Sadhna Sharma

In the present study we have assessed T cell immuno-phenotypes in BCG vaccinated healthy individuals and patients with active pulmonary tuberculosis in response to two latency associated DosR Regulon Proteins Rv2626c and Rv2032. The proteins were shortlisted based on our previous bioinformatics analysis of the 48 DosR Regulon proteins. Both the proteins were seen to increase the percentage of CD4+ and CD8+ memory T cells in patients. Increase in expression of transcription factor T-Bet in response to the proteins suggested that the DosR proteins could be skewing the immune response toward the immune-protective TH1 type. This was confirmed with cell culture supernatant studies for release of TH1 and TH2 cytokines IFN- γ, IL-2, TGF-β, IL-4 and IL-10. A significant increase in frequency of CD4+/IFN-γ+ and CD8+/IFN-γ+T cells in patients was observed in response to both our proteins. This was accompanied with a significant downregulation in regulatory T cell population. Based on our findings of increase in TH1 response and decrease in Treg cells responsible for suppressing the immunity, we project Rv2626c and Rv2032 as antigens capable of inducing a strong immune response against Mycobacterium tuberculosis.

Viscum articulatum Burm. f. (leafless mistletoe) has been used in traditional system of medicines in India, China, Taiwan, Cambodia, Laos, and Vietnam, to treat blood-related diseases and various inflammatory and degenerative diseases including cancer. Anticancer activities of some phytomolecules purified from Viscum articulatum Burm. f. have been tested. However scientific evidence for the anticancerous potential of aqueous extract of V. articularum (VAQE) used in traditional medicine is lacking. To study the antiproliferative and apoptotic effect of VAQE on Jurkat E6.1 and THP1 leukemia cells. The aqueous extract of the whole plant of Viscum articulatum Burm. f. was prepared in phosphate buffer saline. In VAQE, total soluble protein was estimated using Bradford's dye-binding assay; flavonoid content was determined using aluminum chloride colorimetric assay; and phenolic content was estimated following Folin-Ciocalteu colorimetric assay. XTT cell viability assay was used to tes...

Leukemia is among the most aggressive and prevalent human malignant carcinoma. Chemotherapy is the preferred therapeutic strategy; however, recurrence of cancer and non-selective cytotoxicity are the major concerns. Unlike synthetic chemotherapeutic agents, mistletoe ribosome-inactivating protein (RIP) displays anti-tumor function in various types of cancers. However, its effect on leukemia cells is little explored. In this study, we assessed the impact of Viscum articulatum RIP (Articulatin-D) on the survival of acute T-cell leukemia cells and the involved molecular and cellular mechanisms. Cell proliferation assay showed that Articulatin-D suppressed the viability of leukemia cells selectively. We further confirmed that the elevation of mitochondrial membrane potential and exposure of phosphatidylserine are the early events of apoptosis induction in Articulatin-D-treated Jurkat cells. Subsequently, we found that Articulatin-D treatment induces apoptosis in Jurkat cells in a time- and concentration-dependent manner. In conclusion, we provided evidence that Articulatin-D efficiently activates caspase-8 involved in extrinsic pathway of apoptosis induction, which ultimately results in caspase-3-dependent DNA fragmentation of Jurkat cells. Further evaluation of Articulatin-D in cell culture and animal models may provide novel information on selective cytotoxicity to acute T-cell leukemia and its involvement in targeting tumor cell survival pathways.

Tuberculosis is a global health problem especially with the emergence of drug-resistant Mycobacterium tuberculosis strains, creating an urgent need to identify new drug targets. The mycobacterial cell wall is an attractive target for chemotherapeutic agents. Gene products of mymA operon are known to be required for the maintenance of cell wall and play an important role in persistence, thus making them important drug targets. This study was undertaken to biochemically characterize the MymA as a flavin-containing monooxygenase (FMO). Our results established its enzymatic activity in vitro and found that the mycobacterial FMO requires NADPH and FAD as cofactors, similar to other characterized bacterial FMOs. The enzyme follows Michaelis-Menten kinetics to catalyze substrates such as trimethylamine and thiourea. We also propose that MymA could be one of the targets of the antituberculosis drug, isoniazid (INH), which is a cell wall inhibitor. Molecular docking studies revealed that INH targeted NADPH-binding site of the MymA. Further, experimental validation revealed that INH inhibits MymA with the IC50 value of 4.9 μm. Thus, this study characterizes for the first time that MymA is a mycobacterial FMO, which may be a target of INH.

Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, is an intracellular bacterium capable of surviving and persisting within host mononuclear cells. The host response against tubercle bacilli is dominated by fine-tuned interaction of innate and adaptive immune responses. Toll-like receptors (TLRs) play a critical role in the formation of this immune response by facilitating in elaboration of protective T helper type 1 (Th1) cytokines and microbicidal molecules, but the intracellular persistence of M. tuberculosis in the phagosome and processing and presentation of TLR ligands by host antigen-presenting cell leads to continuous and chronic TLR2 signaling. The prolonged stimulation of TLR ultimately results in elaboration of immunosuppressive cytokines and downregulation of antigen presentation by major histocompatibility complex (MHC) class II and therefore becomes beneficial for M. tuberculosis, resulting in its continued survival inside macrophages. A...

Tuberculosis remains a great health threat to the world among infectious diseases particularly with the advent of human immunodeficiency virus and emergence of drug resistant strains. In the light of the inconsistent efficacy imparted by the only currently available pre-exposure vaccine bacillus Calmette-Guerin BCG, the development of an improved TB vaccine is a very high international research priority. Vaccine candidates currently in clinical trials are also pre-exposure vaccines that aim to prevent active tuberculosis during an individual's lifetime. According to World Health Organization approximately a third of the world's population is latently infected with Mycobacterium tuberculosis. Dormancy or latency of Mycobacteria is associated with the formation of granuloma with poorly perfused interior leading to expression of genes which help them survive in this hostile environment. A group of about 50 genes belonging to the DosR regulon also known as latency antigens are expressed by Mycobacteria when they are persisting in the immuno-competent host. An understanding of the immunological effects produced by products of these latency induced genes may help in making a more potent vaccine. Incorporation of latency antigens into improved (live or subunit) vaccines may enhance the impact of these vaccines in which BCG priming can be followed by multisubunit protein boosting. These vaccines could act as post exposure vaccines for containment and prevention of latent TB activation. This heterologous boosting of BCG-primed immunity will be able to stimulate the known immune correlates of protective immunity against M. tuberculosis i.e. TH1 cells (CD4(+) and CD8(+) T cells) mediated immune responses with cytokines such as IFN-γ and TNF-α⋅ In our review we have analysed and compared the immunogenic potential of various latency-associated antigens of the DosR regulon in line with the current strategy of developing a recombinant post exposure booster vaccine.

Immunopathogenesis of tuberculosis needs to be explored in search of a proper vaccine as well as for adjunctive immunotherapy particularly in patients with drug resistant tuberculosis. In tuberculosis, IFN-gamma, a product of T lymphocytes, contributes to protective immunity against M. tuberculosis by activating macrophages to a more effective elimination of these organisms. Interleukin-12 and interleukin-18 are macrophage products that favor the development of Th1 type of protective immune response. Production of these cytokines may not only facilitate granuloma formation and bacillary elimination but may also cause local tissue necrosis and systemic effects such as fever and wasting, due to the release of TNF-alpha into the circulation. The production of anti-inflammatory cytokines such as IL-10, TGF-beta and IL-4 in response to M. tuberculosis may down regulate the immune response and limit tissue injury by inhibiting excessive inflammatory response. These cytokines, if produced in excess, may result in failure to control infection resulting in widely disseminated tuberculosis. It is the balance between the inflammatory and protective immune response that determines the outcome of tuberculosis infection. In that context, increased IFN-y as against reduced TNF-alpha probably suggests a better outcome. Similarly, an effective vaccine has to stimulate a precise combination of T cells and cytokines needed for the many aspects of immune response and a potent immunotherapeutic agent may require to encompass the multiple parameters to be of therapeutic relevance.

The induction of apoptosis of T cells by intracellular pathogen is an attractive hypothesis to explain their persistence in host cells. To test this hypothesis, human monocyte-derived macrophages were infected with Mycobacterium tuberculosis H37Rv and cocultured with autologous T ...

Recent report from our laboratory showed that A549 cells representing alveolar epithelial cells produce chemokine interleukin-8 and nitric oxide (NO) when challenged with Mycobacterium tuberculosis. Interferon-(IFN-) played a critical role in priming these cells to generate NO ...

In view of the presence of a large number of epithelial cells in the alveoli of the lung and their ability to produce various cytokines and chemokines, the possible role of alveolar epithelial cells in the innate immune response to tuberculosis was examined. The human alveolar ...

Summary Sixty children with chronic diarrhoea, age ranging from 9 months to 3 years and 15 normal healthy chil-dren of same age group, all belonging to the low socio-economic families formed the basis of this study. Fifty-six out of these 60 children were undernourished and ...

Mycobacterium tuberculosis is an intracellular pathogen that readily survives and replicates in human macrophages. Host cells have developed various mycobactericidal and immunoregulatory mechanisms, such as the production of nitric oxide and inflammatory cytokines to control intracellular replication of M. tuberculosis. Inducible nitric oxide synthase (iNOS) is transcriptionally under the control of IFN-gamma and TNF-alpha. IL-12 provides a crucial link between activated mononuclear phagocytes and T cells by regulating the production of IFN-gamma. In this study, we investigated the production of nitric oxide (NO), TNF-alpha and IL-12 by the peripheral blood monocytes (PB Mn) of patients suffering from multidrug-resistant tuberculosis (MDR-TB). The cells were infected with M. tuberculosis and stimulated with IFN-gamma or activated with mycobacterial subcellular components. The results were compared with those from cases of newly diagnosed TB and healthy controls. Nitric oxide product...