Emerging role of exosomes as a liquid biopsy tool for diagnosis, prognosis & monitoring treatment response of communicable & non-communicable diseases.

ABSTRACT: From an initial thought of being used as a cellular garbage bin to a promising target for liquid biopsies, the role of exosomes has drastically evolved in just a few years of their discovery in 1983. Exosomes are naturally secreted nano-sized vesicles, abundant in all types of body fluids and can be isolated intact even from the stored biological samples. Being stable carriers of genetic material (cellular DNA, mRNA and miRNA) and having specific cargo (signature content of originating cells), exosomes play a crucial role in pathogenesis and have been identified as a novel source of biomarkers in a variety of disease conditions. Recently exosomes have emerged as a promising 'liquid biopsy tool'and have shown great potential in the field of non-invasive disease diagnostics, prognostics and treatment response monitoring in both communicable as well as non-communicable diseases. However, there are certain limitations to overcome which restrict the use of exosome-based liquid biopsy as a gold standard testing procedure in routine clinical practices. The present review summarizes the current knowledge on the role of exosomes as the liquid biopsy tool in diagnosis, prognosis and treatment response monitoring in communicable and non-communicable diseases and highlights the major limitations, technical advancements and future prospects of the utilization of exosome-based liquid biopsy in clinical interventions.

Dominant negative biologics normalise the tumour necrosis factor (TNF-α) induced angiogenesis which exploits the Mycobacterium tuberculosis dissemination.

BACKGROUND: Tumor necrosis factor (TNF) is known to promote T cell migration and increase the expression of vascular endothelial growth factor (VEGF) and chemokines. The administration of Xpro-1595, a dominant-negative TNF (DN-TNF) engineered to selectively inactivate soluble TNF (solTNF), has been extensively studied and proven effective in reducing TNF production without suppressing innate immunity during infection. The literature also supports the involvement of glutamic acid-leucine-arginine (ELR+) chemokines and VEGF in angiogenesis and the spread of infections. MATERIALS AND METHODS: In this study, we administered Xpro-1595 to guinea pigs to selectively inhibit solTNF, aiming to assess its impact on Mycobacterium tuberculosis (M.tb) dissemination, bacterial growth attenuation, and immunological responses. We conducted immunohistochemical analyses, immunological assays, and colony enumeration to comprehensively study the effects of Xpro-1595 by comparing with anti-TB drugs treated M.tb infected guinea pigs. Throughout the infection and treatment period, we measured the levels of Interleukin-12 subunit alpha (IL-12), Interferon-gamma (IFN-γ), TNF, Tumor growth factor (TGF), and T lymphocytes using ELISA. RESULTS: Our findings revealed a reduction in M.tb dissemination and inflammation without compromising the immune response during Xpro-1595 treatment. Notably, Xpro-1595 therapy effectively regulated the expression of VEGFA and ELR + chemokines, which emerged as key factors contributing to infection dissemination. Furthermore, this treatment influenced the migration of CD4 T cells in the early stages of infection, subsequently leading to a reduced T cell response and controlled proinflammatory signalling, thus mitigating inflammation. CONCLUSION: Our study underscores the pivotal role of solTNF in the dissemination of M.tb to other organs. This preliminary investigation sheds light on the involvement of solTNF in the mechanisms underlying M.tb dissemination, although further in-depth research is warranted to fully elucidate its role in this process.

Mycobacterium bovis induced human tuberculosis in India: Current status, challenges & opportunities.

Tuberculosis (TB) caused by Mycobacterium tuberculosis is a leading cause of human deaths due to any infectious disease worldwide. However, infection of Mycobacterium bovis, primarily an animal pathogen, also leads to the development of 'human tuberculosis'. Infected animals have been considered the major source of M. bovis infection and humans get exposed to M. bovis through close contact with infected animals or consumption of contaminated milk, unpasteurized dairy products and improperly cooked contaminated meat. The information on the global distribution of bovine TB (bTB) is limited, but the disease has been reported from all the livestock-producing middle- and low-income countries of the world. In recent years, there is a renewed interest for the control of bTB to minimize human infection worldwide. In India, while the sporadic presence of M. bovis has been reported in domestic animals, animal-derived food products and human beings from different geographical regions of the country, the information on the national prevalence of bTB and transmission dynamics of zoonotic TB is, however, not available. The present article reviewed published information on the status of M. bovis-induced zoonotic TB to highlight the key challenges and opportunities for intervention to minimize the risk of M. bovis infection in humans and secure optimum animal productivity in India.

Genetic variability in multidrug-resistant Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in North India.

BACKGROUND: Information on the genetic variability of drug resistant isolates of Mycobacterium tuberculosis is of paramount importance to understand transmission dynamics of disease and to improve TB control strategies. Despite of largest number of multidrug-resistant (MDR) tuberculosis cases (1, 30,000; 27% of the global burden), strains responsible for the expansion or development of drug-resistant Mycobacterium tuberculosis infections have been poorly characterized in India. Present study was aimed to investigate the genetic diversity in MDR isolates of Mycobacterium tuberculosis in North India. RESULTS: Spacer oligonucleotide typing (spoligotyping) was performed on 293 clinical MDR isolates of Mycobacterium tuberculosis recovered from cases of pulmonary tuberculosis from North India. Spoligotyping identified 74 distinct spoligotype patterns. Comparison with an international spoligotype database (spoldb4 database) showed that 240 (81.91%) and 32 (10.92%) strains displayed known and shared type patterns, while 21 (7.16%) strains displayed unique spoligotype patterns. Among the phylogeographic lineages, lineage 3 (East African-Indian) was found most predominant lineage (n = 159, 66.25%), followed by lineage 2 (East Asian; n = 34, 14.16%), lineage 1 (Indo-Oceanic; n = 30, 12.50%) and lineage 4 (Euro American; n = 17, 7.08%). Overall, CAS1_DEL (60.41%; SITs 2585, 26, 2694, 309, 381, 428, 1401, 141, 25, 1327) was found most pre-dominant spoligotype pattern followed by Beijing (14.16%; SITs255, 260, 1941, 269) and EAI3_IND (5.00%; SITs 298, 338, 11). The demographic and clinical characteristics were not found significantly associated with genotypic lineages of MDR-M.tuberculosis isolates recovered from pulmonary TB patients of North India. CONCLUSIONS: Present study reveals high genetic diversity among the Mycobacterium tuberculosis isolates and highlights that SIT141/CAS1_Del followed by SIT26/ Beijing lineage is the most common spoligotype responsible for the development and transmission of MDR-TB in North India. The high presence of shared type and unique spoligotype patterns of MDR strains indicates epidemiological significance of locally evolved strains in ongoing transmission of MDR-TB within this community which needs to be further monitored using robust molecular tools with high discriminatory power.

Genetic diversity of Mycobacterium tuberculosis in south coastal Karnataka, India, using spoligotyping.

Background & objectives: Despite high occurrence of tuberculosis in India very little information is available about the genetic diversity of Mycobacterium tuberculosis isolates prevailing in coastal Karnataka, India. Thus, the present study was undertaken to explore the genetic biodiversity of M. tuberculosis isolates prevailing in south coastal region of Karnataka (Udupi District), India. Methods: A total of 111 Mycobacterial isolates were cultured in Lowenstein Jensen (LJ) medium and after obtaining growth, DNA was extracted and spoligotyping was performed. SITVIT WEB database was used to locate families of spoligotypes. Results: On analyzing the hybridization results of all 111 isolates on SITVIT WEB database 57 (51.35%) isolates were clustered into 11 Spoligotype International Types (SIT). The largest cluster of 14 (12.61%) isolates was SIT-48 (EAI1-SOM), followed by SIT-1942 (CAS1-Delhi) with 11 isolates (9.9%) and SIT-11 with seven (6.30%). Moreover, 23 isolates (20.72%) had unique spoligotypes and 31 (27.92%) were orphans. Spotclust analysis revealed that majority (67%) of orphan isolates were variants of CAS (37%) and EAI-5 (34%). Interpretation & conclusions: The present study revealed high biodiversity among the circulating isolates of M. tuberculosis in this region with the presence of mixed genotypes earlier reported from north and south India along with certain new genotypes with unique SITs. The study highlights the need for further longitudinal studies to explore the genetic diversity and to understand the transmission dynamics of prevailing isolates.

Quantitative Proteomic and Phosphoproteomic Analysis of H37Ra and H37Rv Strains of Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of tuberculosis, accounts for 1.5 million human deaths annually worldwide. Despite efforts to eradicate tuberculosis, it still remains a deadly disease. The two best characterized strains of M. tuberculosis, virulent H37Rv and avirulent H37Ra, provide a unique platform to investigate biochemical and signaling pathways associated with pathogenicity. To delineate the biomolecular dynamics that may account for pathogenicity and attenuation of virulence in M. tuberculosis, we compared the proteome and phosphoproteome profiles of H37Rv and H37Ra strains. Quantitative phosphoproteomic analysis was performed using high-resolution Fourier transform mass spectrometry. Analysis of exponential and stationary phases of these strains resulted in identification and quantitation of 2709 proteins along with 512 phosphorylation sites derived from 257 proteins. In addition to confirming the presence of previously described M. tuberculosis phosphorylated proteins, we identified 265 novel phosphorylation sites. Quantitative proteomic analysis revealed more than five-fold upregulation of proteins belonging to virulence associated type VII bacterial secretion system in H37Rv when compared to those in H37Ra. We also identified 84 proteins, which exhibited changes in phosphorylation levels between the virulent and avirulent strains. Bioinformatics analysis of the proteins altered in their level of expression or phosphorylation revealed enrichment of pathways involved in fatty acid biosynthesis and two-component regulatory system. Our data provides a resource for further exploration of functional differences at molecular level between H37Rv and H37Ra, which will ultimately explain the molecular underpinnings that determine virulence in tuberculosis.

TlyA protein of Mycobacterium leprae: a probable bio-marker of active infection.

The extent of pathogenicity of the mycobacterial infections depends on virulence factors that mediate survival inside macrophages. Virulence factors are generally believed to be specific for pathogenic species and mutated/non-functional in nonpathogenic strains. Mycobacterial TlyA can modulate the phagolysosome maturation pathway, immediately after entry into macrophages. Over-expression of open reading frame (ORF) ML1358 (tlyA) in tissues of leprosy patients by partial DNA chip and real time PCR analysis during active infection attracted our interest to explore the properties of this gene at molecular and serological levels, to understand its role in the host. Molecular properties were studied by cloning and expression of the corresponding gene in pASK-iba 43(þ) expression vector in E. coli and bioinformatics tools while sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA were applied to investigate the serological significance of rTlyA protein in different clinical states of leprosy. We observed that TlyA has a close relation among mycobacteria with specific protein domains in slow growing intracellular adapted pathogenic species. The presence of trans-membrane domains indicates its association to the cell membrane. The study revealed its highly significant sero-reactivity (P value , 0·001) in borderline lepromatous (BL) patients, and those with reversal reaction (RR) and erythema nodosum leprosum (ENL). Its role in active infection, association with the cell membrane, presence in pathogenic species and high sero-reactivity, suggested the tlyA gene as a strong disease progression marker.

Analysis of expression profile of mce operon genes (mce1, mce2, mce3 operon) in different Mycobacterium tuberculosis isolates at different growth phases.

BACKGROUND & OBJECTIVES: Mycobacterium tuberculosis (M. tuberculosis) has four homologous mammalian cell entry (mce) operons (mce1-4) that encode exported proteins and have a possible role in the virulence mechanism of this pathogen. The expression of mce operon is considered to be complex and not completely understood. Although expression of mce operon at different in vitro growth phases has been studied earlier, its expression in different M. tuberculosis isolates under different growth phases is not yet studied. The present preliminary study was conducted on a limited number of isolates to know the trend of expression pattern of mce operon genes in different M. tuberculosis isolates under different growth stages. METHODS: In this study, we monitored the transcriptional profile of selected mce operon genes (mce1A, mce1D, mce2A, mce2D, mce3A, mce3C) in different M.tuberculosis isolates (MDR1, MDR2, and sensitive isolate) at early exponential and stationary phases using real-time quantitative PCR. RESULTS: The expression ratio of all selected mce operon genes in all M. tuberculosis isolates was reduced at the initial phase and increased substantially at a later phase of growth. Higher expression of mce1 operon genes was found in all M. tuberculosis isolates as compared to other mce operon genes (mce2 and mce3 operons) at stationary growth phase. INTERPRETATION & CONCLUSIONS: the higher expression of mce operon genes at stationary phase (as compared to early exponential phase) suggested growth phase dependent expression of mce operon genes. This indicated that the mce operon genes might have a role in M. tuberculosis survival and adaptation on the onset of adverse condition like stationary phase. Identification of differentially expressed genes will add to our understanding of the bacilli involved in adaptation to different growth conditions.

Current status of Mycobacterium avium subspecies paratuberculosis infection in animals & humans in India: What needs to be done?

Mycobacterium avium subspecies paratuberculosis (MAP) has emerged as a major health problem for domestic livestock and human beings. Reduced per animal productivity of domestic livestock seriously impacts the economics of dairy farming globally. High to very high bioload of MAP in domestic livestock and also in the human population has been reported from north India. Presence of live MAP bacilli in commercial supplies of raw and pasteurized milk and milk products indicates its public health significance. MAP is not inactivated during pasteurization, therefore, entering into human food chain daily. Recovery of MAP from patients with inflammatory bowel disease or Crohn's disease and animal healthcare workers suffering with chronic gastrointestinal problems indicate a close association of MAP with a number of chronic and other diseases affecting human health. Higher bioload of MAP in the animals increases the risk of exposure to the human population with MAP. This review summarizes the current status of MAP infection in animals as well as in human beings and also highlights the prospects of effective management and control of disease in animals to reduce the risk of exposure to human population.

Early detection of multidrug resistant (MDR) Mycobacterium tuberculosis in a single tube with in-house designed fluorescence resonance energy transfer (FRET) probes using real-time PCR.

Rapid and correct diagnosis is crucial for the management of multidrug resistance (MDR) in Mycobacterium tuberculosis (MTB). The present study aims at rapid diagnosis for identification of multidrug resistance tuberculosis (MDR-TB) using real-time PCR. FRET hybridization probes targeting most prominent four selected codons for rpoB526 and 531 and for katG314 and 315 genes were designed and evaluated on 143 clinical MTB isolates and paired sputa for rapid detection of MDR-TB. The results of real-time PCR were compared with gold standard L-J proportion method and further validated by DNA sequencing. Of the 143 MTB positive cultures, 85 and 58 isolates were found to be 'MDR' and 'pan susceptible', respectively by proportion L-J method. The sensitivity of real-time PCR for the detection of rifampicin (RIF) and isoniazid (INH) were 85.88 and 94.11%, respectively, and the specificity of method was found to be 98.27%. DNA sequencing of 31 MTB isolates having distinct melting temperature (Tm) as compared to the standard drug susceptible H37Rv strain showed 100% concordance with real-time PCR results. DNA sequencing revealed the mutations at Ser531Leu, His526Asp of rpoB gene and Ser315Thr, Thr314Pro of katG gene in RIF and INH resistance cases. This real-time PCR assay that targets limited number of loci in a selected range ensures direct and rapid detection of MDR-TB in Indian settings. However, future studies for revalidation as well as refinement are required to break the limitations of MDR-TB detection.

Application of IS1311 locus 2 PCR-REA assay for the specific detection of 'Bison type' Mycobacterium avium subspecies paratuberculosis isolates of Indian origin.

BACKGROUND & OBJECTIVES: Of the three major genotypes of Mycobacterium avium subspecies paratuberculosis (MAP), 'Bison type' is most prevalent genotype in the domestic livestock species of the country, and has also been recovered from patients suffering from Crohn's disease. Recently, a new assay based on IS1311 locus 2 PCR- restriction endonuclease analysis (REA) was designed to distinguish between 'Indian Bison type' and non-Indian genotypes. The present study investigated discriminatory potential of this new assay while screening of a panel of MAP isolates of diverse genotypes and from different geographical regions. METHODS: A total of 53 mycobacterial isolates (41 MAP and 12 mycobacterium other than MAP), three MAP genomic DNA and 36 MAP positive faecal DNA samples from different livestock species (cattle, buffaloes, goat, sheep and bison) and geographical regions (India, Canada, USA, Spain and Portugal) were included in the study. The extracted DNA samples (n=92) were analyzed for the presence of MAP specific sequences (IS900, ISMav 2 and HspX) using PCR. DNA samples were further subjected to genotype differentiation using IS1311 PCR-REA and IS1311 L2 PCR-REA methods. RESULTS: All the DNA samples (except DNA from non-MAP mycobacterial isolates) were positive for all the three MAP specific sequences based PCRs. IS1311 PCR-REA showed that MAP DNA samples of Indian origin belonged to 'Bison type'. Whereas, of the total 19 non-Indian MAP DNA samples, 2, 15 and 2 were genotyped as 'Bison type', 'Cattle type' and 'Sheep type', respectively. IS1311 L2 PCR-REA method showed different restriction profiles of 'Bison type' genotype as compared to non-Indian DNA samples. INTERPRETATION & CONCLUSIONS: IS1311 L2 PCR-REA method successfully discriminated 'Indian Bison type' from other non-Indian genotypes and showed potential to be future epidemiological tool and for genotyping of MAP isolates.

Current understanding on micro RNAs and its regulation in response to Mycobacterial infections.

MicroRNAs (miRNAs) are evolutionarily conserved, naturally abundant, small, regulatory non-coding RNAs that inhibit gene expression at the post-transcriptional level in a sequence-specific manner. Due to involvement in a broad range of biological processes and diseases, miRNAs are now commanding considerable attention. Although much of the focus has been on the role of miRNAs in different types of cancer, recent evidence also points to a critical role of miRNAs in infectious disease, including those of bacterial origin. Now, miRNAs research is exploring rapidly as a new thrust area of biomedical research with relevance to deadly bacterial diseases like Tuberculosis (caused by Mycobacterium tuberculosis). The purpose of this review is to highlight the current developments in area of miRNAs regulation in Mycobacterial diseases; and how this might influence the diagnosis, understanding of disease biology, control and management in the future.

Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry.

The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ~80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ~250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes.

Atorvastatin Potentially Reduces Mycobacterial Severity through Its Action on Lipoarabinomannan and Drug Permeability in Granulomas.

The majority of preclinical research has shown that Mycobacterium tuberculosis can modify host lipids in various ways. To boost its intramacrophage survival, M. tuberculosis causes host lipids to build up, resulting in the development of lipid-laden foam cells. M. tuberculosis binds to and enters the macrophage via the cell membrane cholesterol. Aggregation of cholesterol in the cell wall of M. tuberculosis and an increase in vascularity at the granuloma site reduce the permeability of rifampicin and isoniazid concentrations. However, very few studies have assessed the effect of statins on drug penetration. Here, we used atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, to observe its effect on the bacterial burden by increasing the drug concentration at the infection site. We looked into how atorvastatin could be used in conjunction with first-line drugs to promote drug permeation. In this study, we detected an accumulation of drugs at the peripheral sites of the lungs and impaired drug distribution to the diseased sites. The efficacy of antituberculosis drugs, with atorvastatin as an adjunct, on the viability of M. tuberculosis cells was demonstrated. A nontoxic statin dosage established phenotypic and normal granuloma vasculature and showed an additive effect with rifampicin and isoniazid. Our data show that statins help to reduce the tuberculosis bacterial burden. Our findings reveal that the bacterial load is connected with impaired drug permeability resulting from lipid accumulation in the bacterial cell wall. Statin therapy combined with antituberculosis medications have the potential to improve treatment in tuberculosis patients. IMPORTANCE Mycobacterium tuberculosis binds to and enters the macrophage via the cell membrane cholesterol. M. tuberculosis limits phagosomal maturation and activation without engaging in phagocytosis. Aggregation of cholesterol in the cell wall of M. tuberculosis and an increase in the vascularity at the granuloma site reduce the permeability of rifampicin and isoniazid concentrations. However, very few studies have assessed the effect of statins on drug penetration, which can be increased through a reduction in cholesterol and vascularity. Herein, we used atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, to observe its effect on bacterial burden through increasing the drug concentration at the infection site. Our main research goal is to diminish mycobacterial dissemination and attenuate bacterial growth by increasing drug permeability.

Whole cell & culture filtrate proteins from prevalent genotypes of Mycobacterium tuberculosis provoke better antibody & T cell response than laboratory strain H 37 Rv.

BACKGROUND & OBJECTIVES: The immune responses to different antigens of Mycobacterium tuberculosis H 37 Rv vary from patient to patient with tuberculosis (TB). Therefore, significant difference might be documented between the H 37 Rv with long histories of passages and recent clinical isolates of M. tuberculosis. In the present study, immune response of TB patients and healthy controls against 39 clinical M. tuberculosis isolates was correlated with laboratory strain H 37 Rv. METHODS: The antibody response was studied coating whole cell extracts and culture filtrate proteins of M. tuberculosis isolates and laboratory strain H 37Rv by enzyme linked immunosorbent assay (ELISA). Lymphoproliferation was studied by incorporation of tritiated thymidine and cytokines (IFN-γ and IL-4) by using commercially available kits. RESULTS: Sero-reactivity to whole cell extract (WCE) of 11 clinical isolates was higher with pooled serum and individual's serum from tuberculosis patients showed significant reactivity (P<0.05) to ten of these isolates using ELISA. Of the WCE of 39 clinical isolates, 10 were found to be potent inducer of lymphoproliferation as well as cytokine secretion (P<0.05) in peripheral blood mononuclear cells from PPD+ healthy controls. Six culture filtrate proteins (CFPs) from these selected clinical isolates were also better inducers of antibody and T-cell response. INTERPRETATION & CONCLUSION: Overall, our results revealed that the clinical isolates belonging to prevalent genotypes; CAS1_Del (ST-26), East African-Indian (ST-11) and Beijing family (ST-1) induced better antibody and T cell responses compared to H 37 Rv laboratory strain. Further studies need to be done to purify and identify the dominant protein (s) using whole cell extract and culture filtrates from these immunologically relevant clinical M. tuberculosis isolates, which will be worthwhile to find out pathogenic factors, potential diagnostic markers and protective molecules for tuberculosis.

Exploring modulations in T-cell receptor-mediated T-cell signaling events in systemic circulation and at local disease site of patients with tubercular pleural effusion: An attempt to understand tuberculosis pathogenesis at the local disease site.

Introduction: T cells are crucial for pathogenesis as well as control for tuberculosis (TB). Although much is known about the signaling pathways which are required for the activation of T cells during acute infection but the way these cells respond during persistent of infection still remained elusive. Therefore, it is rationale to understand T cell activation during tuberculous pleural effusion (TPE), which is similar to bacterial persistency system. Methods: Herein, we will employ T cell receptor (TCR) based approaches for studying events of T cell activation pathways in cells of blood and pleural fluid among patients with TPE. We performed spectrofluorimetric analysis to study effect of M. tuberculosis antigens, ESAT-6 and Ag85A stimulation on intracellular calcium levels, Phosphorylation levels of ZAP-70 (Zeta-chain-associated protein kinase 70), PKC-θ (Protein kinase C theta), Erk1/2 (Extracellular signal-regulated kinase 1 and 2) and p-38 two important members of MAPKs (Mitogen activated Protein kinases) in CD3 and CD28 induced cells of blood and pleural fluid of same patients with TPE by western blotting. Patients with non-TPE were also included as matching disease controls in this study. Results: We observed significantly higher intracellular calcium levels, Phosphorylation levels of ZAP-70, Erk1/2 and p-38 in CD3 and CD28 induced cells of pleural fluid as compared to the blood cells of same patients with TPE. Alteration in the activation of these events has also been noted after stimulation of ESAT-6 and Ag85A. Discussion: Present study demonstrated up-regulated activation of TCR mediated T cell signaling events at local disease site (Pleural fluid) as compared to the blood sample of TB pleurisy patients which could be involved in T-cell dysfunctioning during the progression of the disease and also could be responsible for Th 1 dominance at local disease site in patients with TPE.

Stimulated expression of ELR+ chemokines, VEGFA and TNF-AIP3 promote mycobacterial dissemination in extrapulmonary tuberculosis patients and Cavia porcellus model of tuberculosis.

Pathogenic mycobacteria induce and accelerate blood vessel formation driven by extensive inflammation during granuloma formation, which is a central feature of mycobacterial pathogenesis. Tumor necrosis factor-alpha (TNF-α) enhances the expression of vascular endothelial growth factor (VEGF) and glutamic acid-leucine-arginine (ELR+) chemokines, which are potent inducers of vascularization. Most of the reported research work contends that VEGF growth factor induces neovascularization in human tuberculosis (TB) patients, but the evidence is inconclusive. Considerable ambiguity exists concerning the factors responsible for miliary tuberculosis. To identify such factors, we proposed an alternative explanation that could be found in miliary tuberculosis (MTB) cases. We performed a comparative analysis of angiogenic factors TNF-α, VEGF, and angiogenic ELR+ CXC and CC chemokine ligands in extrapulmonary tuberculosis (EPTB) and pulmonary tuberculosis (PTB) patients. To observe the relationship of these factors with the severity of bacterial burden, guinea pigs were infected with Mycobacterium tuberculosis (M.tb) and levels of the angiogenic factors were examined at different time intervals. Expression of these factors also exhibited a significant positive correlation with bacterial burden in other organs like the spleen, liver, and lymph nodes. We demonstrated statistical data on bacterial burden at different time points following the dissemination of infection in guinea pigs. In this study, we observed that there was a stimulated increase in the expression of ELR+ chemokines and VEGF in EPTB patients as compared to PTB patients. Following increased dissemination, the host immune response clears bacteria from the lungs during disease progression in guinea pigs.