Mussarat Ashraf

CCL2 responses to Mycobacterium tuberculosis are associated with disease severity in tuberculosis.

Background Leucocyte activating chemokines such as CCL2, CCL3, and CXCL8 together with proinflammatory IFNγ, TNFα and downmodulatory IL10 play a central role in the restriction of M. tuberculosis infections, but is unclear whether these markers are indicative of tuberculosis disease severity. Methodology We investigated live M. tuberculosis- and M. bovis BCG- induced peripheral blood mononuclear cell responses in patients with tuberculosis (TB) and healthy endemic controls (ECs, n = 36). TB patients comprised pulmonary (PTB, n = 34) and extrapulmonary groups, subdivided into those with less severe localized extrapulmonary TB (L-ETB, n = 16) or severe disseminated ETB (D-ETB, n = 16). Secretion of CCL2, IFNγ, IL10 and CCL3, and mRNA expression of CCL2, TNFα, CCL3 and CXCL8 were determined. Results M. tuberculosis- and BCG- induced CCL2 secretion was significantly increased in both PTB and D-ETB (p<0.05, p<0.01) as compared with L-ETB patients. CCL2 secretion in response to M. tuberculosis was significantly greater than to BCG in the PTB and D-ETB groups. M. tuberculosis-induced CCL2 mRNA transcription was greater in PTB than L-ETB (p = 0.023), while CCL2 was reduced in L-ETB as compared with D-ETB (p = 0.005) patients. M. tuberculosis –induced IFNγ was greater in L-ETB than PTB (p = 0.04), while BCG-induced IFNγ was greater in L-ETB as compared with D-ETB patients (p = 0.036). TNFα mRNA expression was raised in PTB as compared with L-ETB group in response to M. tuberculosis (p = 0.02) and BCG (p = 0.03). Mycobacterium-induced CCL3 and CXCL8 was comparable between TB groups. Conclusions The increased CCL2 and TNFα in PTB patients may support effective leucocyte recruitment and M. tuberculosis localization. CCL2 alone is associated with severity of TB, possibly due to increased systemic inflammation found in severe disseminated TB or due to increased monocyte infiltration to lung parenchyma in pulmonary disease.

M. leprae inhibits apoptosis in THP-1 cells by downregulation of bad and bak and upregulation of mcl-1 gene expression

BackgroundVirulent Mycobacterium leprae interfere with host defense mechanisms such as cytokine activation and apoptosis. The mitochondrial pathway of apoptosis is regulated by the Bcl-2 family of proteins. Expression of Fas ligand and apoptotic proteins is found in leprosy lesions and M. leprae has been shown to activate pro-apoptotic Bcl-2 genes, Bak and Bax. However, the mechanism by which M. leprae modulates apoptosis is as yet unclear. We investigated expression of apoptotic genes in THP-1 monocytes in response to infection by M. leprae and non-pathogenic M. bovis BCG.ResultsM. leprae did not induce apoptosis in THP-1 cells, while BCG induced a significant loss of cell viability by 18 h post-infection at both (multiplicity of infection) MOI-10 and 20, with an increase by 48 h. BCG-induced cell death was accompanied by characteristic apoptotic DNA laddering in cells. Non-viable BCG had a limited effect on host cell death suggesting that BCG-induced apoptosis was a function of mycobacterial viability. M. leprae also activated lower levels of TNF-alpha secretion and TNF-alpha mRNA expression than BCG. Mycobacterium-induced activation of apoptotic gene expression was determined over a time course of infection. M. leprae reduced Bad and Bak mRNA expression by 18 h post-stimulation, with a further decrease at 48 h. Outcome of cell viability is determined by the ratio between pro- and anti-apoptotic proteins present in the cell. M. leprae infection resulted in downregulation of gene expression ratios, Bad/Bcl-2 mRNA by 39% and Bak/Bcl-2 mRNA by 23%. In contrast, live BCG increased Bad/Bcl-2 mRNA (29 %) but had a negligible effect on Bak/Bcl-2 mRNA. Heat killed BCG induced only a negligible (1–4 %) change in mRNA expression of either Bak/Bcl-2 or Bad/Bcl-2. Additionally, M. leprae upregulated the expression of anti-apoptotic gene Mcl-1 while, BCG downregulated Mcl-1 mRNA.ConclusionThis study proposes an association between mycobacterium-induced apoptosis in THP-1 cells and the regulation of Bcl-2 family of proteins. M. leprae restricts apoptosis in THP-1 cells by downregulation of Bad and Bak and upregulation of Mcl-1 mRNA expression.

ESAT6-Induced IFNγ and CXCL9 Can Differentiate Severity of Tuberculosis

Background Protective responses against Mycobacterium tuberculosis are dependent on appropriate T cell and macrophage activation. Mycobacterial antigen six kDa early secreted antigenic target (ESAT6) and culture filtrate protein 10 (CFP10) can detect M. tuberculosis specific IFNγ responses. However, most studies have been performed in non-endemic regions and to study pulmonary tuberculosis (PTB). We have studied ESAT6 and CFP10 induced cytokine and chemokines responses in PTB and extrapulmonary (EPul) TB. Methodology IFNγ, IL10, CXCL9 and CCL2 responses were determined using an ex vivo whole blood assay system in PTB (n = 30) and EPulTB patients with limited (LNTB, n = 24) or severe (SevTB, n = 22) disease, and in healthy endemic controls (ECs). Responses to bacterial LPS were also determined. Principal Findings ESAT6- and CFP10-induced IFNγ was comparable between ECs and TB patients. Both ESAT6- and CFP10-induced IFNγ secretion was greater in LNTB than PTB. ESAT6-induced CXCL9 was greater in EPulTB as compared with PTB, with an increase in SevTB as compared with LNTB. CFP10-induced CCL2 was higher in PTB than LNTB patients. LPS-stimulated CXCL9 was greatest in SevTB and LPS-induced CCL2 was increased in PTB as compared with LNTB patients. A positive correlation between ESAT6-induced IFNγ and CXCL9 was present in all TB patients, but IFNγ and CCL2 was only correlated in LNTB. ESAT-induced CCL2 and CXCL9 were significantly associated in LNTB while correlation in response to LPS was only present in SevTB. Conclusions ESAT6 induced IFNγ and CXCL9 can differentiate between limited and severe TB infections.

Differential live Mycobacterium tuberculosis-, M. bovis BCG-, recombinant ESAT6-, and culture filtrate protein 10-induced immunity in tuberculosis.

ABSTRACT The high prevalence of Mycobacterium tuberculosis makes it imperative that immune responses to evaluate could be predictive of infection. We investigated live Mycobacterium- and recombinant antigen-induced cytokine and chemokine responses in patients with active tuberculosis (TB) compared with those of healthy controls from an area where TB is endemic (ECs). M. tuberculosis-, M. bovis BCG-, ESAT6-, and culture filtrate protein 10 (CFP10)-induced responses were determined in peripheral blood mononuclear cells from patients with pulmonary TB (n = 38) and ECs (n = 39). The levels of the cytokines gamma interferon (IFN-γ) and interleukin-10 (IL-10) and the chemokines CCL2, CCL3, and CXCL9 were measured. The levels of M. tuberculosis- and BCG-induced IFN-γ secretion were significantly reduced (P = 0.002 and P < 0.01, respectively), while the amount of IL-10 induced by both virulent (P < 0.01) and avirulent (P = 0.002) mycobacteria was increased in patients with TB. The ESAT6-induced IFN-γ responses were increased in the patients with TB (P = 0.013) compared with those in the EC group. When tuberculin skin test (TST)-negative (TST−; induration, <10 mm) and TST-positive (TST+) donors were studied separately, both TST− and TST+ individuals showed increased IFN-γ responses to M. tuberculosis compared with the responses of the patients with TB (P = 0.037 and P = 0.006, respectively). However, only TST+ ECs showed reduced IFN-γ responses to ESAT6 (P = 0.008) compared with the responses of the patients with TB. The levels of M. tuberculosis-induced CCL2 (P = 0.006) and CXCL9 (P = 0.017) were greater in the patients with TB. The levels of CCL3 secretion in response to Mycobacterium and antigen stimulation were comparable between the two groups. While the levels of ESAT6-induced chemokines did not differ between the patients with TB and the ECs, the levels of CFP10-induced CCL2 (P = 0.01) and CXCL9 (P = 0.001) were increased in the patients. These data indicate differential host IFN-γ, CXCL9, and CCL2 responses to live mycobacteria and mycobacterial antigens and have implications for the identification of potential biomarkers of infection which could be used for the diagnosis of TB.

SOCS1 Gene Expression is Increased in Severe Pulmonary Tuberculosis

Suppressors of cytokine signalling (SOCS) molecules inhibit cytokine signalling and may regulate protective immunity in tuberculosis (TB). We investigated the association of SOCS with disease progression in patients with pulmonary TB. For this purpose, we studied peripheral blood mononuclear cells (PBMCs) and T cells from patients with pulmonary TB (TB, n = 33) and healthy endemic controls (EC, n = 15). Cases were stratified into those with moderately advanced (Mod‐PTB) or far advanced disease (Adv‐PTB). Interferon‐gamma (IFN‐γ), SOCS1 and SOCS3 gene expression was determined by RT‐PCR. Statistical analysis was performed using the Mann–Whitney test. Levels of IL6 (P = 0.018) and IL10 (P = 0.013) were found to be elevated in PBMC supernatants from patients with TB as compared with EC. SOCS1 mRNA gene expression in T cells from patients with TB was increased as compared with that of EC (P = 0.02). In addition, levels of SOCS1 mRNA transcripts were found to be elevated in PBMCs of Adv‐PTB as compared with Mod‐PTB (P = 0.008) cases. Our data show that raised SOCS1 levels are associated with increased disease severity in TB. As SOCS1 regulates IFN‐γ‐driven immunity and SOCS1 can be further upregulated by IL6 levels, the increase in SOCS1 in severe disease indicates a mechanism by which mycobacteria impede disease control in TB.

Mycobacterium tuberculosis Sonicate-Induced IFNγ, CXCL10 and IL10 can Differentiate Severity in Tuberculosis.

Improved tools are required to study immunopathogenesis of tuberculosis (TB). Mycobacterium tuberculosis antigen‐stimulated T cell‐based assays can detect TB but are less effective when responses are compromised such as in severe disease. We investigated immune responses to M. tuberculosis whole sonicate (MTBs), recombinant antigens ESAT6 and CFP10 in whole blood cells of healthy endemic controls (EC, n = 42) and patients with pulmonary (PTB, n = 36) or extrapulmonary (ETB, n = 41) disease. Biomarkers of T cell activation (IFNγ) or modulation (IL10) and chemokines, CXCL9, CXCL10 and CCL2, secretion were measured. MTBs, ESAT6 and CFP10 all induced IFNγ responses in TB. ESAT6‐induced IFNγ was elevated in TB as compared with EC. MTBs stimulated the highest IFNγ levels but did not differentiate between TB and EC. However, MTBs‐induced CXCL10 (P = 0.004) was reduced, while IL10 (P < 0.001) was raised in TB as compared with EC. Between sites, MTBs‐induced CCL2 (P = 0.001) and IL10 secretion was higher in PTB than ETB (P < 0.001). In comparison of disease severity, MTBs‐induced IFNγ (P = 0.014) and CXCL10 (P = 0.022) levels were raised in moderate as compared with far advanced PTB. In ETB, MTBs‐induced IL10 levels were greater in less‐severe (L‐ETB) than in severe disseminated (D‐ETB) cases, P = 0.035. Within the L‐ETB group, MTBs‐induced IFNγ was greater in patients with tuberculous lymphadenitis than those with pleural TB (P = 0.002). As immune responses to MTBs were differentially activated in TB of different sites and severity, we propose the utility of MTBs‐induced IFNγ, CXCL10 and IL10 as biomarkers in TB.

Alternate efflux pump mechanism may contribute to drug resistance in extensively drug-resistant isolates of Mycobacterium tuberculosis.

Introduction: Extensively drug-resistant tuberculosis (XDR-TB) has emerged as one of the biggest threats to public health and TB control programs worldwide. XDR-TB is caused by Mycobacterium tuberculosis (MTB) strains resistant to rifampin and isoniazid, as well as to a fluoroquinolone and to at least one injectable aminoglycoside. Drug resistance in MTB has primarily been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, it has also been shown that efflux pumps may play a role in resistance of MTB. Upregulation of drug efflux pumps can decrease the intracellular concentration of drugs and reduce their efficacy. Methods: Whole genome sequencing was performed on 32 XDR-TB clinical isolates. Sequence data were used to investigate SNPs in efflux pump genes as compared with the H37Rv reference genome. Results: Of the XDR MTB strains, eight (21.62%) were wild type for rpsL, rrs (500 region), and gidB genes, but had non-synonymous (ns) SNPs (aspartic acid to histidine) in the drrA efflux pump gene at position 3273138. Three of eight (37.5%) XDR MTB strains, wild type for rpsL, rrs (500 region), gidB, and gyrB genes were phenotypically streptomycin sensitive and five (62.5%) XDR MTB strains were streptomycin resistant, while all XDR MTB strains, wild type for rpsL, rrs, gidB, and gyrB genes were resistant to fluoroquinolone (ofloxacin) and ethambutol. In addition, three XDR MTB strains wild type for rpsL, rrs, gidB, and drrA genes showed nsSNPs (isoleucine to valine) in the major facilitator superfamily, Rv1634 efflux pump gene at position 1839306. Conclusion: Our data show an nsSNP in the drrA efflux pump gene that may result in upregulation of drug efflux mechanisms in MTB strains. It is therefore imperative to understand the mechanism of efflux and its role in drug resistance, which will enable the identification of new drug targets and development of new drug regimens to counteract the drug efflux mechanism of MTB.

Effective testing for pulmonary tuberculosis using Xpert MTB/RIF assay for stool specimens in immunocompetent Pakistani children

Objective/background: Childhood tuberculosis (TB) is largely a paucibacillary disease and difficult to diagnose. It is difficult to obtain a sputum or gastric aspirate (GA) sample, and patients are often undiagnosed and treated empirically. Stool is a noninvasive specimen not usually used for TB testing in Pakistan. We investigated the value of Xpert MTB/RIF to diagnose Mycobacterium tuberculosis (MTB) in children with pulmonary TB cases, by performing comparative testing of GA and stool samples. Method: We recruited 60 children aged 1–15 years, suspected of TB, from the Department of Pediatrics, Civil Hospital, Karachi, Pakistan and The Aga Khan University Hospital, Karachi, Pakistan. All were immunocompetent. Patients had a Kenneth Jones TB score of ≥5. Paired GA/sputum and stool samples were collected for testing. All GA samples were tested by Xpert MTB/RIF assay and MTB culture, while stool was tested by Xpert MTB/RIF. Results: The study participants included 27 males and 23 females with a mean age of 6 years and a mean TB (Kenneth Jones) score of 7. Stool was received in the laboratory within 1–2 days of the GA sample for all but one participant, who expired. The rates of MTB detection were as follows: 22% (11 cases) based on Xpert MTB testing of GA, 21% (10 cases) based on MTB culture of GA, and 21% (10 cases) based on Xpert MTB testing of stool. No rifampicin resistance was detected. Overall, there was concordance between testing of GA and stool. One case had GA with low positive Xpert and positive MTB culture, but negative stool Xpert result. In another case, there was low positive GA Xpert, positive GA MTB culture, and positive stool Xpert. A positive Xpert MTB stool test was associated with a higher TB score (>5) and a greater bacillary load. All 11 cases of TB diagnosed were put on antituberculous therapy and responded well to treatment. Conclusion: Use of Xpert MTB/RIF assay for stool-based diagnosis of pulmonary TB in immunocompetent children is useful in a resource poor setting. This is a valuable and noninvasive diagnostic alternative for the diagnosis of childhood TB and can be adapted by pediatric arms of national TB programs.

BCG vaccination is associated with decreased severity of tuberculosis in Pakistan

Vaccination with Bacille Calmette-Guérin (BCG) is given at birth to protect against tuberculosis (TB) in Pakistan. The country ranks 6th amongst high-burden countries worldwide and has an incidence of 231/100,000 pyopulation. This was a cross-sectional multi-center hospital-based study. TB patients (n=218) with pulmonary (PTB, n=120) or extrapulmonary (ETB, 98) were recruited, and the presence of a BCG vaccination scar was documented. Cases were further classified into minimal, moderate and advanced PTB or less severe (L-ETB) or severe disseminated (D-ETB) disease. The association of age, gender and severity of TB infections with BCG vaccination of the individual TB cases was investigated. No difference was found of the BCG vaccination status of PTB and ETB cases, or in relation to age or gender. Patients under 29years of age comprised the largest group. There were more females with ETB than PTB. The largest group within ETB comprised those with tuberculous lymphadenitis (LNTB, 39%). A significantly greater number of LNTB cases had received BCG vaccinations than had those with pleural (unilateral) TB (p=0.004), and tuberculous meningitis (p=0.027) groups. Also, there were more immunized patients with pulmonary as compared with pleural disease (p=0.001). LNTB represents localized granulomatous disease and the observation of higher vaccination rates in this group suggests that BCG has protected against more severe forms of TB in this high-burden region.

Measurement of ESAT6-induced IFNγ responses adjunct with CXCL9 increases the rate of diagnosis of active tuberculosis in an endemic region

Due to difficulties in direct diagnosis of Mycobacterium tuberculosis infection where site-specific specimens are not available, indirect methods of testing for infection are required. M. tuberculosis early secreted antigen target-6 (ESAT6) induced IFN-γ responses are specific, but do not differentiate between latent and active TB. The use of adjunct biomarkers for TB diagnosis has been proposed, such as the chemokines: CXCL9, CXCL10 and CCL2. ESAT6-induced IFN-γ CXCL9, CXCL10 and CCL2 was measured in whole blood cell supernatants of patients with pulmonary tuberculosis (PTB, n=36) and extrapulmonary TB (ETB, n=31) and compared with healthy endemic controls (EC, n=33). ESAT6-induced IFN-γ responses were positive in 32% of TB cases as compared with 15% of EC cases (p=0.048). ESAT6-induced CXCL9 responses were positive in 42% of TB cases and 15% of EC cases (p=0.006). ESAT6-induced-CXCL10 and -CCL2 responses did not discriminate between TB and EC groups. Measurement of IFN-γ or CXCL9 together diagnosed TB (53%) cases and was significant as compared with EC (p=0.014) cases. IFN-γ and CXCL10 together did not increase the number of TB cases diagnosed. Within TB groups, ESAT6-IFN-γ/CXCL9-based detection increased to 53% in PTB (p=0.031) and 54% in ETB (p=0.021), with comparable diagnosis in less severe extrapulmonary TB (L-ETB, 55%) and severe disseminated extrapulmonary TB (D-ETB, 50%). Given that 47% of TB cases remained undetected, this study shown that ESAT6-induced IFNγ and CXCL9 can support diagnosis, but must be supported by clinical correlation and other relevant investigations.