Cell-specific methylation patterns in TB patients and household contacts
ABSTRACT: Monocytes and granulocytes were isolated from blood of TB patients and household contacts. DNA was isolated and methylation profile was measured using Illumina HumanMethylation450. House-hold contacts were matched to TB patients by gender and age. From each subject, two profiles (monocytes and granulocytes) were collected.
Project description:Monocytes and granulocytes were isolated from blood of TB patients and household contacts. RNA was isolated and gene expression was measured using microarrays. House-hold contacts were matched to TB patients by gender and age. From each subject, two profiles (monocytes and granulocytes) were collected.
Project description:Monocytes and granulocytes were isolated from blood of TB patients and household contacts. RNA was isolated and microRNA expression was measured using microarrays. House-hold contacts were matched to TB patients by gender and age. From each subject, two profiles (monocytes and granulocytes) were collected.
Project description:Monocytes and granulocytes were isolated from blood of TB patients and household contacts. RNA was isolated and microRNA expression was measured using microarrays. Overall design: House-hold contacts were matched to TB patients by gender and age. From each subject, two profiles (monocytes and granulocytes) were collected.
Project description:We sought to develop and characterize a novel paucibacillary model in mice, which develop necrotic lung granulomas following infection with Mycobacterium tuberculosis. Paucibacillary infection was established, recapitulating the sterilizing activities of human LTBI regimens. TNF neutralization led to increased lung bacillary load, disrupted granuloma architecture with expanded necrotic foci and reduced tissue hypoxia, and accelerated animal mortality. TNF-neutralized mouse lungs and sera showed significant upregulation of IFNγ, IL-1β, IL-6, IL-10, CCL2, CCL3, and matrix metalloproteinase genes Six weeks after aerosol-immunization with recombinant M. bovis BCG overexpressing the 30-kilodalton antigen, C3HeB/FeJ mice were aerosol-infected with M. tuberculosis H37Rv. Six weeks later, mice were treated with one of three standard regimens for latent TB infection (LTBI) or TNF-neutralizing antibody. Mouse lungs were analyzed by histology, positron emission tomography/computed tomography, whole-genome microarrays, and RT-PCR. Lungs and sera were studied by multiplex enzyme-linked immunosorbent assays
Project description:Most individuals infected with Mycobacterium tuberculosis can control the infection by forming and maintaining TB granulomas at the local infection foci. However, when the chronic infection (also known as latency) becomes active, the caseous center of TB granuloma enlarges, and it liquefies and cavitates, ultimately releasing bacilli into airway. Deciphering how genes are regulated within TB granulomas will help to understand the granuloma biology. Therefore, we performed genome-wide microarray on caseous human pulmonary TB granulomas and compared with normal lung tissues. Laser capture microdissection (LCM) was used to dissect out caseous granulomas from TB patients' lung tissues, excluding uninvolved areas. Total RNA were isolated from LCM-derived materials and used for microarray. As a control, parenchyma from normal lung tissues was prepared in the same manner as caseous granulomas. Sample GSM501252, Caseum 2-C, is missing a CEL file.
Project description:Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly impact on human and animal health worldwide. Mycobacterial life cycle is complex and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa) are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB). In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals when compared to uninfected controls are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB-) and M. bovis-infected young (TB+) and adult animals with different infection status [TB lesions localized in the head (TB+) or affecting multiple organs (TB++)]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls but protein levels increased as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggested that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recover to limit pathogen multiplication and promote survival that also facilitates pathogen transmission.
Project description:Infection with Mycobacterium tuberculosis (M. tb) is initiated when an aerosol droplet carrying a few bacilli is inhaled into an alveolus. Alveolar epithelial cells (AEC) (type II and type I) are among the first cells encountered by the infecting bacteria and greatly outnumber macrophages in the alveolus. M. tb replicates dramatically (>20,000 fold) in a “non-migrating” compartment in the lung prior to the development of the cell-mediated immune response in aerosol-infected mice (Wolf AJ, 2008). M. tb DNA has been found in type II AEC in autopsied lung tissue of latently infected individuals (Hernandez-Pando R et. al, 2000; Barrios-Payan J et. al 2012), and M. tb-infected AEC in broncho-alveolar lavage (BAL) and in sputum samples from TB patients indicates the infection of these cells in active as well as latent human TB (Eum SY et. al, 2010). M. tb has been shown to infect and replicate in the human type II AEC line, A549, and passaging of M. tb through A549 increases M. tb invasiveness (Bermudez LE et.al, 2002). In this work, we have used DNA microarray analysis to investigate the transcriptome of M. tb replicating in type II AEC (A549) compared to M. tb grown logarithmically in Middlebrook 7H9 broth media in order to identify M. tb adaptations to this intracellular environment as well as M. tb mechanisms/factors contributing to M. tb replication and increased invasiveness in primary infection. The global gene expression of M. tb H37Rv replicating in A549 cells at 72 hr was compared to that of M. tb grown to log phase in Middlebrook 7H9 media.
Project description:Mycobacterium tuberculosis (M. tb), the cause of tuberculosis (TB), utilizes the blood circulation to spread systemically and establish infection, and the risk of developing active TB (pulmonary and extrapulmonary) is significantly increased in individuals infected with human immunodeficiency virus (HIV). In this work, we have used DNA microarray analysis to investigate the transcriptome of M. tb replicating in human whole blood from both HIV-negative and HIV-positive donors compared to M. tb grown in Middlebrook 7H9 broth media in order to identify M. tb adaptations to this host environment as well as M. tb mechanisms/factors contributing to increased active and disseminated TB during M. tb/HIV co-infection. We compared the global gene expression of M. tb H37Rv replicating in whole blood from 6 HIV- and 6 HIV+ individulas at 96 hr to M. tb grown to log phase in Middlebrook 7H9 media.
Project description:For further identify the differentiation between latent and clinical tuberculosis (TB), we employed whole genome microarray expression profiling to study genes with significant expression change in peripheral CD4+T cells between healthy control, latent tuberculosis (LTB) and clinical tuberculosis (TB). Our experiment included 4 groups: healthy donor (HD), latent TB1 (LTB1) with low IFN-gamma release level, latent TB2 (LTB2) with high IFN-gamma release level, and tuberculosis (TB) with high IFN-gamma release level. Human peripheral blood mononuclear cells were collected, from which CD4+T cells were isolated. Total RNA of each individuals of each group was extracted from peripheral CD4+T cells. One μg of RNA mixture, pooled equivalently by each individual total RNA of each group, was administrated microarray test. Compared with HD, through analyzing enriched-Gene Ontology (GO) terms and KEGG pathways of each group, we found peripheral CD4+T cells might had different ability for mycobacterium tuberculosis infection in LTB1, LTB2 and TB. Finally we detected that TNFSF13/APRIL and TNFSF13B/BAFF was significant up-regulation in both CD4+T cells and serum of TB by real time PCR and ELISA, respectively. Peripheral CD4+ T cells were purified by positive selection using magnetic beads (BD IMagTM anti-human CD4 Particles-DM, BD Biosciences). Total RNA was extracted from CD4+ T cells (CD4-RNA) of LTB1, LTB2 and TB patients and healthy controls by RNeasy Mini Kit (QIAGEN).Then, RNA quality was checked and mixed with an equal quantity of each individual. The number of included-individuals in each group was 11 for HD, 11 for LTB1, 12 for LTB2 and 11 for TB.