Comparison of whole blood and PBMC assays for T-cell functional analysis

BACKGROUND: Tuberculosis remains the foremost cause of morbidity and mortality, more than any other single infectious disease in the world. Cell mediated immune response plays a crucial role in the control of tuberculosis. Therefore, measuring cell mediated immune response against the antigens is having a vital role in understanding the pathogenesis of tuberculosis, which will also help in the diagnosis of and vaccination for tuberculosis. FINDINGS: The aim of the present study was to compare and optimize the assay conditions to measure the cell mediated immune response against M. tuberculosis specific antigens. Because the conventional PBMC assays (due to requirement of large volume of blood sample) are unable to screen more number of antigens within the same blood sample. So, here we have compared 6 days culture supernatants of 1:5 and 1:10 diluted blood and PBMCs from healthy laboratory volunteers, to assess the proliferative response of T lymphocytes and secreted IFN-γ levels against purified recombinant antigen of M. tuberculosis (MPT51, Rv3803c), crude antigens of M. tuberculosis (PPD) and mitogen (PHA). CONCLUSIONS: We have observed good correlation between each assay and also the mean difference of these assays did not reach the statistical significance (p > 0.05). From these results, we conclude that 1:10 diluted whole-blood cultures can be well-suited as an alternative assay to measure cytokine production and lymphocyte proliferation in comparison to the conventional PBMC assays. Moreover, 1:10 diluted blood assays require less volume of blood when compared to PBMC assays which will be useful particularly in paediatric and field studies in endemic countries, where blood volume is a limiting factor

Role of a Putative Alkylhydroperoxidase Rv2159c in the Oxidative Stress Response and Virulence of Mycobacterium tuberculosis

Mycobacterium tuberculosis, which causes tuberculosis, is one of the leading infectious agents worldwide with a high rate of mortality. Following aerosol inhalation, M. tuberculosis primarily infects the alveolar macrophages, which results in a host immune response that gradually activates various antimicrobial mechanisms, including the production of reactive oxygen species (ROS), within the phagocytes to neutralize the bacteria. OxyR is the master regulator of oxidative stress response in several bacterial species. However, due to the absence of a functional oxyR locus in M. tuberculosis, the peroxidase stress is controlled by alkylhydroperoxidases. M. tuberculosis expresses alkylhydroperoxide reductase to counteract the toxic effects of ROS. In the current study, we report the functional characterization of an orthologue of alkylhydroperoxidase family member, Rv2159c, a conserved protein with putative peroxidase activity, during stress response and virulence of M. tuberculosis. We generated a gene knockout mutant of M. tuberculosis Rv2159c (MtbΔ2159) by specialized transduction. The MtbΔ2159 was sensitive to oxidative stress and exposure to toxic transition metals. In a human monocyte (THP-1) cell infection model, MtbΔ2159 showed reduced uptake and intracellular survival and increased expression of pro-inflammatory molecules, including IL-1β, IP-10, and MIP-1α, compared to the wild type M. tuberculosis and Rv2159c-complemented MtbΔ2159 strains. Similarly, in a guinea pig model of pulmonary infection, MtbΔ2159 displayed growth attenuation in the lungs, compared to the wild type M. tuberculosis and Rv2159c-complemented MtbΔ2159 strains. Our study suggests that Rv2159c has a significant role in maintaining the cellular homeostasis during stress and virulence of M. tuberculosis

Immunological and proteomic analysis of preparative isoelectric focusing separated culture filtrate antigens of Mycobacterium tuberculosis

Isolation of the secreted proteins and studying the immune response they induce is an essential prerequisite for understanding the pathogenesis of M. tuberculosis. In this study, preparative liquid-phase isoelectric focusing was used for the separation of culture filtrate protein (CFP) of M. tuberculosis. This procedure resolved culture filtrate proteins into 20 fractions with a pI range of 2.59 to 12.9. These 20 fractions were subjected to immunological analysis in healthy laboratory volunteers from our endemic area. Eleven fractions (Fractions 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, and 19) showed increased interferon gamma (IFN-γ) secretion and 5 fractions induced increased proliferative response, when compared to unfractionated CFP. In the 11 fractions which showed increased IFN-γ secretion, mass spectrometric analysis identified 19 different proteins. Apart from the already reported immunodominant antigens like FbpB, CFP-10 and ESAT-6, two new T cell antigens (AcpM and PpiA) were also identified in the immunologically active fractions. Immunoinformatic analysis showed that PpiA was predicted to bind more number of class I and class II HLA alleles compared with the immunodominant ESAT-6 and CFP-10. Population coverage calculations also showed that PpiA protein (85%) had a higher population coverage compared with ESAT-6 (79%) and CFP-10 (77%). This result shows that the PpiA protein has a potential to be a novel T cell antigen