella and Parabacteroides. Lactobacilli, Lachnospiraceae and Muribaculaceae family members were depleted upon inflammation. In summary, chronic inflammation aggravates colonic tumorigenesis under mismatch repair deficiency and is associated with a shift in microbiota composition.

Evaluating Influenza A virus vaccines in a dirty mouse model better mimics the human immune response

Abstract Current seasonal influenza A virus (IAV) vaccines often exhibit reduced efficacy to seasonal strains and offer limited protection to novel pandemics. A number of therapies, including vaccines, that are successful in mice often fail to translate to humans. This could be due to housing animals in specific pathogen free (SPF) conditions. Humans are exposed to a variety of natural host pathogens that SPF mice are protected from. Unlike SPF mice, the immune system of pet store mice more closely recapitulates the immune system of humans. When SPF mice are co-housed with pet store mice harboring natural mouse pathogens, termed dirty mice, these co-housed mice obtain phenotypes observed in the human immune system. In order to determine if dirty mice are an improved mouse model for vaccine development/testing, we sought to study the adaptive immune response to IAV in dirty mice. We infected SPF housed mice or dirty mice with IAV and assessed viral replication, clearance and adaptive immune responses. When we assessed memory responses to IAV in control or dirty mice, we observed altered B and T cell responses in dirty mice. These data demonstrate the importance of studying IAV in the dirty mouse model to facilitate more effective vaccine development and testing before human studies are performed.

Microbial exposure increases the magnitude and diversity of peritoneal inflammatory cell infiltrates and cytokine storm during sepsis

Abstract Sepsis – defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection - remains a major healthcare burden and cause of death in the United States and worldwide. While there has been a vast expansion in knowledge regarding the impact of sepsis on the immune system, virtually all preclinical sepsis research to date has relied on using mice housed under specific pathogen-free (SPF) conditions. Environmental pathogen exposure is one important difference between basic human and laboratory mouse biology that must be considered when using mice to evaluate immune system fitness. Interestingly, B6 mice that have been cohoused with pet store mice (to permit microbial transfer and immune system maturation; “CoH” mice) exhibit exaggerated acute hyperinflammation, morbidity, and mortality during cecal ligation and puncture (CLP)-induced polymicrobial sepsis compared to SPF B6 mice. To understand the mechanism of this increased acute response, we examined peritoneal immune cell composition in SPF and CoH mice before and after sepsis induction. CoH mice have more ‘large’ MHC IIloF4/80hi and ‘small’ MHC IIhiF4/80lo peritoneal macrophages (LPM and SPM) and neutrophils than SPF mice at baseline. Upon the induction of polymicrobial-induced peritonitis, there was increased recruitment of neutrophils, monocytes, T cells, and NK cells into the peritoneum of CoH mice vs. SPF mice. This increased immune cell influx correlated with increased CXCL1 and CCL2 in peritoneal lavage samples from CoH mice. CoH mice devoid of neutrophils or LPM exhibit reduced cytokine storm and improved outcomes after sepsis induction. These data suggest the cells of the innate immune system govern the magnitude of the acute septic response.

Dirty Mice Have Altered Influenza A Virus Clearance And Lesions Compared To SPF Mice: A Proposed Model For Study Of Vaccine Response In Humans

Influenza A virus (IAV) is a seasonal pathogen that causes annual morbidities and mortalities. Current vaccines often exhibit reduced efficacy to seasonal strains and offer limited protection to novel pandemics. Additionally, vaccines that are successful in mice often fail to translate to humans. This could be due to the nature of the mouse model housed in specific pathogen free (SPF) conditions. Humans are exposed to a variety of pathogens that SPF mice are protected from. Unlike SPF mice, the immune system of pet store mice more closely recapitulates the immune system of humans. When SPF mice are co‐housed with pet store mice harboring natural mouse pathogens, termed dirty mice, these mice obtain phenotypes observed in the human immune system. In order to determine if dirty mice are an improved mouse model for vaccine development/testing, we sought to study IAV disease in dirty mice. We infected control or dirty mice with IAV and assessed viral clearance and pathology. We observed reduced IAV clearance and enhanced airway pathology characterize by denuded and sloughed/necrotic airway epithelium in dirty mice. These data demonstrate the importance of studying IAV in the dirty mouse model to facilitate more effective vaccine development and testing to improve translation to humans.Support or Funding InformationNIH

Degeneration of the intestinal microbial community in PI3Kγ-knockout mice

Background and aimPI3Kγ is closely related to inflammation and cardiovascular diseases and thus, PI3Kγ inhibitors are candidate drugs for the treatment of these disorders. Considering the potential effect of the intestinal microbiome on inflammation and cardiovascular diseases, this study aimed to identify characteristics of the intestinal microbial community under PI3Kγ deficiency, to help reveal the potential influence of PI3Kγ inhibitors mediated by the microbial community. MethodsExon 2 of the PI3Kγ gene was knocked out in a Balb/c mouse by using single-guide RNAs. Homozygous PI3Kγ-knockout (PI3Kγ–/–) mice were obtained by embryo transfer and hybridization. PI3Kγ–/– and wild-type (WT) mice were raised in the same specific pathogen-free conditions until 8 weeks of age. Then, colonic tissues and feces from the middle segment of the colon were collected and analyzed by Illumina MiSeq sequencing. Differences in intestinal microbial community between the PI3Kγ–/– and WT mice were detected by bioinformatics analysis. ResultsThe richness and alpha diversity of the colonic microbial community were decreased in PI3Kγ–/– mice. The alpha diversity of the microbial community in feces did not differ between PI3Kγ–/– and WT mice. The beta diversity of the microbial community in feces of PI3Kγ–/– mice was obviously different from that in WT mice, whereas the within-group variation in Bray-Curtis distances of the mucosal microbial community was significantly decreased in PI3Kγ–/– mice. The topological structure of the species-related network of the colonic microbial community in PI3Kγ–/– mice was more polarized. Finally, we predicted that PI3Kγ deficiency might affect the synthesis of some antibiotics, bile acid, and thiamine through effects on the microbial community. ConclusionsPI3Kγ dysfunction led to degeneration of the intestinal microbial community and might alter the synthesis of some antibiotics, bile acids, and thiamine. The usage of PI3Kγ inhibitors for inflammation and cardiovascular diseases might lead to knock-on effect on our organism through intestinal microbiota.

Exogenous GM1 Ganglioside Attenuates Ketamine-Induced Neurocognitive Impairment in the Developing Rat Brain

A prolonged exposure to ketamine triggers significant neurodegeneration and long-term neurocognitive deficits in the developing brain. Monosialotetrahexosylganglioside (GM1) can limit the neuronal damage from necrosis and apoptosis in neurodegenerative conditions. We aimed to assess whether GM1 can prevent ketamine-induced developmental neurotoxicity. Postnatal day 7 (P7) rat pups received 5 doses of intraperitoneal ketamine (20 mg/kg per dose) at 90-minute intervals for 6 hours. Cognitive functions, determined by using Morris water maze (MWM) including escape latency (at P32-36) and platform crossing (at P37), were compared among the ketamine-exposed pups treated with or without exogenous GM1 (30 mg/kg; n = 12/group). The effect of GM1 on apoptosis in hippocampus was determined by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining and activated caspase 3 measurement. The hippocampal expression of brain-derived neurotrophic factor (BDNF), along with the phosphorylation of protein kinase B (AKT) and extracellular signal-related kinases 1 and 2 (ERK1/2), was detected by western blotting (n = 6/group). Anti-BDNF antibody (2 μg per rat) administered before GM1 treatment was applied to determine the neuroprotective mechanisms of GM1. The rats receiving ketamine exposure experinced cognitive impairment in MWM test compared to the control rats, indicated by prolonged escape latency at P34 (P = .006), P35 (P = .002), and P36 (P = .005). However, in GM1-pretreated rats, ketamine exposure did not induce prolonged escape latency. The exogenous GM1 increased the platform-crossing times at P37 (3.00 ± 2.22 times vs 5.40 ± 1.53 times, mean ± standard deviation; P = .041) and reduced the hippocampal TUNEL-positive cells and cleaved-caspase 3 expression in ketamine-exposed young rats. Ketamine decreased BDNF expression and phosphorylation of AKT and ERK in the hippocampus, whereas exogenous GM1 blocked these ketamine-caused effects. However, for the ketamine-exposed rat pups receiving exogenous GM1, compared to immunoglobulin Y (IgY) isotype control, the BDNF-neutralizing antibody treatment counteracted the exogenous GM1-induced improvement of the escape latency at P36 (41.32 ± 12.37 seconds vs 25.14 ± 8.97 seconds, mean ± standard deviation; P = .036), platform-crossing times at P37 (2.16 ± 1.12 times vs 3.92 ± 1.97 times, mean ± standard deviation; P < .036), apoptotic activity, as well as AKT and ERK1/2 phosphorylation in the hippocampus of ketamine-challenged young rats. Our data suggest that the exogenous GM1 acts on BDNF signaling pathway to ameliorate the cognitive impairment and hippocampal apoptosis induced by ketamine in young rats. Our study may indicate a potential use of GM1 in preventing the cognitive deficits induced by ketamine in the young per se.

Microbiota Suppresses Innate Type 2 Immune Responses in the lungs in Mice

Microbiota has been known to promote generation of regulatory T cells and prevent development of autoimmune diseases. However, our knowledge is limited regarding the role for microbiota in allergic airway diseases.The goal of this project was to address this question directly by using a mouse model. C57BL/6 mice were housed in a germ-free (GF) condition or specific pathogen-free (SPF) condition during pregnancy and after birth. The steady-state immune status was analyzed, and innate type 2 immune responses in the lungs were compared by exposing them intranasally (i.n.) to extract of fungus Alternaria. At steady-state, the total numbers of CD3+T cells and B220+B cells in the lungs were lower in GF mice as compared to SPF mice. On the other hand, plasma levels of total IgE and IgG1 were elevated in GF mice as compared to SPF mice. When naïve mice were exposed i.n, to Alternaria extract, GF mice produced significantly higher amounts of type 2 cytokines in the lungs. No differences were observed in Alternaria-induced IL-33 release or IL-33-induced innate type 2 response in two conditions. In contrast, lung levels of IL-10 were significantly lower at steady-state and after Alternaria exposure in GF mice as compared to SPF mice. Microbiota affects the steady-state levels of lung lymphocytes and suppresses IgE antibody production and innate type 2 responses to inhaled allergen(s). Lung IL-10, but not IL-33, is likely involved in regulating these responses and protecting animals from developing allergic airway diseases.

Reprint of: Manipulation of microbiota reveals altered callosal myelination and white matter plasticity in a model of Huntington disease

Structural and molecular myelination deficits represent early pathological features of Huntington disease (HD). Recent evidence from germ-free (GF) animals suggests a role for microbiota-gut-brain bidirectional communication in the regulation of myelination. In this study, we aimed to investigate the impact of microbiota on myelin plasticity and oligodendroglial population dynamics in the mixed-sex BACHD mouse model of HD. Ultrastructural analysis of myelin in the corpus callosum revealed alterations of myelin thickness in BACHD GF compared to specific-pathogen free (SPF) mice, whereas no differences were observed between wild-type (WT) groups. In contrast, myelin compaction was altered in all groups when compared to WT SPF animals. Levels of myelin-related proteins were generally reduced, and the number of mature oligodendrocytes was decreased in the prefrontal cortex under GF compared to SPF conditions, regardless of genotype. Minor differences in commensal bacteria at the family and genera levels were found in the gut microbiota of BACHD and WT animals housed in standard living conditions. Our findings indicate complex effects of a germ-free status on myelin-related characteristics, and highlight the adaptive properties of myelination as a result of environmental manipulation.

Cystatin C promotes cognitive dysfunction in rats with cerebral microbleeds by inhibiting the ERK/synapsin Ia/Ib pathway.

Although higher serum level of cystatin C (CysC) was observed in patients with cerebral microbleeds, its associated role in the disease has not been elucidated. In this work, a rat model of cerebral microbleeds was created with the aim of investigating effects of CysC on cognitive function in rats with cerebral microbleeds and the underlying mechanism. Serum samples of patients with cerebral microbleeds and healthy people of the same age were collected. Levels of cystatin C expression in these samples were measured using CysC kits. Moreover, 48 spontaneously hypertensive rats (SHRs) bred under specific pathogen-free (SPF) conditions were randomly divided into 4 groups: sham surgery control group (sham), model group (CMB), model + empty vector control group (CMB + vehicle), and model + cystatin C overexpression group (CMB + CysC). Expression levels of CysC in hippocampus of rats in each group were measured by western blot analysis. The Y-maze was used to evaluate cognitive function of rats. Hippocampal long-term potentiation (LTP) in rats was assessed by the electrophysiological assay. Alterations in levels of p-ERK1/2 and p-synapsin Ia/b proteins associated with cognitive function were identified by western blot analysis. The serum levels of CysC in patients with cerebral microbleeds were significantly upregulated (P<0.001). After injection of CysC, its expression levels in rat hippocampus were significantly increased (P<0.001), which enhanced the decline in learning and memory function, as well as the decrease of LTP in the rat model of cerebral microbleeds (P<0.001). Western blot results showed that injection of CysC further reduced the levels of p-ERK1/2 and p-synapsin Ia/b in the rat model of microbleeds (P<0.001). CysC was up regulated in serum of patients with cerebral microbleeds. It promoted cognitive dysfunction in rats with microbleeds by inhibiting ERK/synapsin Ia/Ib pathway.

Skin Associated Staphylococcus Aureus Contributes to Disease Progression in CTCL

It has been proposed that bacteria play a direct role in progression of cutaneous T cell lymphoma (CTCL), although definitive evidence is missing, and the underlying mechanism of how microbes contribute to disease progression remains unknown. The skin of CTCL patients is frequently colonized with Staphylococcus aureus (S. aureus) strains and infections with hospital and community associated strains of S. aureus are a frequent cause of morbidity and mortality among patients with advanced CTCL. Here we provide a comprehensive analysis of the association between CTCL and S. aureus colonization, and use our unique pre-clinical animal model of CTCL to determine the cause-effect relationship between skin-associated S. aureus and CTCL progression. To understand the relationship between bacterial colonization and CTCL we collected skin swabs from active lesions, unaffected skin and nares of CTCL patients to perform S. aureus cultures and 16S rRNA gene sequencing. Skin swabs of psoriasis patients and healthy donors served as controls. The frequency of S. aureus colonization determined by culture based techniques revealed that &amp;gt;65% of advanced stage patients had S. aureus present at lesional/tumor sites, while corresponding sites in patients with psoriasis and in healthy controls rarely had detectable S. aureus. Colonization rates correlated positively with the disease stage. Unbiased, 16s sequencing based analysis of the skin microbiome from advanced CTCL patients revealed that the overall skin microbiome of these patients is distinct from that of healthy individuals and patients with psoriasis. A lower phylogenetic diversity and significantly higher relative abundance of Staphylococcus species was found in CTCL patients. To determine the causal relationship between skin flora and progression of CTCL we used our mouse model of CTCL and assessed disease progression in both conventionally housed specific-pathogen-free (SPF) conditions and in germ free (GF) isolators using a standardized clinical score. The CD4CreSTAT3stopfl/+ mice express a hyper-active STAT3C mutant protein selectively in T lymphocytes and virtually all mutant mice develop T cell infiltration in the epidermis causing skin lesions resembling CTCL, by eight months of age. In contrast to the SPF housed animals, GF mice remained disease free or developed only a mild phenotype (clinical score 1 out of 5) after 11 months of follow-up. Notably, when GF CD4CreSTAT3stopfl/+ mice were transitioned to SPF conditions they all developed advanced disease. Finally, we examined the role of T cell antigen receptor (TCR) signaling in mediating the transformation of T lymphocytes. R26STAT3Cstopfl/+CD4Cre Rag2KO OTII mice express only OVA-specific TCRs. T cells from R26STAT3Cstopfl/+ CD4Cre Stim1fl/fl mice express a normal TCR repertoire, but exhibit defective T cell receptor signaling due to compromised calcium influx. Both strains failed to develop typical skin lesions, suggesting an essential role for TCR interaction with tumor microenvironment and microbial antigens in the pathogenesis of CTCL. In conclusion, we demonstrate a strong correlation between CTCL staging and rates of S. aureus colonization. Our study supports a cause-effect relationship between skin flora and CTCL oncogenesis. We propose that CTCL represents an antigen driven malignancy. Further studies using mono-colonization with single bacterial strains are needed to further interrogate the role of specific bacteria. Disclosures Hymes: Celgene: Consultancy. Odum:Micreos human Health B.V: Consultancy. Geskin:Merck: Other: Supported/Contracted Research; UpToDate: Patents &amp; Royalties: Royalty, Receipt of Intellectual Property Rights / Patent Holder; Mallinckrodt: Consultancy, Honoraria, Other: Supported/Contracted Research; Helsinn: Consultancy, Honoraria, Other: Supported/Contracted Research; Stratpharma: Other: Supported/Contracted Research; Medivir: Consultancy, Honoraria; Medscape: Speakers Bureau; Actelion: Other: Supported/Contracted Research.

The Influence of Ketogenic Diets on Psoriasiform-Like Skin Inflammation

The Influence of Ketogenic Diets on Psoriasiform-Like Skin Inflammation

The absence of interleukin 10 affects the morphology, differentiation, granule content and the production of cryptidin-4 in Paneth cells in mice.

Paneth cells (PCs) are specialized epithelial cells of the small bowel that contain multiple secretory granules filled with antimicrobial peptides and trophic factors, which are essential for the control of the microorganisms growth and maintaining intestinal integrity. Alterations in their function are associated with an imbalance of the normal microbiota, gastrointestinal infections and inflammatory processes, such as Crohn’s disease (CD). One of the most common murine models for studying CD is IL-10-/- mouse. IL-10-/- mice when housed in conventional conditions and take contact with commensal microorganisms develop an acute enterocolitis mediated by a Th1 immune response. Even though, alterations in PCs function are related to CD, they had not been characterized yet in this mouse model. Here we show that in specific pathogen free conditions IL-10-/- mice have aberrant granules and a large number of immature PCs at the bottom of the crypt in the ileum of IL-10-/- mice before developing intestinal inflammation, along with a reduced expression of Indian Hedgehog. In addition, IL-10-/- Paneth cells presented a reduced expression of cryptidin-4, and a heterogeneous distribution of lysozyme+ granules. The alterations in the maturation of the PCs at the bottom of the crypt were not modified after the colonization by the conventional microbiota. On the other hand, depletion of microbiota altered the phenotype, but did not normalize PCs. Our results suggest that IL-10 could be necessary for the integrity of PCs. Moreover, our results help to explain why IL-10-/- mice develop enterocolitis in response to microorganisms.