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GNPS - Fecal microbiota transplant rescues mice from human pathogen mediated sepsis by restoring systemic immunity

ABSTRACT: A defined four-member pathogen community isolated from a patient with lethal sepsis was injected directly into the peritoneum (IP) of mice. To test the protective effects of fecal microbiota transplantation (FMT) in this model, immediately upon injection of PC and then once more 14 hours post injection, FMT or an autoclaved FMT (AC-FMT) was administered via enema. Cecal contents were collected approximately 20 hours post injection of PC and short chain fatty acids were extracted from the cecal contents using diethyl ether (Fisher Scientific), derivatized using N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide with 1% tert-Butyldimethylchlorosilane (Sigma). Acetate, butyrate, and propionate levels were measured by GC-MS.

INSTRUMENT(S): Agilent Single Quad GCMS (5977A Single Quad and 7890B GC)

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: John Alverdy

PROVIDER: MSV000085077 | GNPS | Tue Mar 10 15:48:00 GMT 2020

REPOSITORIES: GNPS

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Project description:Background: Vitamin D deficiency is associated with cardiac hypertrophy and heart failure, and vitamin D therapy prevents the progression of cardiac hypertrophy in animal models. Here, we examine whether vitamin D therapy regresses pre-existing cardiac hypertrophy, and prevents the progression to heart failure. Methods and Results: When male Dahl salt-sensitive (DSS) rats are fed a high salt (HS) diet, all rats develop cardiac hypertrophy after 5 weeks (H). Thereafter, rats were treated with vehicle (V), paricalcitol (PC, an active vitamin D analog at 200ng, IP 3x/wk), enalapril (EP, 90ug/day), and PC+EP. All groups were continued on the HS diet and evaluated after 4 weeks of therapy. The PC and PC+EP, but not the V and EP-only groups, showed significant regression of pre-existing cardiac hypertrophy. The signs of decompensated heart failure were evident in the vehicle-treated group; these heart failure parameters significantly improved with PC, EP or PC+EP therapy. The expression of PKCe, which is regulated by Ca2+ and known to stimulate cardiac hypertrophy, was significantly increased in the vehicle group, and PC, EP or PC+EP effectively decreased PKCe activation. We also observed normalization of genetic alterations during progression to heart failure with PC treatment. Conclusions: PC treatment resulted in both the regression of pre-existing cardiac hypertrophy, and the attenuation of the progression to heart failure, compared to improvement in progression to heart failure by EP alone. These beneficial findings in the heart were associated with inhibition of PKCe activation, and reversal of gene alterations. Male Dahl salt-sensitive rats (Harlan Sprague–Dawley, Somerville, NJ) were bred and fed a normal diet until 6 weeks of age. To generate pressure overload cardiac hypertrophy, they were then fed a high salt (6%NaCl) diet for the next 5 weeks. Data for baseline hypertrophic group (H) was obtained at the end of 11 weeks. Among H group animals, they were divided as follows and treated for an additional 4 weeks: 1) continuation of the HS diet with vehicle injection (H+V); 2) continuation of the HS diet with paricalcitol (19-nor-1,25-(OH)2 D2) (PC) (200ng IP 3x/wk) injection (H+PC); 3) continuation of the HS diet with low dose enalapril (EP), an angiotensin-converting enzyme inhibitor, infusion via osmotic pump and vehicle injection (H+EP+V); and 4) continuation of the HS diet with low dose EP infusion via osmotic pump and PC (200ng IP 3x/wk) injection (H+EP+PC). PC was prepared with 95% propylene glycol and 5% ethyl alcohol solution and administered three times a week on Monday, Wednesday, and Friday for 4 consecutive weeks. Vehicle groups received vehicle injections on the same schedule. Two groups of rats were implanted with pumps to deliver EP for 4 weeks. Since the reduction in blood pressure (BP) from high doses of EP would have effects on cardiac hypertrophy and progression to heart failure, we used low dose EP at 90ug/day, a maximum dose that did not significantly decrease BP in these rats, to study the effects of EP and PC that are independent of BP.

Project description:The aim of this experiment was to develop an approach for the assessment of immunotoxicity in zebrafish embryos (Danio rerio) using transcriptomic profiling. Clobetasol propionate, a highly potent synthetic corticosteroid, which is known to have immunosuppressive effects on vertebrates including zebrafish, was used for continuous exposure from fertilization until extraction. As immunotoxicity can only be comprehensively assessed when the effect is observed during an acute infection, the embryos were injected with a mixture of different pathogen-associated molecular patterns (PAMPs) consisting of Pam3CSK4 (synthetic lipopeptide), poly(I:C) (synthetic viral dsRNA) and flagellin (isolated from Bacillus subtilis). Functional gene expression analysis from samples obtained by different kinds and combinations of treatments in relation to appropriate controls is used to identify pathways that are upregulated by artificial infection and suppressed by treatment with clobetasol propionate. In a modified version of the zebrafish embryo toxicity test (OECD 236), 20 fertilized eggs were exposed to a sub-lethal concentration (250 nM) of clobetasol propionate for 48 hours under semi-static conditions. The test comprised of three treatments with clobetasol propionate and two negative control (NC) groups and was performed in triplicates. At 46 hours post fertilization (hpf), the eggs were manually dechorinated. At 48 hpf, the embryos were immobilized by anaesthetization using 200 mg/mL tricaine. Using microinjection, 8 nL of the mixed PAMPs (0.33 µg/mL Pam3CSK4, 0.33 µg/mL poly(I:C) and 0.033 µg/mL flagellin) were injected directly into the embryos bloodstream. One of each exposure groups was injected with PAMPs, while another one was injected with ultrapure water to control for effects introduced by the injection process. One control group remained completely untreated. After injection, the embryos were further incubated for 3 hours before 10 embryos were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit (Macherey-Nagel). RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before coding RNA was purified (PolyA selection with TruSeq RNA Library Prep Kit v2) and sequenced on an Illumina HiSeq 4000 System (Illumina) in 50 bp single read mode. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.

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GNPS - Fecal microbiota transplant rescues mice from human pathogen mediated sepsis by restoring systemic immunity

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