Tubercolosis and healthy infected patients PBMC_TB_vs_Pool_LTBI
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ABSTRACT: Comparison of PBMC tuberculosis patients and healthy infected control donors via an reference RNA pool from non-infected controls Keywords: Case control study
Project description:RNA-sequencing was performed on baseline blood samples from HIV-infected and HIV-uninfected asymptomatic adults with recent household exposure to an index case of infectious pulmonary tuberculosis (TB) and with detectable Mtb DNA in PBMC. Additional sequencing was also performed on follow-up blood samples from HIV-infected participants following completion of isoniazid preventative therapy.
Project description:Differences in the activity of monocytes/macrophages, important target cells of Mycobacterium tuberculosis, might influence tuberculosis progression. With the purpose of identifying candidate genes for tuberculosis susceptibility we infected with M. tuberculosis monocytes from both, healthy elders (a tuberculosis susceptibility group) and elderly tuberculosis patients, and performed a microarray experiment. We detected 78 differentially expressed transcripts and confirmed these results by quantitative PCR of selected genes. We found that monocytes from tuberculosis patients showed similar expression patterns of these genes regardless of whether they were obtained from younger or elder patients. Only one of the detected genes corresponded to a cytokine: IL-26, a member of the IL-10 cytokine family that we found downregulated in infected monocytes from tuberculosis patients. We have analyzed total RNA from Mycobacterium tuberculosis infected monocytes. We have isolated CD14+ cells (monocytes) from peripheral blood mononuclear cells by magnetic separation, and infected them for 4 days with 1 bacterium per monocyte. Blood donors were 7 elderly patients with pulmonary tuberculosis (average age: 83 years; sex: 3 men and 4 women) and 8 non-tuberculous volunteers (81 years, 6 men and 2 women).
Project description:Bovine tuberculosis (BTB) caused by Mycobacterium bovis continues to cause substantial losses to global agriculture and has significant repercussions for human health. The advent of high throughput genomics has facilitated large scale gene expression analyses that present a novel opportunity for revealing the molecular mechanisms underlying chronic mycobacterial infection. Using this approach, we have previously shown that innate immune genes in peripheral blood mononuclear cells (PBMC) from BTB-infected animals are repressed in vivo in the absence of exogenous stimulation. In the present study, functional genomics methods were used to examine the immune response of PBMC from BTB-infected (n = 6) and healthy control (n = 6) cattle to stimulation with bovine purified protein derivative (PPDb) in vitro. PBMC were harvested before, and at 3h and 12h post in vitro stimulation with PPDb. Gene expression changes were catalogued within each group using a reference hybridization design and a targeted immunospecific cDNA microarray platform (BOTL-5) with 4,800 spot features representing 1,391 genes
Project description:Primary human monocytes were isolated from four healthy human blood donors. Monocytes were isolated from PBMC buffy coats using plastic adherence for 4 hours. Monocytes were allowed to differentiate into macrophages over a period of 1 week. Macrophages from each of the 4 donors were split into two groups - uninfected and infected with Mycobacterium tuberculosis (Mtb). Cells in the infection group were infected with Mtb for 48 hours at a multiplicity of infection (MOI) of 10. Following incubation, uninfected and infected cells were harvested for RNA. RNA was used for next-generation RNA sequencing. Raw RNA sequencing data was processed using a HISAT2, Stringtie, Ballgown, DESeq2 pipeline. Processed data was used to measure differential expression between uninfected and infected macrophages.
Project description:Genome wide DNA methylation profiling of PBMC from South African patients either infected with HIV only or coinfected with HIV and tuberculosis (TB). The Illumina Infinium 27k Human DNA methylation Beadchip was used to obtain DNA methylation profiles from PBMC samples. Samples included 19 HIV patients and 20 HIV/TB co-infected patients.
Project description:Differences in the activity of monocytes/macrophages, important target cells of Mycobacterium tuberculosis, might influence tuberculosis progression. With the purpose of identifying candidate genes for tuberculosis susceptibility we infected with M. tuberculosis monocytes from both, healthy elders (a tuberculosis susceptibility group) and elderly tuberculosis patients, and performed a microarray experiment. We detected 78 differentially expressed transcripts and confirmed these results by quantitative PCR of selected genes. We found that monocytes from tuberculosis patients showed similar expression patterns of these genes regardless of whether they were obtained from younger or elder patients. Only one of the detected genes corresponded to a cytokine: IL-26, a member of the IL-10 cytokine family that we found downregulated in infected monocytes from tuberculosis patients.
Project description:HIV-infected slow-progressors (SP) represent a heterogeneous group of subjects who spontaneously control HIV infection without treatment for several years while showing moderate signs of disease progression. Here we determined the frequency of SP subjects who showed loss of HIV control within our Canadian Cohort of HIV+ Slow-Progressors and identified the proinflammatory cytokine IL-32 as a robust biomarker for control failure. Total cellular RNA was extracted from PBMC from 5 donors at two time points ('V1' before and 'V2' after loss of control, and only Long Term Non Progressors (LTNP) were part of the case study), along with 2 control Leuka914 and Aging106032 samples.
Project description:Genome wide DNA methylation profiling of PBMC from South African patients either infected with HIV only or coinfected with HIV and tuberculosis (TB). The Illumina Infinium 27k Human DNA methylation Beadchip was used to obtain DNA methylation profiles from PBMC samples. Samples included 19 HIV patients and 20 HIV/TB co-infected patients. Bisulphite converted DNA were hybridised to the Illumina Infinium 27k Human Methylation Beadchip v1.2
Project description:Tuberculosis Immune Reconstitution Inflammatory Syndrome (TB-IRIS) frequently complicates combined anti-retroviral therapy (ART) and anti-tubercular therapy in HIV-1 co-infected tuberculosis (TB) patients. The immunopathological mechanism underlying TB-IRIS is incompletely defined. Differential transcript abundance in PBMC from IRIS and control patients stimulated with heat killed H37Rv was determined by microarray
Project description:Tuberculosis Immune Reconstitution Inflammatory Syndrome (TB-IRIS) frequently complicates combined anti-retroviral therapy (ART) and anti-tubercular therapy in HIV-1 co-infected tuberculosis (TB) patients. The immunopathological mechanism underlying TB-IRIS is incompletely defined. Differential transcript abundance in PBMC from IRIS and control patients stimulated with heat killed H37Rv was determined by microarray Blood samples were collected during longitudinal observational studies of TB-IRIS patients and controls (both groups HIV-infected patients placed on antiretroviral treatment). PBMC were stimulated with heat killed H37Rv and RNA extracted.