Pathogen-free Conditions Research Articles

Early-life polyphenol intake promotes Akkermansia growth and increase of host goblet cells in association with the potential synergistic effect of Lactobacillus

Mounting evidence suggests a critical role of gut microbiota in human colon health. Early life is a key developmental growth period, especially for building up gut microbiota and strengthening the colonic barrier. The connection between host colon and gut microbiota especially during early life is an area of increasing interest to researchers, also polyphenols improve host health through modulating this complex relationship. Postweaning (three-week-old) and adult (six-week-old) mice kept under specific pathogen-free conditions were used to investigate how early-life grape polyphenols supplementation influence the association between host colon and gut microbiota. Before grape polyphenols supplementation, postweaning mice had a higher original absolute abundance of Lactobacillus compared to adult mice. A 2-week grape polyphenols supplementation significantly boosted the abundance of Akkermansia and Lactobacillus and increased Lactobacillus-secreted lactate in postweaning mice. Early-life grape polyphenols supplementation also promoted the bloom of goblet cells and mucin 2, which benefitted both Akkermansia growth and colonic barrier. Moreover, the grape polyphenols-modulated bone morphogenetic protein (BMP), Notch and Wnt3 pathways triggered the bloom of goblet cells in GPs-administrated postweaning mice, and the increase in lactate could modulate those pathways. Meanwhile, adult mice were not affected by grape polyphenols supplementation. These results suggested that early-life polyphenol supplementation promoted Akkermansia growth and colonic barrier, which was in association with the sufficient abundance of Lactobacillus during early life. This study also indicated that Lactobacillus interact with Akkermansia through changing the physiology of host colonic goblet cells.

Heme Oxygenase-1 Protects Bone Marrow Mesenchymal Stem Cells Against Iron Overload through Decreasing ROS and Promoting IL-10 Generation

Background:Iron overload caused by frequent transfusions of suspended red blood cells which is of major concern in hematological disease. Heme oxygenase-1(HO-1) is well known as a crucial factor to cells against oxidative stress. In this study, significant upregulation of HO-1 was observed in bone marrow mesenchymal stem cells (BMMSCs) in the early phase of IO and predicted good prognosis in mice.Aim: Given that the role of HO-1 in protecting (BMMSCs) against damage from IO was still unknown. In the study, we investigated the effects and mechanisms of iron overload-related ROS by inducing IL-10 by regulating HO-1.METHODS:Male C57BL/6Ly5.2 (Ly45.2) mice were housed under specific pathogen-free conditions and allowed to acclimate for 2 weeks before experiments. The mice were housed (5 individuals per cage) and used at a weight of approximately 20.0 g. Thirty male mice were recipient mice, and another five were competitive mice. The thirty-five mice were randomly divided into four groups: (a) a control group; (b) an IO group (25 mg/ml); (c) an IO + hemin group and (d) an IO + ZnPP group. The depositions of iron in the liver, spleen and BM were assessed using HE staining and BM iron staining. Then, the expression of HO-1 and IL-10 were examined by Western blotting and q-PCR. Cell proliferation was measured by CCK-8 assay. The quantity of Ferritin, Serum iron, Hepcidin and IL-10 in the blood serum from iron overload cell were detected by ELISA in mouse model and iron overload patients. Finally, the levels of apoptotic rate, intracellular ROS and labile iron pool (LIP) were analyzed by flow cytometer.Result :We found that HO-1 was upregulated in the excess iron culture of BMMSCs in the early phase. Meanwhile, the apoptosis rate of IO BMMSCs was increased by 3.0 times compared with that of CON group. On the contrary, the apoptotic rate of BMMSCs down-regulated by transcription of si-HO-1 virus increased by 7.12 times compared to the CON group. In addition, the apoptosis rate of HO-1 overexpressing BMMSCs was significantly decreased in BM-1 virus transfected with HO-1 virus. Next, we also observed LIP in IO BMMSCs transfected with HO-1 virus was significantly lower than IO BMMSCs in un-transfected group. It suggested that HO-1 up-regulated the BMMSCs was protected from IO damage by decreasing intracellular iron content in vitro.To get an insight into the mechanism involved in cells protection from IO by HO-1 upregulation. We further explore production of ROS and secrection of IL10. As HO-1 was reported to have ability to reduce ROS, and IL-10 could promote excretion of intracellular iron. The results showed the ROS level in IO BMMSC was improved by 5.94 times compared with that of CON group, and the level of ROS in IO BMMSCs transcribed with si-HO-1 and HO-1 virus increased by 6.18 and 4.53 times, respectively. And IL-10 was increased significantly in the HO-1 up-regulated group.Next, we used U0126 (an ERK pathway inhibitor) to co-culture with IO BMMSCs that transcribed HO-1 virus. We observed the secretion of ferritin and the expression of IL-10 were consistent with the BMMSCs concentration changed. In vivo, we found that the serum iron and ferritin levels in the peripheral blood of IO mice were reduced by 1.92 and 1.3 times through ELISA analysis. The levels of IL-10 and ferritin were detected by ELISA. The results of deposition detection in bone marrow were consistent with the in vitro model. Finally, from November 2015 to December 2016, 12 patients diagnosed iron overload were enrolled. Including 6 males and 6 females, aged 44 to 77 years old. By q-PCR, we also found that iron overload in BMMSCs with high expression of HO-1 and IL-10 was reduced in serum ferritin after deferoxamine treatment. Therefore, we hypothesized that HO-1 overexpression of iron overload patients with better prognosis.Conclusions: we hypothesized that HO-1 overexpression of iron overload patients with better prognosis. The results of this study allow for further study of mechanisms of IO-related disease and provide new therapeutic targets for the treatment of IO patients. DisclosuresNo relevant conflicts of interest to declare.

Deficiency in CD4 T Cells Leads to Enhanced Postpartum Internal Carotid Artery Vasoconstriction in Mice: The Role of Nitric Oxide.

The risk of postpartum (PP) stroke is increased in complicated pregnancies. Deficiency in CD4 T cell subsets is associated with preeclampsia and may contribute to PP vascular disease, including internal carotid artery (ICA) stenosis and stroke. We hypothesized that CD4 T cell deficiency in pregnancy would result in ICA dysregulation, including enhanced ICA vasoconstriction. We characterized the function, mechanical behavior, and structure of ICAs from C57BL/6 (WT) and CD4 deficient (CD4KO) mice, and assessed the role of NO in the control of ICA function at pre-conception and PP. WT and CD4KO mice were housed under pathogen-free conditions, mated to same-strain males, and allowed to litter or left virgin. At 3 days or 4 weeks PP, mice were euthanized. The responses to phenylephrine (PE), high K+ and acetylcholine (ACh) were assessed in pressurized ICAs before and after NOS inhibition. Passive lumen diameters were measured at 3–140 mmHg. eNOS and iNOS expression as well as the presence of T cells were evaluated by immunohistochemistry. Constriction of WT ICAs to PE was not modified PP. In contrast, responses to PE were significantly increased in ICAs from PP as compared to virgin CD4KO mice. Constriction to high K+ was not enhanced PP. ICAs from WT and CD4KO mice were equally sensitive to ACh with a significant rightward shift of dose-response curves after L-NNA treatment. NOS inhibition enhanced PE constriction of ICAs from WT virgin and PP mice. Although a similar effect was detected in ICAs of virgin CD4KO mice, no such changes were observed in vessels from PP CD4KO mice. Passive arterial distensibility at physiological levels of pressure was not modified at PP. ICA diameters were significantly increased in PP with no change in vascular wall thickness. Comparison of eNOS expression in virgin, 3 days and 4 weeks PP revealed a reduced expression in ICA from CD4 KO vs. WT PP vessels which reached significance at 4 weeks PP. iNos expression was similar and decreased over the PP period in vessels from WT and CD4KO mice. Dysregulation of the CD4 T cell population in pregnancy may make ICA vulnerable to vasospasm due to decreased NO-dependent control of ICA constriction. This may lead to cerebral hypoperfusion and increase the risk of maternal PP stroke.

Visualization of Respiratory Commensal Bacteria in Context of Their Natural Host Environment.

Commensal microbes are an integral component of mammalian physiology. 16S rRNA gene-specific next generation sequencing from DNA of total organs, swabs or lavages has revolutionized the characterization of bacterial communities in virtually every ecological niche of the body. Culturomics, next allowed the isolation and characterization of commensal bacteria in the lab and the establishment of artificial communities of bacteria, which were eventually reintroduced in model organisms. Spatial organization of microbiota within a given host environment is critical to the physiological or pathological phenotypes provoked by commensal microbiota. In situ hybridization (ISH) is a complementary technique to sequencing and culturing to visualize the presence of individual bacterial operational taxonomic unit (OTUs) in context of the colonized organ. We recently applied highly sensitive in situ RNA hybridization to detection of commensal bacteria in low abundance respiratory tract samples of mice housed under specific pathogen free conditions. This technique allows species-specific detection of living bacteria using RNAScopeTM technology, while preserving the natural environment of the organ. We here provide a detailed step-by-step protocol describing the detection of commensal lung bacteria in respiratory tissue.

Vaccination of Algerian Local Rabbits with Precocious Strains of Eimeria magna and Eimeria media

The present study was conducted to assess the safety and the efficacy of a vaccine containing the Algerian precocious strains of Eimeria magna and Eimeria media used separately or together against rabbit coccidiosis. The samples consisted of 56 young rabbits reared in specific pathogen-free conditions. Following the challenge inoculation, statistically significant decreases in oocyst excretion were noticed in the vaccinated rabbits with the precocious strain of Eimeria magna, Eimeria media, and both species leading toa good immune response acquired by the vaccination associated with a good growth rate. Moreover, there was a statistically significant increase in oocyst output following the challenge in all challenged groups. Unlike the vaccinated groups, the challenged groups showed poor weight gains. More than 50% of the young rabbits from all the challenged groups presented diarrhea. Consequently, these precocious strains constitute good candidates for mono or polyvalent anticoccidial vaccines in the future.

Amelanotic B16-F10 Melanoma Compatible with Advanced Three-Dimensional Imaging Modalities

Amelanotic B16-F10 Melanoma Compatible with Advanced Three-Dimensional Imaging Modalities

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.

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.

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.

Evaluation of a cystatin-like protein of Trichinella spiralis for serodiagnosis and identification of immunodominant epitopes using monoclonal antibodies

An antigenic cystatin-like protein (Ts-CLP) selected from cDNA library of intestinal infective larvae at 6 h post-infection, was expressed by prokaryotes in the form of a histidine-tagged protein (rTs-CLP). The fusion protein was purified by an on-column refolding procedure using Ni-NTA affinity chromatography. An indirect rTs-CLP ELISA was developed using 270 known negative serum samples from commercial swine maintained under non-special pathogen free conditions. Based on the distribution of the signal-to-positive (S/P) ratio, a cut-off value was set at 0.30. Using this cut-off value, rTs-CLP ELISA was evaluated using sera from swine experimentally infected with 1000 and 50,000 muscle larvae of Trichinella spiralis. Specific IgG antibodies were detectable by rTs-CLP ELISA as soon as 17 days post-infection (dpi), but the commercial ELISA kit based on excretory-secretory (ES) antigens did not permit detection before 21 dpi. Three monoclonal antibodies (McAbs) against Ts-CLP (designated 1H9, 6B5 and 7F8) were obtained by screening with both rTs-CLP ELISA and ES ELISA methods. Two dominant epitopes recognized by McAbs were determined by analysis with overlapping fusion peptides and synthetic peptides. One epitope 39 HEALFSSDLKQESGV 53 was recognized by 1H9 and 6B5, and the other epitope 178 REALFSSDSKEQSGV 192 was recognized by 7F8. The generation of McAbs against Ts-CLP and the characterization of the two dominant epitopes provide a foundation for the development of a specific early serodiagnostic strategy for T. spiralis infection.

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

Rice CaM-binding transcription factor (OsCBT) mediates defense signaling via transcriptional reprogramming

The mutant allele of rice calmodulin-binding transcription activator OsCBT, oscbt-1, exhibits broad-spectrum resistance against rice pathogens. Previously, we reported that the strong resistance of the oscbt-1 mutant to pathogens was conferred by a constitutive upregulation of defense-related genes even under pathogen-free conditions. We also found strong induction of the hypersensitive response as a reaction to pathogen invasion. The results suggest that OsCBT acts as a negative regulator of basal resistance to pathogen attack. To identify the transcriptional network regulated by OsCBT, we compared global gene expression profiles between wild-type (WT) and oscbt-1 rice plants grown under pathogen-free conditions. The results of a 3′ tiling microarray revealed that in oscbt-1 plants, 81 genes are upregulated and 200 genes are downregulated when compared with the WT. A gene ontology analysis showed that differentially regulated genes in oscbt-1 were very closely associated with “death” GO term in a biological process category, and “catalytic activity” and “binding” GO terms in a molecular function category. A MapMan analysis indicated that the functions of these genes were associated with plant responses to biotic stress. Moreover, the results from quantitative real-time PCR in the oscbt-1 mutant showed a significant effect on the gene expression patterns of the fungal pathogen response. Our results suggested that the OsCBT regulates a rice defense response by modulating the expressions of various defense-related genes.

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.

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.

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.