Culture Filtrate Antigen Research Articles

Differential activation of innate and adaptive lymphocytes during latent or active infection with Mycobacterium tuberculosis.

Most individuals infected with Mycobacterium tuberculosis (Mtb) have latent tuberculosis (TB), which can be diagnosed with tests (such as the QuantiFERON-TB Gold test [QFT]) that detect the production of IFN-γ by memory T cells in response to the Mtb-specific antigens 6 kDa early secretory antigenic target EsxA (Rv3875) (ESAT-6), 10 kDa culture filtrate antigen EsxB (Rv3874) (CFP-10), and Mtb antigen of 7.7 kDa (Rv2654c) (TB7.7). However, the immunological mechanisms that determine if an individual will develop latent or active TB remain incompletely understood. Here we compared the response of innate and adaptive peripheral blood lymphocytes from healthy individuals without Mtb infection (QFT negative) and from individuals with latent (QFT positive) or active TB infection, to determine the characteristics of these cells that correlate with each condition. In active TB patients, the levels of IFN-γ that were produced in response to Mtb-specific antigens had high positive correlations with IL-1β, TNF-α, MCP-1, IL-6, IL-12p70, and IL-23, while the proinflammatory cytokines had high positive correlations between themselves and with IL-12p70 and IL-23. These correlations were not observed in QFT-negative or QFT-positive healthy volunteers. Activation with Mtb-soluble extract (a mixture of Mtb antigens and pathogen-associated molecular patterns [PAMPs]) increased the percentage of IFN-γ-/IL-17-producing NK cells and of IL-17-producing innate lymphoid cell 3 (ILC3) in the peripheral blood of active TB patients, but not of QFT-negative or QFT-positive healthy volunteers. Thus, active TB patients have both adaptive and innate lymphocyte subsets that produce characteristic cytokine profiles in response to Mtb-specific antigens or PAMPs. These profiles are not observed in uninfected individuals or in individuals with latent TB, suggesting that they are a response to active TB infection.

Vaccination inducing durable and robust antigen-specific Th1/Th17 immune responses contributes to prophylactic protection against Mycobacterium avium infection but is ineffective as an adjunct to antibiotic treatment in chronic disease

ABSTRACT Mycobacterium avium complex (MAC) causing pulmonary disease in humanshas emerged worldwide. Thus, effective strategies simultaneously aiming to prevent MAC infection and accelerate therapeutic efficacy are required. To this end, subunit vaccine-induced protection against a well-defined virulent Mycobacterium avium (Mav) isolate was assessed as a preventative and therapeutic modality in murine models. Mav-derived culture filtrate antigen (CFA) was used as a vaccine antigen with glucopyranosyl lipid A stable emulsion (GLA-SE) or GLA-SE plus cyclic-di-GMP (GLA-SE/CDG), and we compared the immunogenicities, protective efficacies and immune correlates. Interestingly, CFA+GLA-SE/CDG immunization induced greater CFA-specific Th1/Th17 responses in both the lung and spleen than among the tested groups. Consequently, protective efficacy was optimally achieved with CFA+GLA-SE/CDG by significantly reducing bacterial loads along with long-lasting maintenance of antigen-specific Th1/Th17 cytokine-producing multifunctional T cell responses and relevant cytokine productions. Thus, we employed this subunit vaccine as an adjunct to antibiotic treatment. However, this vaccine was ineffective in further reducing bacterial loads. Collectively, our study demonstrates that strong Mav CFA-specific Th1/Th17 responses are critical for preventative protection against Mav infection but may be ineffective or even detrimental in an established and progressive chronic disease, indicating that different approaches to combating Mav infection are necessary according to vaccination purposes.

Reduced thyroxine production in young household contacts of tuberculosis patients increases active tuberculosis disease risk.

In the current study, we followed 839 household contacts (HHCs) of tuberculosis (TB) patients for 2 years and identified the factors that enhanced the development of TB. Fourteen of the 17 HHCs who progressed to TB were in the 15- to 30-year-old age group. At baseline (the “0“ time point, when all the individuals were healthy), the concentration of the thyroid hormone thyroxine (T4) was lower, and there were increased numbers of Tregs in PBMCs of TB progressors. At baseline, PBMCs from TB progressors stimulated with early secretory antigenic target 6 (ESAT-6) and 10 kDa culture filtrate antigen (CFP-10) produced less IL-1α. Thyroid hormones inhibited Mycobacterium tuberculosis (Mtb) growth in macrophages in an IL-1α–dependent manner. Mtb-infected Thra1PV/+ (mutant thyroid hormone receptor) mice had increased mortality and reduced IL-1α production. Our findings suggest that young HHCs who exhibit decreased production of thyroid hormones are at high risk of developing active TB disease.

Isolation and Purification of Low Molecular Weight Proteins from Culture Filtrate of Mycobacterium Tuberculosis Strain C.

In the last couple of years, a number of new and rapid tests for the diagnosis of Tuberculosis (TB) have been developed based on the low molecular weight antigens from Mycobacterium tuberculosis (Mtb) culture supernatant. This study aimed to isolate and purify low molecular weight antigens secreted by Mtb strain C for diagnostic purpose. The secretory proteins from culture filtrate of Mtb were extracted using ammonium sulphate precipitations and sephadex-G50 gel chromatography. The obtained antigen fractions were analyzed for their protein concentrations and approximate molecular weight using Lowry method and SDS-PAGE (12.5%), respectively. DOT-ELISA and Western blot assay was performed to confirm the presence of purified low molecular weight proteins isolated from Mtb using sera from pulmonary tuberculosis patients (polyclonal antibodies). During chromatography, low molecular weight proteins were separated, that was approximately 0.7 mg/ml of the total proteins (1.662 mg/ml). The purified protein fractions in molecular weight range of 14 kDa-41kDa appeared during SDS-PAGE analysis. The chromatographic band fraction in the weight range of 30-41 kDa was identified in the TB patients’ sera using Western blotting. The low molecular weight proteins in the culture filtrate of Mtb strain C were purified using ammonium sulphate and chromatography. These fractions were confirmed using Western blotting. The obtained results might support the hypothesis that the Mtb culture filtrate antigens could be used as a rapid and sensitive assay for the detection of patients with pulmonary TB.

Preliminary Study on the Secretory Performance of Mycobacterium tuberculosis Hypoxic Response Protein 1 (Rv2626c)

To verify the secretory ability of the hypoxic response protein 1 (HRP1) encoded by Mycobacterium tuberculosis (Mtb) Rv2626c. The target gene attached with His tag was amplified from the genome of Mtb standard virulence strain H37Rv. The recombinant plasmid contained the above amplified product was constructed and electroporated into Mycobacterium smegmatis (Ms) (MC 2155) to construct a recombinant strain. Protein expression was induced under heat condition, and the expression of protein from the culture filtrates and the bacterial lysates was detected afterward. The 10 kDa culture filtrate antigen (CFP-10) (Ms) and CFP-10 (Mtb) were used as positive controls, and the cytoplasmic protein heat shock protein 65 (GroEL2) (Mtb) was used as negative controls. The HRP1, GroEL2 (Mtb), CFP-10 (Mtb) and CFP-10 (Ms) were successfully amplified by PCR from recombinant plasmid, and sequencing results of the recombinant plasmid is right, confirming the successful construction of the recombinant plasmid. The recombinant Ms was successfully constructed and it could express the proteins GroEL2 (Mtb), HRP1, CFP-10 (Mtb) and CFP-10 (Ms). The target protein HRP1 was detected in both of the lysate and the culture filtrate of the recombinant strain by Western blot, which was consistent with the positive control CFP-10. The negative control GroEL2 (Mtb) was only detected in the bacterial lysate, but not detected in the culture filtrate. The protein HRP1 encoded by Mtb Rv2626c can be secreted out of Ms by the secretion system of Ms. It may be a secreted protein and play an important role in the pathogenesis of Mtb.

Experimental exposure of Burkholderia pseudomallei crude culture filtrate upregulates PD-1 on T lymphocytes.

Burkholderia pseudomallei is the causative agent for melioidosis. Because of its intracellular nature, the bacterium is capable of replicating within a plethora of eukaryotic cell lines. B. pseudomallei can remain dormant within host cells without symptoms for years, causing recrudescent infections. Here, we investigated the pathogenesis mechanism behind the suppression of T cell responses by B. pseudomallei . Peripheral blood mononuclear cells (1×106 cells/well) isolated by Ficoll Paque (Sigma-Aldrich) density gradient centrifugation were incubated with optimized concentrations of bacterial crude culture filtrate antigens (CFAs) (10 ug ml−1) and heat-killed bacteria [1 : 10 multiplicity of infection (m.o.i.)]. Following incubation, cells were investigated for surface expression of coinhibitory molecules by flow cytometry. We found that B. pseudomallei induced the upregulation of programmed death 1 (PD-1), a molecule responsible for T cell exhaustion, on T cells in vitro following exposure to crude CFAs of B. pseudomallei . This upregulation of PD-1 probably contributes to poor immune surveillance and disease pathogenesis.

Influence of Cdx2 and TaqI Gene Variants on Vitamin D3 Modulated Intracellular Chemokine Positive T-Cell Subsets in Pulmonary Tuberculosis

PurposeWe studied the effect of 1,25(OH)2D3 (vitamin D3) on intracellular chemokine-positive T-cell subsets in whole blood cultures of healthy controls and patients with pulmonary tuberculosis. MethodsGenotyping was performed by the polymerase chain reaction–restriction fragment length polymorphism method. The regulatory role of the Cdx2 and 3ʹUTR TaqI gene variants on chemokine-positive T-cell subsets was studied from culture filtrate antigen stimulated with or without vitamin D3 treated whole blood cultures of 60 healthy controls and 50 patients with pulmonary tuberculosis. FindingsVitamin D3 significantly suppressed monocyte chemoattractant protein 1, macrophage inhibitory protein (MIP)–1α, MIP-1β, regulated on activation, normal T-cell expressed and secreted (RANTES), and interferon-γ inducible protein 10 (IP-10)–positive T-cell subsets compared with culture filtrate antigen stimulated cells without vitamin D3 treatment. In the Cdx2 AA genotype, vitamin D3 decreased MIP-1α, MIP-1β, and RANTES-positive T cells compared with the GG genotype. Whereas in the TaqI tt genotype, decreased MIP-1β and RANTES and increased IP-10–positive T cells were observed compared with the TT genotype in vitamin D3 treated cells (p < 0.05). ImplicationsThis study suggests that vitamin D3 may regulate the chemokine-positive T cells through the Cdx2 AA and TaqI tt genotypes. This could be helpful to regulate chemokine-mediated inflammatory response during active disease condition. Hence, vitamin D3 supplementation along with tuberculosis drugs may be useful for faster recovery from the disease.

Regulatory role of CCL5 (rs2280789) and CXCL10 (rs56061981) gene polymorphisms on intracellular CCL5 and CXCL10 expression in pulmonary tuberculosis

Genetic variations in chemokine genes influence the chemoattractive properties of T cells which may be associated with outcome of infections. In present study, we have investigated the regulatory role played by In1.1T/C (rs2280789) polymorphism of CCL5 and −135G/A (rs56061981) polymorphism of CXCL10 gene on intracellular CCL5 and CXCL10 expression in T cells. Whole blood cell cultures were stimulated with culture filtrate antigen (CFA) and infected with live M. tuberculosis were used for intracellular CCL5 and CXCL10 expression using flow cytometry. Genotyping was performed using polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP). Significantly higher expression of CCL5 expressing CD3+ and CD3+ CD8+ T cells were observed in HCs with In1.1TT genotype compared to C allele carrier (TT+TC) under unstimulated and CFA induced cultures (p<0.05). In −135G/A (rs56061981) polymorphism, PTB patients with GG genotype showed a significantly decreased expression of CD3+ CXCL10+ and CD3+ CD4+ CXCL10+ T cells compared to A allele carrier (GA+AA) under unstimulated, CFA induced and M. tuberculosis infected cultures (P<0.05). The present study suggest that TT genotype of CCL5 In1.1T/C (rs2280789) polymorphism play an important role to increased CCL5 expression in T cell which may enhanced Th1 immunity and help in protection against tuberculosis. The study also suggests GG genotype of CXCL10 −135G/A (rs56061981) polymorphism decreased CXCL10 expression in T cells which may have defective recruitment of mononuclear cells at the site of infection as well granuloma formation and in turn contribute to progression of TB.

Immunotoxic effect of thiamethoxam in immunized mice with Brucella abortus cultural filtrate antigen.

Aim:This study was planned for determination the toxic effect of thiamethoxam (TMX) in immunized mice with Brucella abortus culture filtrate antigen (CFBAgs) (as a vaccine) and its role of TMX on decrease activity of B. abortus antigen on eliciting of humoral and cellular immunity.Materials and Methods:To achieve these goals 60 female mice were used, 7-8 weeks age, they were divided equally into three groups (20 in each group) and treated as follows: 1st group: Mice were immunized with CFBAgs intraperitoneally in two doses, 2 weeks intervals with (protein concentration 2 mg\\ml), 2nd group: Mice immunized as in the 1st group and was administrated orally with 1/10 lethal dose 50% of TMX (83.7 mg/kg B.W.) for 4 weeks daily, 3rd group was administrated orally with 0.3 ml normal saline served as a control group. At day 28 post immunization (PI) delayed type hypersensitivity (skin test) was done, and serum samples were collected at day 30 (PI) for detection of passive hemagglutination test (PHA); interferon gamma (IFN-γ) which was done by enzyme-linked immunosorbent assay test in addition to phagocytes assay.Results:The results of skin test post injection with soluble antigen of B. abortus intradermally showed a high significantly mean values at p≤0.05 of footpad skin thickness in the 1st group of mice which recorded (0.51±0.002 mm) as compared with the 2nd group of mice which showed (0.08±0.002 mm) after 24 h; the mean values of skin thickness were declined in the 1st mice (0.46±0.002) and 2nd mice (0.070±0.001) at 48 h; control group showed a negative results. These results were agreed with results of serum levels of IFN-γ (pg/ml) that showed that a significant increase the vaccinated 1st group (406.36±1.52), than those values in the 2nd group (151.61±0.89) and negative result in 3rd group (46.47±0.60), in addition to results of PHA test which showed a significant increase in antibody titer in the 1st group (139±12.16) with low level of serum antibody in the 2nd group (7.66±0.33). Phagocytic ratio results in the 1st group showed an increase to reach (18.55±0.44) than a ratio in the 2nd group (13.24±0.32) and the control group (5.46±0.25).Conclusion:It was concluded that TMX induced suppression of humoral and cellular immune responses in immunized mice with CFBAgs.

Multifunctional T Cell Response to DosR and Rpf Antigens Is Associated with Protection in Long-Term Mycobacterium tuberculosis-Infected Individuals in Colombia.

Multifunctional T cells have been shown to be protective in chronic viral infections. In mycobacterial infections, however, evidence for a protective role of multifunctional T cells remains inconclusive. Short-term cultures of peripheral blood mononuclear cells stimulated with the Mycobacterium tuberculosis RD1 antigens 6-kDa early secretory antigenic target (ESAT6) and 10-kDa culture filtrate antigen (CFP10), which are induced in the early infection phase, have been mainly used to assess T cell multifunctionality, although long-term culture assays have been proposed to be more sensitive than short-term assays for assessment of memory T cells, which are essential for long-term immunity. Here we used a long-term culture assay system to study the T cell immune responses to the M. tuberculosis latency-associated DosR antigens and reactivation-associated Rpf antigens, compared to ESAT6 and CFP10, in patients with pulmonary tuberculosis (PTB) and household contacts of PTB patients with long-term latent tuberculosis infection (ltLTBI), in a community in which M. tuberculosis is endemic. Our results showed that the DosR antigens Rv1737c (narK2) and Rv2029c (pfkB) and the Rv2389c (rpfD) antigen of M. tuberculosis induced higher frequencies of CD4+ or CD8+ mono- or bifunctional (but not multifunctional) T cells producing interferon gamma (IFN-γ) and/or tumor necrosis alpha (TNF-α) in ltLTBI, compared to PTB. Moreover, the frequencies of CD4+ and/or CD8+ T cells with a CD45RO+ CD27+ phenotype were higher in ltLTBI than in PTB. Thus, the immune responses to selected DosR and Rpf antigens may be associated with long-term latency, correlating with protection from M. tuberculosis reactivation in ltLTBI. Further study of the functional and memory phenotypes may contribute to further discrimination between the different states of M. tuberculosis infections.

Regulatory role of Cdx-2 and Taq I polymorphism of vitamin D receptor gene on chemokine expression in pulmonary tuberculosis

Vitamin D receptor (VDR) gene variants have been shown to be regulating the immune response in tuberculosis. We studied the regulatory role of VDR promoter Cdx-2 and 3′UTR TaqI gene variants on chemokine levels from culture filtrate antigen (CFA) stimulated with or without 1,25(OH)2D3 treated peripheral blood mononuclear cells of 50 pulmonary tuberculosis patients (PTB) and 51 normal healthy controls (HCs). In CFA with 1,25(OH)2D3 treated cultures, the MIP-1α, MIP-1β, RANTES levels were significantly decreased in Cdx-2 AA genotype compared to GG genotype, while a significantly increased MIG level was observed in Cdx-2 AA genotype (p<0.05). In TaqI polymorphism, tt genotype significantly decreased MIP-1β and RANTES levels compared to TT genotype. Moreover, a significantly increased level of IP-10 and MIG was observed in TaqI tt genotype compared with TT genotype (p<0.05). The results suggests that the 1,25(OH)2D3 may alter the chemokine response through the VDR polymorphic variants during infection.

Effect of vitamin D3 on chemokine levels and regulatory T-cells in pulmonary tuberculosis

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] the active form of vitamin D3 acts as an immunomodulator in various immune cells. The present study is aimed to study the effect of 1,25(OH)2D3 on chemokine levels and regulatory T-cells in 51 healthy controls (HCs) and 50 pulmonary tuberculosis (PTB) patients. Peripheral blood mononuclear cells were cultured with culture filtrate antigen (CFA) of Mycobacterium tuberculosis in the presence or absence of 1,25(OH)2D3 at 10−7M concentration for 72h and the percentage positive regulatory T-cell subsets were studied using flow cytometry. The chemokine levels were estimated in the culture supernatants by ELISA. 1,25(OH)2D3 significantly upregulated the frequency of regulatory T-cell subsets while suppressed the production of chemokine levels in CFA stimulated cultures of HCs and PTB patients (p<0.05). Correlation analysis revealed a significant negative correlation between CD4+Foxp3+ regulatory T-cells and MCP-1, MIP-1β and IP-10 in CFA stimulated with 1,25(OH)2D3 treated cells (p<0.05). The results suggested that 1,25(OH)2D3 upregulated regulatory T-cells and act as anti-inflammatory by downregulating chemokine levels which could be beneficial to protect the host from inflammation and tissue damage during infection.

Chitin enhances serum IgE in Aspergillus fumigatus induced allergy in mice

Aspergillus fumigatus (A. fumigatus) is a ubiquitous fungus that activates, suppresses or modulates the immune response by changing its cell wall structure and by secreting proteases. In this study, we show that chitin acts as an adjuvant in a murine model of A. fumigatus protease induced allergy. The mice were immunised intraperitoneally with A. fumigatus culture filtrate antigen either with or without chitin and were subsequently challenged with the culture filtrate antigen intranasally. Alum was used as an adjuvant control. Compared to alum, chitin induced a weaker inflammatory response in the lungs, measured as the total cell efflux in BAL, EPO and chitinase production. However, chitin enhanced the total IgE, specific IgE and specific IgG1 production as efficiently as alum. Pre-treatment with chitin but not with alum depressed the concentration of the Th2 cytokines IL-4 and IL-13 in BAL fluid. These results shows that chitin, in spite of a reduction of the Th2 cytokine levels in the lungs, enhanced the total and specific IgE production in A. fumigatus culture filtrate induced allergy.

1,25-Dihydroxy vitamin D3 downregulates pro-inflammatory cytokine response in pulmonary tuberculosis.

1,25-Dihydroxy vitamin D3 [1,25(OH)2D3] is a potent immunomodulator and regulates various immune responses to Mycobacterium tuberculosis (Mtb). The present study aimed to understand the effect of 1,25(OH)2D3 on pro-inflammatory cytokine response to Mtb antigen. Peripheral blood mononuclear cells from 42 healthy controls (HCs) and 42 pulmonary tuberculosis (PTB) patients were cultured with culture filtrate antigen (CFA) of Mtb with and without 1,25(OH)2D3 at 10(-7)M concentration for 72 h. The levels of IL-1α, IL-1β, TNF-α, TNF-β, IL-17 and IL-23 were estimated in the culture supernatants by ELISA. 1,25(OH)2D3 significantly suppressed all the CFA induced pro-inflammatory cytokines (p<0.05) studied except IL-1β in both HCs and PTB patients. Among the PTB patients, the observed suppression was visible both in patients with and without cavitary tuberculosis. The present study results suggest that 1,25(OH)2D3 downregulates the production of pro-inflammatory cytokines and may control the exacerbated inflammatory response that may protect the host from excessive tissue damage at the site of infection.

Backbone assignment and Secondary Structure of Intrinsically Unstructured Culture Filtrate Antigen protein (CFP10) from Mycobacterium Tuberculosis

Backbone assignment and Secondary Structure of Intrinsically Unstructured Culture Filtrate Antigen protein (CFP10) from Mycobacterium Tuberculosis

Cryptic species of Paracoccidioides brasiliensis: impact on paracoccidioidomycosis immunodiagnosis.

We aimed to evaluate whether the occurrence of cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii, has implications in the immunodiagnosis of paracoccidioidomycosis (PCM). Small quantities of the antigen gp43 were found in culture filtrates of P. lutzii strains and this molecule appeared to be more variable within P. lutzii because the synonymous-nonsynonymous mutation rate was lower, indicating an evolutionary process different from that of the remaining genotypes. The production of gp43 also varied between isolates belonging to the same species, indicating that speciation events are important, but not sufficient to fully explain the diversity in the production of this antigen. The culture filtrate antigen AgEpm83, which was obtained from a PS3 isolate, showed large quantities of gp43 and reactivity by immunodiffusion assays, similar to the standard antigen (AgB-339) from an S1 isolate. Furthermore, AgEpm83 was capable of serologically differentiating five serum samples from patients from the Botucatu and Jundiaí regions. These patients had confirmed PCM but, were non-reactive to the standard antigen, thus demonstrating an alternative for serological diagnosis in regions in which S1 and PS2 occur. We also emphasise that it is not advisable to use a single antigen preparation to diagnose PCM, a disease that is caused by highly diverse pathogens.

Purification of Antigen 85 Complex of Mycobacterium tuberculosis

Serodiagnosis of tuberculosis using purified native antigens is one of the approaches for the early detection of TB. The Antigen 85 complex consisting of Ag 85A, 85B and 85C are the most abundant antigens in the culture supernatant of mycobacteria. It has also been shown to be the immunodominant (the property of an antigenic determinant that causes it to be responsible for the major immune response in a host) antigens of CFA (culture filtrate antigens – proteins secreted by mycobacteria into the culture medium). This protocol gives the details for purification of the Ag85 complex from CFA and also the method of isolation of the individual components of the complex by column chromatography.

Effect of vitamin D3 on chemokine expression in pulmonary tuberculosis

1,25 Dihydroxy vitamin D3 (vitamin D3) is an immunomodulator and its deficiency has been associated with susceptibility to tuberculosis. We have studied the immunoregulatory role of vitamin D3 on various chemokine expression in pulmonary tuberculosis. Peripheral blood mononuclear cells obtained from 21 pulmonary tuberculosis (PTB) patients and 24 healthy controls (HCs) were cultured for 48h with culture filtrate antigen (CFA) of Mycobacterium tuberculosis with or without vitamin D3 at a concentration 1×10−7M. The relative mRNA expression of monocyte chemoattractant protein-1 (MCP-1, CCL2), macrophage inflammatory protein-1α (MIP-1α, CCL3), macrophage inflammatory protein-1β (MIP-1β, CCL4), and regulated upon-activation, normal T cell-expressed and secreted (RANTES, CCL5) and IFN-γ inducible protein-10 (IP-10, CXCL10) chemokines were estimated from 48h old macrophages using real-time polymerase chain reaction (RT-PCR). The culture supernatants were used to estimate the various chemokines including monokine induced by IFN-γ (MIG, CXCL9) levels using cytometric bead array. In HCs, vitamin D3 significantly suppressed the MCP-1 mRNA expression of CFA stimulated cells (p=0.0027), while no such effect was observed in PTB patients. Vitamin D3 showed no significant effect on MIP-1α, MIP-1β and RANTES in both the study groups. The CFA induced IP-10 mRNA and protein expression was significantly suppressed by vitamin D3 in both the study groups (p<0.05). A similar suppressive effect of vitamin D3 was observed with MIG protein in healthy controls (p=0.0029) and a trend towards a suppression was observed in PTB patients. The suppressive effect of vitamin D3 is more prominent in CXC chemokines rather than CC chemokines. This suggests that vitamin D3 may down regulate the recruitment and activation of T-cells through CXC chemokines at the site of infection and may act as a potential anti-inflammatory agent.

Monocytes from tuberculosis patients that exhibit cleaved caspase 9 and denaturalized cytochrome c are more susceptible to death mediated by Toll‐like receptor 2

Experimental models have shown that lipoproteins from Mycobacterium tuberculosis induce apoptosis via Toll-like receptor 2 (TLR2) in the THP-1 cell line and in monocyte-derived macrophages from healthy volunteers. We found an increased percentage of circulating monocytes in patients with tuberculosis (TB) in comparison to healthy controls. Patients with TB showed a higher TLR2 and TLR4 expression density on monocytes, and a higher proportion of TLR2(+) monocytes, as well as increased serum tumour necrosis factor-α level. In culture, monocytes from TB patients were more susceptible to death than monocytes from healthy controls. Moreover, death-susceptible monocytes were positive to both TLR2 and TLR4 at the start of culture. Freshly obtained monocytes from TB patients exhibited cleaved caspase 9 and denaturalized cytochrome c. For levels of caspase 8, apoptosis-regulating signal kinase 1, and phospho-p38 mitogen-activated protein kinase there was no difference between samples from TB patients and from healthy controls. The culture filtrate antigen extract from M.tuberculosis H37Rv strain induced the death of monocytes from patient with TB after a 4-hr incubation, which was abrogated by neutralizing antibodies for TLR2 but not TLR4. Similarly, Pam3CSK4, a synthetic agonist triacylated ligand to TLR2, also induced the death of monocytes, although it did not increase levels of cleaved caspase 9. Our findings suggest that monocytes from TB patients are more susceptible to death, probably through mitochondrial damage, and that cell death increases in the presence of mycobacterial antigen by a TLR2-dependent pathway.

The influence of BCG vaccine strain on mycobacteria-specific and non-specific immune responses in a prospective cohort of infants in Uganda.

Globally, BCG vaccination varies in efficacy and has some non-specific protective effects. Previous studies comparing BCG strains have been small-scale, with few or no immunological outcomes and have compared TB-specific responses only. We aimed to evaluate both specific and non-specific immune responses to different strains of BCG within a large infant cohort and to evaluate further the relationship between BCG strain, scarring and cytokine responses. Infants from the Entebbe Mother and Baby Study (ISRCTN32849447) who received BCG-Russia, BCG-Bulgaria or BCG-Denmark at birth, were analysed by BCG strain group. At one year, interferon-gamma (IFN-γ), interleukin (IL)-5, IL-13 and IL-10 responses to mycobacteria-specific antigens (crude culture filtrate proteins and antigen 85) and non-mycobacterial stimuli (tetanus toxoid and phytohaemagglutinin) were measured using ELISA. Cytokine responses, scar frequency, BCG associated adverse event frequency and mortality rates were compared across groups, with adjustments for potential confounders. Both specific and non-specific IFN-γ, IL-13 and IL-10 responses in 1341 infants differed between BCG strain groups including in response to stimulation with tetanus toxoid. BCG-Denmark immunised infants showed the highest cytokine responses. The proportion of infants who scarred differed significantly, with BCG scars occurring in 52.2%, 64.1% and 92.6% of infants immunised with BCG Russia, BCG-Bulgaria and BCG-Denmark, respectively (p<0.001). Scarred infants had higher IFN-γ and IL-13 responses to mycobacterial antigens only than infants without a scar. The BCG-Denmark group had the highest frequency of adverse events (p=0.025). Mortality differences were not significant. Both specific and non-specific immune responses to the BCG vaccine differ by strain. Scarring after BCG vaccination is also strain-dependent and is associated with higher IFN-γ and IL-13 responses to mycobacterial antigens. The choice of BCG strain may be an important factor and should be evaluated when testing novel vaccine strategies that employ BCG in prime-boost sequences, or as a vector for other vaccine antigens.