Whole genome transcriptional profiling of reconstructed human epidermis treated with 5% lactic acid compare to negative control as treated with water
ABSTRACT: Lactic acid is used to check skin sensitivity of human. However transcriptional profiling has not been done yet. This study was targeted to understand the transcriptional changes occur in human skin upon treatment with lactic acid.
Project description:GDAP1 is a mitochondrial fission factor and mutations in GDAP1 cause Charcot-Marie-Tooth disease. Gdap1 knockout mice, mimicking genetic alterations of patients suffering from severe CMT forms, develop an age-related, hypomyelinating peripheral neuropathy. We used microarrays to determine changes in the expression profiles in the peripheral nervous system before a phenotype was detectable in the animal model (2 month of age). To seek changes in gene expression patterns of Gdap1-/- mice, we isolated motoneurons by laser dissection and took sciatic nerve lysates of two-month-old mice and purified RNA of five mice. Based on the amount and quality of the isolated RNA we selected three samples per tissue (sciatic nerve lysate and motoneruons) and genotype (GDAP1-/- or wild type). Note: the probe sets for Gdap1 lie within exon 4 and exon 6, thus no apparent loss of Gdap1 mRNA expression is present in Gdap1-/- animals.
Project description:Changes in Treg function are difficult to quantify due to the lack of Treg-exclusive markers in humans and the complexity of functional experiments. We sorted naive and memory human Tregs and conventional T cells, and identified genes that identify human Tregs regardless of their state of activation. We developed this Treg signature using Affymetrix human genome U133A 2.0 microarrays. To generate Tregs and Tconvs in multiple states of activation, naïve (CD4+CD25hiCD45RA+) and memory (CD4+CD25hiCD45RA-) Tregs, and naïve (CD4+CD25-CD45RA+) and memory (CD4+CD25-CD45RA-) Tconvs were sorted from blood of 7 healthy adults and RNA was isolated ex vivo or after stimulation for 40h, promoting activation-induced FOXP3 in Tconvs. The gene-expression profile of the eight cell subsets was analyzed. 7 adult healthy control samples were sorted into 4 subsets: naïve (CD4+CD25hiCD45RA+) and memory (CD4+CD25hiCD45RA-) Tregs, and naïve (CD4+CD25-CD45RA+) and memory (CD4+CD25-CD45RA-) Tconvs. These were used for RNA ex vivo and after 40h stimulation with anti-CD3/CD28 beads to induce an activation phenotype.
Project description:Objective: Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS), characterized by a global increasing incidence driven by relapsing-remitting disease in females. p38 MAP kinase (MAPK) has been described as a key regulator of inflammatory responses in autoimmunity, but its role in the sexual dimorphism in MS or MS models remains unexplored. Methods: Toward this end, we used experimental autoimmune encephalomyelitis (EAE), the principal animal model of MS, combined with pharmacologic and genetic inhibition of p38 MAPK activity and transcriptomic analyses. Results: Pharmacologic inhibition of p38 MAPK selectively ameliorated EAE in female mice. Conditional deletion studies demonstrated that p38α signaling in macrophages/myeloid cells, but not T cells or dendritic cells, recapitulated this sexual dimorphism. Analysis of CNS inflammatory infiltrates showed that female, but not male mice lacking p38α in myeloid cells exhibited reduced immune cell activation compared with controls, while peripheral T cell priming was unaffected in both sexes. Transcriptomic analyses of myeloid cells revealed differences in p38α-controlled transcripts comprising female- and male-specific gene modules, with greater p38α dependence of pro-inflammatory gene expression in females. Interpretation: Our findings demonstrate a key role for p38α in myeloid cells in CNS autoimmunity and uncover important molecular mechanisms underlying sex differences in disease pathogenesis. Taken together, our results suggest that the p38 MAPK signaling pathway represents a novel target for much needed disease modifying therapies for MS WT vs. p38alphaCKO macrophages, male vs. female
Project description:Some commensal bacteria stimulate the immune system but do not present specific antigenicity. Such adjuvant effects have been reported for the bacterial species Lactobacillus plantarum. To study in vivo human responses to L. plantarum, a randomised double-blind placebo-controlled cross-over study was performed. Healthy adults were provided preparations of living and heat-killed L. plantarum bacteria, biopsies were taken from the intestinal mucosa and altered transcriptional profiles were analysed. Transcriptional profiles of human epithelia displayed striking differences upon exposure to living L. plantarum bacteria harvested at different growth phases. Modulation of NF-κB-dependent pathways was central among the major altered cellular responses. This unique in vivo study shows which cellular pathways are associated with the induction of immune tolerance in mucosal tissues towards common adjuvanticity possessing lactobacilli. Keywords: mucosal response of healthy adult humans to lactic acid bacteria This study was set up according to a randomised double-blind cross-over placebo-controlled design. It contains transcriptional profiles from biopsies from 8 healthy individuals after oral intake of three different growth stages of Lactobacillus plantarum or placebo control. In total, this study includes data from 8 individuals x 4 treatments=32 arrays.
Project description:Mechanisms controlling the proliferative activity of neural stem/progenitor cells (NSPCs) play a pivotal role to ensure life-long neurogenesis in the mammalian brain. How metabolic programs are coupled with NSPC activity remains unknown. Here we show that fatty acid synthase (FASN), the key enzyme of de novo lipogenesis, is highly active in adult NSPCs and that conditional deletion of FASN in NSPCs impairs adult neurogenesis. The rate of de novo lipid synthesis and subsequent proliferation of NSPCs is regulated by Spot14, a gene we found to be selectively expressed in low proliferating adult NSPCs. Spot14 reduces the availability of malonyl-CoA, which is an essential substrate for FASN to fuel lipogenesis. Thus, we here identified a functional coupling between the regulation of lipid metabolism and adult NSPC proliferation. 6 samples were analyzed. Spot14+: Spot14 GFP positive mouse neural stem cell, 3 biological rep Spot14-: Spot14 GFP negative mouse neural stem cell, 3 biological rep
Project description:Probiotic bacteria, specific representatives of bacterial species that are a common part of the human microbiota, are proposed to deliver health benefits to the consumer by modulation of intestinal function via largely unknown molecular mechanisms. To explore in vivo mucosal responses of healthy adults to probiotics, we obtained transcriptomes in an intervention study following a double-blind placebo-controlled cross-over design. In the mucosa of the proximal small intestine of healthy volunteers, probiotic strains from the species Lactobacillus acidophilus, L. casei and L. rhamnosus each induced differential gene regulatory networks and pathways in the human mucosa. Comprehensive analyses revealed that these transcriptional networks regulate major basal mucosal processes, and uncovered remarkable similarity to response profiles obtained for specific bioactive molecules and drugs. This study elucidates how intestinal mucosa of healthy humans perceive different probiotics and provides avenues for rationally designed tests of clinical applications. Keywords: mucosal response of healthy adult humans to lactic acid bacteria This study was set up according to a randomised double-blind cross-over placebo-controlled design. It contains transcriptional profiles from biopsies from 7 healthy individuals after oral intake of three different Lactobacillus species or placebo control. In total, this study includes data from 7 individuals x 4 treatments=28 arrays.
Project description:Brain perivascular cells have been recently identified as new mesodermal cell type of the human brain. These cells reside in the perivascular niche and were shown to have mesodermal and – to a lesser extend – tissue-specific differentiation potential. Mesenchymal stem cells (MSCs) are widely discussed for the use in cell therapy in many neurological disorders. Therefore it is of importance to better understand the “intrinsic” MSC population of the human brain. Here we systematically characterized adult human brain-derived pericytes during in vitro expansion and differentiation and compared these cells to fetal and adult human brain-derived NSCs and adult human bone marrow derived MSCs. We found that adult human brain pericytes can be isolated from hippocampal as well as cortical white matter, are – in contrast to adult human NSCs – easily expandable in monolayer cultures and show high similarities to human bone marrow-derived MSCs both regarding surface marker expression and whole transcriptome analysis. Human brain pericytes differentiated only in negligible amounts into neuroectodermal cell types using various differentiation conditions but efficiently differentiated into mesodermal progenies. Thus bone marrow-derived MSCs resemble human brain pericytes and might be therefore very interesting for possible autologous NPC-based treatment strategies, cell therapeutic approaches of neurological diseases. For the gene expression microarray analysis we used the Affymetrix U133A chips The whole procedure was performed following the manufacturer's standard protocol (Affymetrix, Santa Clara, CA). For the data processing, normalization was calculated with the GCRMA (GC content corrected Robust Multi-array Analysis) algorithm. Data post-processing and graphics was performed with in-house developed functions in Matlab. 17 samples were analyzed fNSC, Neural Stem Cell, 2 replicates ANPC-hip, adult Neuroprogenitor - Cell Hippocampus, 3 replicates ANPC-wm, adult Neuroprogenitor Cell - White Matter, 3 replicates ABPMC-hip, adult Brain Perivascular Mesodermal Cell - Hippocampus, 3 replicates ABPMC-wm, adult Brain Perivascular Mesodermal Cell - White Matter, 3 replicates MSC, Mesenchymal Stem Cell, 3 replicates
Project description:Exhausted T cells express multiple co-inhibitory molecules that impair their function and limit immunity to chronic viral infection. Defining novel markers of exhaustion is important both for identifying and potentially reversing T cell exhaustion. Herein, we show that the ectonucleotidse CD39 is a marker of exhausted CD8+ T cells. CD8+ T cells specific for HCV or HIV express high levels of CD39, but those specific for EBV and CMV do not. CD39 expressed by CD8+ T cells in chronic infection is enzymatically active, co-expressed with PD-1, marks cells with a transcriptional signature of T cell exhaustion and correlates with viral load in HIV and HCV. In the mouse model of chronic Lymphocytic Choriomeningitis Virus infection, virus-specific CD8+ T cells contain a population of CD39high CD8+ T cells that is absent in functional memory cells elicited by acute infection. This CD39high CD8+ T cell population is enriched for cells with the phenotypic and functional profile of terminal exhaustion. These findings provide a new marker of T cell exhaustion, and implicate the purinergic pathway in the regulation of T cell exhaustion. CD8+ T cells from subjects with HCV infection were sorted and pelleted and re-suspended in TRIzol (Invitrogen). RNA extraction was performed using the RNAdvance Tissue Isolation kit (Agencourt). Concentrations of total RNA were determined with a Nanodrop spectrophotometer or Ribogreen RNA quantification kits (Molecular Probes/Invitrogen). RNA purity was determined by Bioanalyzer 2100 traces (Agilent Technologies). Total RNA was amplified with the WT-Ovation Pico RNA Amplification system (NuGEN) according to the manufacturer's instructions. After fragmentation and biotinylation, cDNA was hybridized to HG-U133A 2.0 microarrays (Affymetrix).
Project description:This experiment compares the transciptional changes in antigen specific murine CD8 T cells (P14 T cells) after exposure in vivo to dendritic cells (DC) pulsed with low dose cognate peptide (1uM KAVYNFATC), high dose cognate peptide (100uM KAVYNFATC) or no antigen. Splenic dendritic cells were freshly isolated, peptide pulsed, washed and then adoptively transferred s.c. to the right footpad of C57BL/6 hosts. After 18h, freshly isolated P14 CD8 T cells were labelled with CMFDA and adoptively transferred iv. Two hours after T cell transfer, anti-L selectin antibody was given iv. At 12 and 24 hours, recipients were sacrificed and The right popliteal LN was harvested at 12 or 24h post T cell transfer and a single cell suspension was created and stained with PE CD4, B220 and CD19 (dump channel). Cells were then sorted on a FacsARIA for being non-doublets, CMFDA positive and dump channel negative. This experiment compares the transciptional changes in antigen specific murine CD8 T cells (P14 T cells) after exposure in vivo to dendritic cells (DC) pulsed with low dose cognate peptide (1uM KAVYNFATC), high dose cognate peptide (100uM KAVYNFATC) or no antigen. Splenic dendritic cells were freshly isolated, peptide pulsed, washed and then adoptively transferred s.c. to the right footpad of C57BL/6 hosts. After 18h, freshly isolated P14 CD8 T cells were labelled with CMFDA and adoptively transferred iv. Two hours after T cell transfer, anti-L selectin antibody was given iv. At 12 and 24 hours, recipients were sacrificed and The right popliteal LN was harvested at 12 or 24h post T cell transfer and a single cell suspension was created and stained with PE CD4, B220 and CD19 (dump channel). Cells were then sorted on a FacsARIA for being non-doublets, CMFDA positive and dump channel negative. The experiment was conducted for 39 samples out of which 35 passsed transcriptional quality control tests. The phenotypic distribution for the 35 samples includes: (1) high dose (100uM KAVYNFATC ) cognate peptide pulsed samples harvested at 12h post T cell transfer: 6 biological replicates (2) high dose (100uM KAVYNFATC ) cognate peptide pulsed samples harvested at 24h post T cell transfer: 7 biological replicates (3) low dose (1uM KAVYNFATC) cognate peptide pulsed samples harvested at 12h post T cell transfer: 6 biological replicates (4) low dose (1uM KAVYNFATC) cognate peptide pulsed samples harvested at 24h post T cell transfer: 9 biological replicates (5) no antigen pulsed samples harvested at 12h post T cell transfer: 3 biological replicates (6) no antigen pulsed samples harvested at 24h post T cell transfer: 4 biological replicates.
Project description:Mutations in BRCA1 and BRCA2 genes confer an increased lifetime risk for breast and ovarian cancer. Ovarian cancer risk can be decreased by risk-reducing salpingo-oophorectomy (RRSO). Studies on RRSO material have altered the paradigm of serous ovarian cancer pathogenesis. The purpose of this study was to identify candidate genes possibly involved in pathogenesis of serous ovarian cancer by carrying out a microarray analysis of differentially expressed genes in BRCA1/2- mutation positive ovarian and fallopian tube epithelium derived from RRSO surgery. Freshly frozen ovarian and fallopian tube samples from nine BRCA1/2 mutation carriers scheduled for RRSO were prospectively collected in comparison with five mutation-negative control patients undergoing salpingo-oophorectomy for benign indications. Microarray analysis of genome-wide gene expression was performed on ovarian and fallopian tube samples from BRCA1/2 and control patients. The validation of microarray data was performed by quantitative real-time polymerase chain reaction (qRT-PCR) in selected cases of RRSO samples, and also high grade serous carcinoma samples collected from patients with BRCA phenotype. From 22,733 genes, 454 transcripts were identified that were differentially expressed in BRCA1/2 mutation carriers when statistically compared to controls pooling all ovarian and fallopian tube samples together. Of these, 299 genes were statistically significantly downregulated and 155 genes were upregulated. Differentially expressed genes in BRCA1/2 samples reported here might be involved in serous ovarian carcinogenesis and provide interesting targets for further studies. Both fallopian tube and ovarian samples were collected from each BRCA1/2 mutation carrier resulting in eighteen mutation positive adnexal samples. Both fallopian tube and ovarian control samples were collected from one control patient while either ovarian or fallopian tube sample was available from four control patients, respectively, resulting in 6 adnexal control samples. High quality RNA was available from nine BRCA1/2-mutation positive ovarian and eight BRCA1/2-mutation positive fallopian tube samples and from three control ovarian and three control fallopian tube samples.