Potential of adjunctive Mycobacterium w (MIP) immunotherapy in reducing the duration of standard chemotherapy against tuberculosis.

INTRODUCTION: The need to shorten the treatment duration in tuberculosis has always been felt. Immunotherapy in combination with chemotherapy has been considered a promising approach for this purpose into tuberculosis. We studied the adjuvant immunotherapeutic activity of Mycobacterium indicus pranii (MIP or Mw) in combination with conventional chemotherapy using guinea pig of pulmonary tuberculosis infected with Mycobacterium tuberculosis H37Rv via aerosol. METHODS: Experimental animals treated with standard chemotherapy and immunotherapy (MIP) separately and in combination of both. Guinea pig lungs evaluated following infection and subsequent therapy at predefine time point. Various cytokine mRNA expressions levels were quantified by quantitative reverse transcriptase PCR at the 4th, 8th and 12th week post-infection of M. tuberculosis. RESULTS: We determined the time required for bacterial clearance from guinea pig lungs. Standard chemotherapy (RvCh) compared to the animals where chemotherapy plus Mw immunotherpay (RvChMwT) was given. It took 12 weeks to achieve bacterial clearance in the RvCh group while this was achieved in 8 weeks in RvChMwT group. Pro-inflammatory cytokines (IFN-γ, IL-2, IL-12p35 and TNF-α) level were higher in RvCh, RvChMwT and RvMwT group, while the IL-10 and TGF-ß were suppressed. CONCLUSION: Cytokine expression level showed that Mw in conjunction with chemotherapy enhances the effect of pro-inflammatory cytokines (such as, IFN-γ, IL-2, IL-12 and TNF-α) and reduces the production and effect of anti-inflammatory cytokines (like IL-10 and TGF-ß) thereby restoring the pro-inflammatory / anti-inflammatory cytokines balance. Thus, the present study indicates that subject to rigorous testing by other parameters, Mw (MIP) as adjunct immunotherapy has potential for reducing treatment duration.

Rapid detection of rifampicin resistance in Mycobacterium tuberculosis by high-resolution melting curve analysis.

SETTING: High-resolution melting curve analysis (HRMA) can be used to screen for mutations in genes without the need for specific probes, with low turnaround time and high cost-effectiveness. OBJECTIVE: To detect the sensitivity and specificity of a line-probe assay (LPA) and HRMA in comparison with BACTEC™ MGIT™ 960 for the detection of rifampicin (RMP) resistance. DESIGN: A total of 219 Mycobacterium tuberculosis isolates tested by MGIT 960 for RMP susceptibility were tested with HRMA and LPA. Discordant samples were processed for sequencing of the RMP resistance-determining region (RRDR) of the rpoB gene. RESULTS: HRMA identified 93 of 103 (90.3%) isolates that were resistant and 113/116 (97.4%) that were susceptible on MGIT 960, with a sensitivity and specificity of respectively 90.3% and 97.4%. HRMA identified 117/119 (98.3%) LPA-susceptible and 94/100 (94%) resistant isolates, with 98.3% specificity and 94% sensitivity. Two isolates that were susceptible on LPA but resistant on HRMA showed silent mutations at 539 and 541 codons on sequencing, while 6 isolates that were susceptible on HRMA but resistant on LPA showed D516V (n = 4) and H526C/D (n = 2) mutations. Four isolates (3.9%) that were resistant on MGIT were susceptible on all three genotypic methods, which could be due to mutations outside the RRDR or efflux pumps. CONCLUSION: HRMA shows good potential as a rapid screening tool for the detection of drug resistance.

Expression of CXCL10 (IP-10) and CXCL11 (I-TAC) chemokines during Mycobacterium tuberculosis infection and immunoprophylaxis with Mycobacterium indicus pranii (Mw) in guinea pig.

Mycobacterium indicus pranii (earlier known as Mycobacterium w) has been used as an immunmodulatory agent in leprosy and tuberculosis by mediating the release of various cytokines and chemokines. CXCL10 (IP-10) and CXCL11 (I-TAC) chemokines are involved in T-cell migration and stimulation of natural killer cells in Mycobacterium tuberculosis infection. In this study, the effect of heat killed M. indicus pranii (alone and in conjunction with chemotherapy) on disease progression was determined by colony forming units (CFUs) in guinea pig lung following their aerosol infection and the expression levels of CXCL10 and CXCL11 were studied by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) and in situ RT-PCR. Four groups of animals included; infection only (Rv), immunoprophylaxis (RvMw), chemotherapy (RvCh) and combination of immunoprophylaxis with chemotherapy (RvChMw). In the group where immunoprophylaxis was given in combination with chemotherapy, the CFU counts reduced significantly at 4th week post-infection as compared to animals that received immunoprophylaxis or chemotherapy alone. At the same time, all groups of animals had elevated expression of CXCL 10 which was significantly high only in animals that received Mw with or without chemotherapy. Unlike to CXCL 10, study demonstrated suppressed expression CXCL 11 in both immunoprophylaxis as well as chemotherapy groups that became up-regulated in synergistic response of immunoprophylaxis and chemotherapy. Taken together, data indicates that the expression of CXCL10 and CXCL11 positively correlates with anti-tubercular treatment (at least with combination of immunoprophylaxis and chemotherapy). Therefore, prior immunization with Mw appears to be a good immunomodulator for release of chemokines and augments the effect of chemotherapy.

In vitro induction of the ubiquitous 60 and 70KD heat shock proteins by pesticides monocrotophos and endosulphan in Musca domestica: potential biomarkers of toxicity.

This study has investigated the effect of two highly toxic pesticides, monocrotophos (organophosphate) and endosulphan (organochlorine), on the inducibility of two major heat shock proteins, the HSP60 and HSP70, essential for cell survival, in the house fly Musca domestica. The LC50 values of the two pesticides for larva and adult (monocrotophos: 0.05 ppm for larva and 0.025 ppm for adult; endosulphan: 15 ppm for larva and 2 ppm for adult) revealed monocrotophos to be potentially more toxic than endosulphan. The relative susceptibility (lethality) of adult to either of these pesticides appeared much higher than that of larva. The expression patterns of HSP60 and HSP70 were analysed in various larval and adult tissues, exposed to varying sub-acute doses of monocrotophos (0.00010 ppm - 0.00075 ppm for larva and 0.00010 ppm - 0.00050 ppm for adult) and endosulphan (0.5ppm - 2.0ppm for larva and 0.01ppm-0.10 ppm for adult). The immunoblots revealed significant correlation between the pattern of HSP's expression and the pesticides-induced tissue injury/ mortality, visualized by trypan blue dye exclusion test. Both the pesticides caused significant induction of these HSPs in a tissue and dose­dependent manner, suggesting their importance as molecular indicators (biomarker) in the assessment of cellular toxicity caused by endosulphan and monocrotophos.

In vitro induction of 60-kDa and 70-kDa heat shock proteins by endosulphan and monocrotophos in sheep blowfly Lucilia cuprina.

The effect of two most commonly used and highly toxic organic pesticides, endosulphan (organochlorine) and monocrotophos (organophosphate), was studied in a blowfly, Lucilia cuprina, to test whether these pesticides induce the stress response and, if so, whether the intensity of the response, in terms of induction of heat shock proteins (HSPs), HSP60 and HSP70 in particular, is pesticide concentration dependent. The in vitro exposure of larval and adult tissues to varying concentrations of these pesticides (endosulphan: 1.0-4.0 ppm for larva and 0.05-0.50 ppm for adult; monocrotophos: 0.0005-0.0050 ppm for larva and 0.0001-0.0010 ppm for adult) revealed that both compounds were able to induce the expression of HSP60 and HSP70 proteins. Western blot analysis of these HSPs indicated that the induction of expression was tissue-specific. The trypan blue staining of pesticide-exposed tissues demonstrated monocrotophos to exert more severe effect than endosulphan, as the former compound induced both HSP60 and HSP70 significantly at a much lower concentration than that of the later. The pattern of expression of these HSPs, in general, appeared in direct correlation with the pesticide concentration. Gut tissues were found relatively more sensitive to pesticide toxicity than other tissues, as revealed by trypan blue staining, and hence, they might serve as primary targets for early detection of pesticide toxicity. The results indicated that either of these HSPs or both could serve as a potential biomarker toward assessment and monitoring of toxicity induced by these pesticides.