Savita Kulkarni

Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology

BackgroundThe Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database.ResultsThe fourth international spoligotyping database, SpolDB4, describes 1939 shared-types (STs) representative of a total of 39,295 strains from 122 countries, which are tentatively classified into 62 clades/lineages using a mixed expert-based and bioinformatical approach. The SpolDB4 update adds 26 new potentially phylogeographically-specific MTC genotype families. It provides a clearer picture of the current MTC genomes diversity as well as on the relationships between the genetic attributes investigated (spoligotypes) and the infra-species classification and evolutionary history of the species. Indeed, an independent Naïve-Bayes mixture-model analysis has validated main of the previous supervised SpolDB3 classification results, confirming the usefulness of both supervised and unsupervised models as an approach to understand MTC population structure. Updated results on the epidemiological status of spoligotypes, as well as genetic prevalence maps on six main lineages are also shown. Our results suggests the existence of fine geographical genetic clines within MTC populations, that could mirror the passed and present Homo sapiens sapiens demographical and mycobacterial co-evolutionary history whose structure could be further reconstructed and modelled, thereby providing a large-scale conceptual framework of the global TB Epidemiologic Network.ConclusionOur results broaden the knowledge of the global phylogeography of the MTC complex. SpolDB4 should be a very useful tool to better define the identity of a given MTC clinical isolate, and to better analyze the links between its current spreading and previous evolutionary history. The building and mining of extended MTC polymorphic genetic databases is in progress.

Drug Resistant Clinical Isolates of Mycobacterium tuberculosis from Different Genotypes Exhibit Differential Host Responses in THP-1 Cells

Mycobacterium tuberculosis (MTB) persistently infects and survives within the host macrophages. Substantial genotypic variation exists among MTB strains which correlate with their interactions with the host. The present study was designed to establish a correlation, if any, between infection and induction of innate immune response by genetically diverse drug resistant MTB isolates from India. For this purpose, three clinical isolates from ancient and modern lineages, along with H37Ra and H37Rv were evaluated for intracellular growth, phagocytic index, induction of proinflammatory cytokines and apoptosis following infection in THP-1 cell line. A wide variation in the induction of cytokines was revealed subsequent to infection with different strains. EAI-5 strain from ancient lineage 1, induced higher proinflammatory responses, higher apoptosis and moderate intracellular growth compared to other strains, in contrast, for Beijing strain of modern lineage 2, all three parameters were lowest among the clinical isolates. Further, the responses induced by LAM-6 from modern lineage 4 were at a moderate level, similar to the laboratory strain H37Rv which also belongs to lineage 4. Thus, these profiles were specific to their respective lineages and/or genotypes and independent of their drug resistance status. Further, a positive correlation, among TNF-α, IL-1β, IL-6 and IL-12 induced in infected THP-1 cells was demonstrated. In addition, induction of all pro-inflammatory cytokines correlated well with the host cell apoptosis. A positive correlation was observed between phagocytic index in the category of ‘>10 bacilli/cell’ and induction of apoptosis, only for virulent strains, indicating that initial accumulation of MTB strains inside the host cell may be an important determining factor for different innate responses.

Use of polymerase chain reaction in the diagnosis of abdominal tuberculosis

Background:  Early diagnosis and prompt treatment of abdominal tuberculosis is vitally important as it greatly reduces disease and treatment related morbidity and even mortality in extreme cases. A polymerase chain reaction (PCR) test was evaluated for its feasibility as a diagnostic tool in abdominal tuberculosis (TB) in the Indian scenario.

G1-4A, a Polysaccharide from Tinospora cordifolia Inhibits the Survival of Mycobacterium tuberculosis by Modulating Host Immune Responses in TLR4 Dependent Manner

Rapid emergence of drug resistance in Mycobacterium tuberculosis (MTB) is a major health concern and demands the development of novel adjunct immunotherapeutic agents capable of modulating the host immune responses in order to control the pathogen. In the present study, we sought to investigate the immunomodulatory effects of G1-4A, a polysaccharide derived from the Indian medicinal plant Tinospora cordifolia, in in-vitro and aerosol mouse models of MTB infection. G1-4A treatment of MTB infected RAW264.7 macrophages significantly induced the surface expression of MHC-II and CD-86 molecules, secretion of proinflammatory cytokines (TNF-α, IL-β, IL-6, IL-12, IFN-γ) and nitric oxide leading to reduced intracellular survival of both drug sensitive (H37Rv) as well as multi drug resistant strains (Beijing and LAM) of MTB, which was partially attributed to G1-4A induced NO production in TLR4-MyD88 dependent manner. Similarly, bacillary burden was significantly reduced in the lungs of MTB infected BALB/c mice treated with G1-4A, with simultaneous up-regulation of the expression of TNF-α, INF-γ and NOS2 in the mouse lung along with increased levels of Th1 cytokines like IFN-γ, IL-12 and decreased levels of Th2 cytokine like IL-4 in the serum. Furthermore, combination of G1-4A with Isoniazid (INH) exhibited better protection against MTB compared to that due to INH or G1-4A alone, suggesting its potential as adjunct therapy. Our results demonstrate that modulation of host immune responses by G1-4A might improve the therapeutic efficacy of existing anti-tubercular drugs and provide an attractive strategy for the development of alternative therapies to control tuberculosis.

Mycobacterium tuberculosis H37Rv infected THP-1 cells induce epithelial mesenchymal transition (EMT) in lung adenocarcinoma epithelial cell line (A549).

Chronic infections of Mycobacterium tuberculosis (MTB) cause oxidative stress, TLR activation and production of inflammatory cytokines and thus can create an environment reinforcing tumorigenesis, progression and metastasis. Epidemiological studies have established a relation between lung cancer and tuberculosis but cellular mechanism is still poorly understood. In present study, we have shown for the first time that MTB infection in human monocytic cell line (THP-1) enhances invasion and induces EMT characteristics in lung adenocarcinoma cell line (A549) during co-culture. After co-culture with MTB infected THP-1 cells A549 cells exhibited morphological and molecular signatures of EMT. During co-culture, expression of inflammatory cytokines like TNF-α, IL-1β and IL-6 was enhanced in the microenvironment of A549 cells in comparison to single culture of A549 cells. Using pharmacological inhibitors of JNK (SP-600125) and p38 MAPK (SB-203580), we demonstrated the involvement of JNK and p38 MAPK in MTB induced EMT induction in A549 cells. To the best of our knowledge this is the first report demonstrating the role of MTB infection in induction of metastasis associated EMT in lung cancer.

Activation of murine macrophages by G1-4A, a polysaccharide from Tinospora cordifolia, in TLR4/MyD88 dependent manner

Abstract Macrophages are centrally placed in the innate immune system and their activation is crucial to the generation of appropriate immune response in the event of any pathogenic invasion, tumorigenesis or other human diseases. Many plant derived polysaccharides are known to activate macrophages. In the present study, effects of G1‐4A, a polysaccharide derived from Tinospora cordifolia, on the activation of macrophages were investigated. Our data demonstrated the up regulation of expression of TNF‐&agr;, IL‐&bgr;, IL‐6, IL‐12, IL‐10 and IFN‐&ggr; in RAW 264.7 cell line and peritoneal macrophages after G‐14A treatment. Nitric oxide levels were also enhanced along with up‐regulation of NOS2 expression in murine macrophages post G1‐4A treatment. Further, G1‐4A treatment up‐regulated the surface expression of MHC‐II and CD‐86 in macrophages. Using siRNA against TLR4, MyD88 and anti‐TLR4 blocking antibodies, we established that G1‐4A activated macrophages by classical pathway in TLR4‐MyD88 dependent manner. Additionally, G1‐4A treatment activated p38, ERK and JNK MAPKs in macrophages. Using pharmaceutical inhibitors of above MAPKs we concluded that G1‐4A activates the macrophages by activation of p38, ERK and JNK MAPKs in RAW264.7 macrophages. Thus our data suggests the activation of macrophages by classical pathway after treatment of G1‐4A. HighlightsG1‐4A treatment activates murine macrophages through classical pathway.G1‐4A mediated macrophage activation is TLR4/MyD88 dependent.ERK, JNK and p38 MAPK were involved in G1‐4A mediated macrophage activation.

Allium sativum constituents exhibit anti-tubercular activity In vitro and in RAW 264.7 mouse macrophage cells infected with Mycobacterium tuberculosis H37Rv

Background: Long duration of treatment, side-effects of currently used anti-tubercular drugs and emergence of drug-resistant forms of Mycobacterium tuberculosis (MTB) warrants the need to develop new drugs to tackle the scourge of tuberculosis (TB). Garlic is an edible plant reported to have anti-tubercular activity. However, previous researches on anti-tubercular effect of garlic were focused mostly on preliminary in vitro screening. Objective: To identify constituents responsible for anti-tubercular activity of thiosulfinate-derivative rich extract of garlic (GE) and to evaluate activity of the most active constituent in RAW 264.7 mouse macrophage cells infected with M. tuberculosis H37Rv (MTBH). Materials and Methods: In the present study, we have isolated eight compounds from GE by flash chromatography. The isolated compounds were characterized by 1H nuclear magnetic resonance spectroscopy, liquid chromatography-mass spectrometry and Fourier transform infrared spectroscopy. Individual isolates and GE were screened for activity against MTBH by Resazurin Microtitre Plate Assay (REMA). Results: Anti-tubercular activity of GE was superior to that of isolates when evaluated by REMA, possibly due to synergism amongst the constituents of GE. Cytotoxicity of GE was evaluated in RAW 264.7 mouse macrophage cells and it was observed that GE had a favorable selectivity index (>10). Therefore, anti-tubercular activity of GE was further evaluated by intracellular macrophage infection model. GE demonstrated concentration-dependent activity in macrophages infected with MTBH. Conclusion: This is the first report on intracellular anti-tubercular activity of any extract of garlic or its components. Appreciable intracellular anti-tubercular activity of GE in macrophages combined with low cytotoxicity makes it a suitable candidate for further development as an anti-tubercular agent. Abbreviations used: TB: Tuberculosis, MTB: Mycobacterium tuberculosis, MTBH: Mycobacterium tuberculosis H37Rv, GE: Thiosulfinate-derivative rich extract of garlic, REMA: Resazurin Microtitre Plate Assay, VD: Vinyldithiin, CFU: Colony forming unit, 1H NMR: 1H nuclear magnetic resonance spectroscopy, FT-IR: Fourier transform-infrared spectroscopy, LC-MS: Liquid chromatography-mass spectrometry, IC50: Concentration required to inhibit the cells by 50%, ANOVA: Analysis of variance.

A multi-analyte immunoassay for thyroid related analytes

ABSTRACT We describe the development and validation of multianalyte immunoassays (MAIA) for three analytes, viz., thyroxine (T4), thyroid stimulating hormone (TSH), and thyroglobulin (Tg) essential for assessment of thyroid function but having widely varying molecular weights. Using polycarbonate (PC) track-etched membranes (TEM) as an immobilization support and 125I as the tracer, both competitive assay for T4 and non-competitive assay for TSH and Tg were performed on the same TEM. MAIA was found to be highly sensitive and precise with clinically useful working range and correlated very well with individual analyte immunoassays. While we have demonstrated this assay format with radiotracer, it can be used with non-isotopic tracers equally well.

Limulus Amebocyte Lysate Testing: Adapting It for Determination of Bacterial Endotoxin in 99mTc-Labeled Radiopharmaceuticals at a Hospital Radiopharmacy

A bacterial endotoxin test (BET) is required to detect or quantify bacterial endotoxin that may be present in radiopharmaceutical preparations. The test uses Limulus amebocyte lysate, which, in the presence of bacterial endotoxin and divalent calcium ions, causes the formation of a coagulin gel. 99mTc-labeled radiopharmaceuticals have chelating ligands such as diethylene triamine pentaacetic acid (DTPA), ethylene dicysteine (EC), L,L-ethyl cysteinate dimer (ECD), N-[2,4,6-trimethyl-3 bromoacetanilid] iminodiacetic acid (mebrofenin), dimercapto succinic acid-III (DMSA-III), dimercapto succinic acid-V (DMSA-V), and several others, which form a coordination complex with Na-99mTc-O4 in the presence of reducing agents. During BET by the gel-clot method, the free sulfhydryl (–SH) and carboxyl (–COOH) in some of the chelating agents in the final 99mTc-labeled radiopharmaceuticals decrease the free divalent calcium ion concentration, which in turn inhibits coagulin gel formation. This study was designed using the premise that addition of calcium chloride solution to the reaction mixture would nullify this effect. Methods: We present here the data obtained from BET assay analysis of 99mTc-labeled radiopharmaceuticals and the cold kits from which they are made (EC, ECD, methoxyisobutylisonitrile, DTPA, mebrofenin, methylene diphosphonic acid [MDP], DMSA-III, and DMSA-V) using 2 different dilutions, maximum valid dilution (MVD) and half maximum valid dilution (MVD/2), with and without the addition of calcium chloride at a final concentration of 300 μM. Results: It was observed that at MVD and MVD/2 all of the 99mTc-labeled kits exhibited interference in coagulin gel formation with the exception of 99mTc-methoxyisobutylisonitrile, 99mTc-MDP, 99mTc-mebrofenin, and 99mTc-ECD. However, only the cold kits of methoxyisobutylisonitrile and MDP did not show inhibition. An addition of calcium chloride solution nullified this interference at both MVD and MVD/2 in all of the 99mTc-labeled radiopharmaceuticals in which interference was observed. Conclusion: In practice, Limulus amoebocyte lysate testing is not a method of choice for 99mTc-labeled radiopharmaceuticals because these radiopharmaceuticals exhibit interference. However, our study proves the hypothesis that the addition of calcium chloride can circumvent this problem. The addition of calcium chloride provides an enhanced biologic quality control testing option for the final formulation of 99mTc-labeled radiopharmaceuticals at the hospital radiopharmacy end.

Inhibition of Intracellular Survival of Multi Drug Resistant Clinical Isolates of Mycobacterium tuberculosis in Macrophages by Curcumin

Curcuma longa commonly known as turmeric has been used in Indian Ayurvedic medicine as a constituent to treat various disorders. It is by now clear that principal curcuminoid of turmeric; curcumin, a yellow pigment, is responsi- ble for these beneficiary activities. The aim of the present study was to evaluate anti-mycobacterial effect of curcumin (CMN) on intracellular growth of MDR clinical isolates of Mycobacterium tuberculosis (MTB). Curcumin was evaluated for its efficacy to inhibit the intracellular growth of MTB H37Rv and two MDR clinical isolates in Raw 264.7 cell line us- ing CFU assay. Resazurin microtiter plate assay (REMA) was used to evaluate its direct anti-mycobacterial activity. Curcumin, though did not show direct anti-mycobacterial activity against three MTB strains, exhibited dose dependent in- hibition of intracellular growth for MTB H37Rv as well as two MDR clinical isolates. These results suggest that CMN could be a potential candidate for future, novel adjunctive anti-TB therapy.