Culture Filtrate Antigen Research Articles

Purification of 38kDa Antigen of Mycobacterium tuberculosis by Two Dimensional Preparative Electrophoresis

Serodiagnosis of tuberculosis using purified native antigens is one of the approach for the early detection of TB. This protocol gives the details for growing Mycobacterium tuberculosis in liquid culture and one proven way to get a good yield of the total secreted antigens in the culture supernatant. In addition the procedure for purification of the 38 kDa antigen from the total culture filtrate antigens is also explained. 38 kDa is one of the specific antigen for Mycobacterium tuberculosis complex and in this particular study it was used as one of the antigens for serodiagnosis of different category of tuberculosis patients.

Evaluation and Identification of in vitro Cellular Immune Response to Culture Filtrate Antigens of M. tuberculosis Culture. Implications for Vaccine Design

In the current study, fractions of culture filtrate proteins isolated at different time periods from M. tuberculosis sputum culture were evaluated for T cell activity (ADA, IFN-γ, TNF-α, & IL-12) using in vitro PBMC model. These isolated fractions were later on partially characterized by antibody detection assay using panel of six M. tuberculosis H37RV antigens (Ag85B, ESAT-6, CFP-10, GroES, 45 KD, and Hsp 16). Our results suggested us that PBMCs induced with culture filtrate proteins particularly those secreted towards later phase of growth curve of M. tuberculosis culture have good potential T cell activity as compared to BCG vaccine. On partial characterization we found levels of all secretary antigens increased towards later phase fractions (fraction C). Moreover on further evaluating individual purified M. tuberculosis H37RV antigens for T cell activity, we found that PBMCs induced with these antigens induces good T-cell response but not as consistent as fraction C. Thus to conclude, Culture Filtrate Proteins of M. tuberculosis sputum culture are important T-cell targets, and thus potential of such culture filtrate proteins may be further explored in near future for development of effective vaccination strategies for improving efficacy of currently available TB vaccine.

Rv3615c is a highly immunodominant RD1 (Region of Difference 1)-dependent secreted antigen specific for Mycobacterium tuberculosis infection

The 6-kDa early secretory antigenic target of Mycobacterium tuberculosis (ESAT-6) and the 10-kDa culture filtrate antigen (CFP-10), encoded in region of difference 1 (RD1) and secreted by the ESAT-6 system 1 (Esx-1) secretion system, are the most immunodominant and highly M. tuberculosis (MTB)-specific antigens. These attributes are responsible for their primary importance in tuberculosis (TB) immunodiagnosis and vaccine development. Rv3615c [Esx-1 substrate protein C (EspC)], encoded outside RD1, is similar in size and sequence homology to CFP-10 and ESAT-6, suggesting it might be a target of cellular immunity in TB. Using ex vivo enzyme-linked immunospot- and flow cytometry-based cytokine-secretion assay, we comprehensively assessed cellular immune responses to EspC in patients with active TB, latently infected persons, and uninfected bacillus Calmette-Guérin (BCG)-vaccinated controls. EspC was at least as immunodominant as ESAT-6 and CFP-10 in both active and latent TB infection. EspC contained broadly recognized CD4(+) and CD8(+) epitopes, inducing a predominantly CD4(+) T-cell response that comprised functional T-cell subsets secreting both IFN-γ and IL-2 as well as functional T-cell subsets secreting only IFN-γ. Surprisingly, T-cell responses to EspC were as highly specific (93%) for MTB infection as responses to ESAT-6 and CFP-10, with only 2 of 27 BCG-vaccinated controls responding to each antigen. Using quantitative proteomics and metabolically labeled mutant and genetically complemented MTB strains, we identified the mechanism of the specificity of anti-EspC immunity as the Esx-1 dependence of EspC secretion. The high immunodominance of EspC, equivalent to that of ESAT-6 and CFP-10, makes it a TB vaccine candidate, and its high specificity confers strong potential for T-cell-based immunodiagnosis.

Comparison of Three Immunodiagnostic Assays for Antemortem Detection of Mycobacterium Bovis Stimulation in Domestic Cats

Mycobacterium bovis causes disease in numerous mammalian species including humans, thus making research, surveillance, and control important in the eradication of tuberculosis. Domestic cats are susceptible to multiple mycobacterial species including Mycobacterium bovis; however, their role in the epidemiology of bovine tuberculosis is not fully documented. The current study was an evaluation of the immune response in specific pathogen-free (SPF) cats stimulated with sensitinogen, a heat-killed M. bovis product, using the rapid test, multiple antigen print immunoassay (MAPIA), and bovine-purified protein derivative (bPPD) single skin test. Six cats were inoculated with sensitinogen subcutaneously on days 0 and 24; 2 noninoculated cats and 49 non-SPF cats were controls. Serial serum samples were collected during 135 days and assayed for M. bovis antibodies by rapid test and MAPIA. On day 123, bPPD skin test was performed and read at 48 and 72 hr. The bPPD test at 72 hr had a mean skin thickness of 0.3 mm for stimulated cats and 0.1 mm for controls. Rapid test identified 4 of 6 stimulated cats after bPPD injection. The MAPIA detected antibody against MPB83, 16/83, 16 kDa, and M. bovis culture filtrate (MBCF) antigens. All assays differentiated between stimulated and control cats; however, 7 of 49 non-SPF control cats had a reaction for either antigen MBCF or 16/83. These preliminary studies show potential for antemortem detection of M. bovis among domestic cats. Additional studies to better characterize virulent M. bovis infection in cats would be of value.

Clinical study of bronchial inhalation challenge in summer-type hypersensitivity pneumonitis induced by Trichosporon cutaneum

To evaluate the methods and criteria of judgement for the bronchial antigen inhalation challenge test, the test was performed with culture filtrate antigen of serotype I and II of Trichosporon cutaneum in 18 patients with summer-type hypersensitivity pneumonitis from 15 families. The quantity of 15 mg of culture filtrate antigen was adequate, and had no side effects. In the tests, 17 of 18 patients showed a positive reaction to both or either, serotype of antigen. In 36 performances of inhalation, there were 21 positive reactions and 15 negative reactions. According to the criteria of judgment for inhalation challenge test, the positive response rates of observation items were 75% for symptoms and signs, and 51% for laboratory data. Items with a high positive rate were cough, crepitant rales, and decrease of PaO2. On the other hand, low positive rates were observed for decrease of DLco, VC and positive CRP. Items with both high sensitivity and high specificity were cough, crepitant rales and decrease of PaO2. The low positive rate of decreased DLco was due to insufficient improvement before inhalation challenge. It was concluded that our methods and criteria of judgment for bronchial inhalation challenge test are useful.

Immunological and proteomic analysis of preparative isoelectric focusing separated culture filtrate antigens of Mycobacterium tuberculosis

Isolation of the secreted proteins and studying the immune response they induce is an essential prerequisite for understanding the pathogenesis of M. tuberculosis. In this study, preparative liquid-phase isoelectric focusing was used for the separation of culture filtrate protein (CFP) of M. tuberculosis. This procedure resolved culture filtrate proteins into 20 fractions with a pI range of 2.59 to 12.9. These 20 fractions were subjected to immunological analysis in healthy laboratory volunteers from our endemic area. Eleven fractions (Fractions 5, 6, 7, 8, 9, 10, 11, 13, 15, 16, and 19) showed increased interferon gamma (IFN-γ) secretion and 5 fractions induced increased proliferative response, when compared to unfractionated CFP. In the 11 fractions which showed increased IFN-γ secretion, mass spectrometric analysis identified 19 different proteins. Apart from the already reported immunodominant antigens like FbpB, CFP-10 and ESAT-6, two new T cell antigens (AcpM and PpiA) were also identified in the immunologically active fractions. Immunoinformatic analysis showed that PpiA was predicted to bind more number of class I and class II HLA alleles compared with the immunodominant ESAT-6 and CFP-10. Population coverage calculations also showed that PpiA protein (85%) had a higher population coverage compared with ESAT-6 (79%) and CFP-10 (77%). This result shows that the PpiA protein has a potential to be a novel T cell antigen.

Biomarker Changes Associated with Tuberculin Skin Test (TST) Conversion: A Two-Year Longitudinal Follow-Up Study in Exposed Household Contacts

BackgroundA high prevalence (50–80%) of Tuberculin Skin Test Positivity (TST+ ≥10 mm indurations) has been reported in TB endemic countries. This pool forms a huge reservoir for new incident TB cases. However, immune biomarkers associated with TST conversion are largely unknown. The objective of this study was to identify immune biomarkers associated with TST conversion after acute Mycobacterium tuberculosis (MTB) exposure.Methodology/Principal FindingsA 24 month longitudinal study was carried out in a recently MTB exposed cohort of household contacts (HC = 93; 75% TST+). Control group consisted of unexposed community controls (EC = 59; 46%TST+). Cytokine secretion was assessed in whole blood cultures in response to either mycobacterial culture filtrate (CF) antigens or mitogens (PHA or LPS) using Elisa methodology. Compared to the EC group, the HC group at recruitment (Kruskal-Wallis Test) showed significantly suppressed IFN γ (p = 0.0001), raised IL-10 (p = 0.0005) and raised TNF α (p = 0.001) in response to CF irrespective of their TST status. Seventeen TST-HC, showed TST conversion when retested at 6 months. Post TST conversion (paired t tests) significant increases were observed for CF induced IFN γ (p = 0.038), IL-10 (p = 0.001) and IL-6 (p = 0.006). Cytokine responses were also compared in the exposed HC group with either recent infection [(TST converters (N = 17)] or previous infection [TST+ HC (N = 54)] at 0, 6, 12 and 24 months using ANOVA on repeated measures. Significant differences between the exposed HC groups were noted only at 6 months. CF induced IFN γ was higher in previously infected HC group (p = 0.038) while IL-10 was higher in recently infected HC group (p = 0.041). Mitogen induced cytokine secretion showed similar differences for different group.Conclusions/SignificanceOur results suggest that TST conversion is associated with early increases in IFN γ and IL-10 responses and precedes latency by several months post exposure.

Effect of 1, 25 dihydroxyvitamin D3 on matrix metalloproteinases MMP-7, MMP-9 and the inhibitor TIMP-1 in pulmonary tuberculosis

Matrix metalloproteinases (MMPs) play a vital role in the pathogenesis of several inflammatory diseases including tuberculosis through tissue remodeling. 1, 25(OH)(2)D(3) has several well recognized biological functions including suppression of MMP production. The influence of 1, 25(OH)(2)D(3) on MMP-7, MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1), production was studied in 43 pulmonary tuberculosis (PTB) patients and 44 healthy controls (HC). Peripheral blood mononuclear cells (PBMCs) were cultured with culture filtrate antigen (CFA) of Mycobacterium tuberculosis (MTB) and live MTB with or without 1, 25(OH)(2)D(3) (10(-7) M) for 48 h and the culture supernatants were assayed for MMP-7, MMP-9, TIMP-1 and cytokines IFN-gamma and TNF-alpha using ELISA. In HC and PTB, the levels of MMP-7, MMP-9 and TIMP-1 were not altered by CFA and live MTB stimulation in both groups. However, a significant decrease in the spontaneous production of MMP-7 (p=0.007), and an increase in MMP-9 (p=0.07) and TIMP-1 (p=0.0001) were observed in PTB patients as compared to HC. Vitamin D(3) significantly reduced the MMP-7 (p=0.0001) and MMP-9 (p=0.0001) and increased the TIMP-1 (p=0.005) level in antigen stimulated and unstimulated cultures of PTB as compared to HC. A significant positive correlation between MMP-9 and IFN-gamma was observed in unstimulated cultures of both HC (p=0.05) and PTB patients (p=0.0007). The present study suggests that 1, 25(OH)(2)D(3) suppresses the production of MMPs and enhances the level of TIMP-1 in tuberculosis. The present study suggests that 1, 25(OH)(2)D(3) may probably play an important role in the pathological process in tuberculosis by downregulating the levels of MMPs and upregulating the levels of TIMPs.

Development of an Immunochromatographic Test for Rapid Serodiagnosis of Human Pythiosis

Human pythiosis is an emerging and life-threatening infectious disease caused by the fungus-like organism Pythium insidiosum. High rates of morbidity and mortality for patients with pythiosis are exacerbated by the lack of early diagnosis and an effective treatment. Here, we developed and evaluated an immunochromatographic test (ICT) for the diagnosis of human pythiosis, in comparison to a standard serological test of immunodiffusion (ID). Culture filtrate antigen of P. insidiosum was used to detect human anti-P. insidiosum antibody. Sheep anti-human immunoglobulin G-colloidal gold conjugate was used to generate an ICT signal. Thirty-three sera from patients with vascular (n = 27), ocular (n = 4), and cutaneous (n = 2) pythiosis and 181 control sera from healthy blood donors (n = 100), as well as patients with a variety of infectious (n = 56) and noninfectious (n = 25) diseases, were included in the test evaluation. The turnaround time for generating a result by the ICT was less than 30 min, while that for ID was approximately 24 h. Based on the results for all sera of pythiosis patients and the control groups, the ICT showed 88% sensitivity and 100% specificity and ID showed 61% sensitivity and 100% specificity. By both tests, false-negative results for sera from all ocular pythiosis patients were obtained. In addition, the ID test yielded false-negative results for sera from eight patients with vascular pythiosis and one patient with cutaneous pythiosis. It was concluded that the ICT is a rapid, user-friendly, and reliable serological test for the early diagnosis of vascular and cutaneous pythiosis.

Development of a Slide Latex Agglutination Assay for Identification and Confirmation of Burkholderia pseudomallei from Cultures

Culture and biochemical assay remain as “gold standard” for identification and confirmation of B. pseudomallei. However confirmation by API 20NE biochemical assay is time consuming as the identification can only be done 24 hours onwards up to a week. Consequently, treatment of the patients suspected for melioidosis can be delayed and increases mortality rate as death can occur within 24 hours onset of the disease. Therefore there is a need to develop an assay that can shorten the time for identification and confirmation of B. pseudomallei from cultures. In this study, a rapid slide agglutination assay was developed using a specific monoclonal antibody raised against culture filtrate antigen of B. pseudomallei in Balb-C mice. The purified monoclonal antibody was tagged onto 0.8 um latex particles by means of passive adsorption. Latex agglutination assay was performed on a total of 45 B. pseudomallei strains and additional of five other Burkholderia species on glass microscope slide. Cross reactivity studies were performed on control panel strains which consisted of various gram-negative and gram-positive organisms. The latex agglutination assay was able to identify correctly 44 of 45 B. pseudomallei strains with a remarkable sensitivity of 97.8% and specificity of 100%. No cross reaction was observed with the control panel strains. In addition, latex agglutination assay was also performed on various B. pseudomallei antigen and compared with the developed assay. It was found that the developed latex agglutination assay is useful in identification and confirmation of B. pseudomallei from cultures.

Regulatory role of 1, 25-dihydroxyvitamin D 3 and vitamin D receptor gene variants on intracellular granzyme A expression in pulmonary tuberculosis

Vitamin D receptor (VDR) genotypes have been shown to be associated with differential susceptibility or resistance to tuberculosis. The influence of FokI, BsmI, ApaI and TaqI variants of VDR gene on 1, 25(OH) 2 D 3 modulated granzyme A expression of cytotoxic lymphocytes induced by culture filtrate antigen (CFA) of Mycobacterium tuberculosis was studied in 40 pulmonary tuberculosis (PTB) patients and 49 normal healthy subjects (NHS) by flow cytometry. In both the study groups, addition of 1, 25(OH) 2 D 3 (10 − 7 M) significantly reduced the percentage of granzyme A positive cells in both unstimulated (NHS, p < 0.0001; PTB, p = 0.02) and stimulated culture conditions (CFA, NHS, p < 0.0001; PTB, p = 0.0001) which correlated positively with the IFN-γ levels (unstimulated, p = 0.01; CFA stimulated, p = 0.004) in NHS. The ApaI aa genotype and bbaaTT extended genotype were associated with a significantly decreased percentage of granzyme A positive cells in NHS ( p < 0.05). Our results suggest that 1, 25(OH) 2 D 3 suppresses granzyme A probably by down-regulating Th1 cytokine response. Moreover, the VDR gene variants might regulate cytotoxic T-cell response via 1, 25(OH) 2 D 3 mediated suppression of granzyme A expression in tuberculosis.

Whole Blood Interferon-Gamma Responses to Mycobacterium tuberculosis Antigens in Young Household Contacts of Persons with Tuberculosis in Uganda

BackgroundDue to immunologic immaturity, IFN-γ-producing T cell responses may be decreased in young children compared to adults, thus we hypothesized that IFN-γ responses to mycobacterial antigens in household contacts exposed to Mycobacterium tuberculosis (Mtb) would be impaired in young children relative to adults. The objective of this study was to compare whole blood IFN-γ production in response to mycobacterial antigens between children and adults in Uganda.Methodology/Principal FindingsWe studied household contacts of persons with culture-positive pulmonary tuberculosis (TB) enrolled in a cohort study conducted in Kampala, Uganda. Whole blood IFN-γ production in response to Mtb culture-filtrate antigens was measured by ELISA and compared between infants (<2 years old, n = 80), young children (2 <5 years old, n = 216), older children (5 <15 years old, n = 443) and adults (≥15 years old, n = 528). We evaluated the relationship between IFN-γ responses and the tuberculin skin test (TST), and between IFN-γ responses and epidemiologic factors that reflect exposure to Mtb, and the effect of prior BCG vaccination on IFN-γ responses. Young household contacts demonstrated robust IFN-γ responses comparable to those of adults that were associated with TST and known risk factors for infection. There was no effect of prior BCG immunization on the IFN-γ response.Conclusions/SignificanceYoung children in a TB endemic setting can mount robust IFN-γ responses generally comparable to those of adults, and as in adults, these responses correlated with the TST and known epidemiologic risk factors for Mtb infection.

Antibody repertoire against culture filtrate antigens in wild house mice infected withMycobacterium bovisBCG

Wild house mice (Mus domesticus) captured in a Flemish pigsty were infected intravenously with 4 x 10(6) variable units of Mycobacterium bovis BCG and examined by Western blot analysis for IgG secretion against BCG culture filtrate (CF) antigens. Wild mice showed a marked individual variation in antibody pattern when tested 4, 6 and 8 weeks after infection. Some animals reacted to a wide range of antigens and others only to a limited number. Most wild mice recognized preferentially antigens with molecular weight of 24 kD, 32 kD, 37-38-40 kD, 65 kD and 82 kD, i.e. the major CF antigens known to be recognized by sera from BCG-infected inbred laboratory strains, BALB/c, DBA/2, CBA/Ca and C57BL/6. The 32-kD fibronectin-binding protein and the 65-kD heat-shock protein appeared as very immunodominant in wild mice. Furthermore, about 20-25% of the mice reacted strongly with a unique antigen of 35 kD estimated molecular weight, to which the tested inbred laboratory mice did not respond. Monitoring the size of the bacterial population in the spleen indicated that the BCG inoculum did not replicate in wild mice, suggesting that the Bcgr allele is expressed in this population.

Immune Response to Nocardia brasiliensis Extracellular Antigens in Patients with Mycetoma

The ability of culture-filtrate proteins to induce a cellular immune response in infected mice and humans was investigated. A crude extract culture filtrate of Nocardia brasiliensis (CFA) and five semi-purified CFA fractions (P1, P2, P3, P4, P5) were used to stimulate BALB/c mice spleen-cell cultures. The animals were divided into three groups: the first group was infected with 1 x 10(7) CFU of N. brasiliensis in the footpad, the second group was immunized with heat-killed bacteria, and the third was injected with sterile saline. IFN-gamma, IL-1alpha, and IL-4 concentrations were determined in culture supernatants. Protein fractions eliciting IFN-gamma production in mice, as well as the CFA, were used to stimulate IFN-gamma production and in vitro cell proliferation assays with peripheral blood mononuclear cells of patients with actinomycetoma by N. brasiliensis, individuals with pulmonary tuberculosis, and healthy controls. In mice, CFA and three of the protein fractions (P3, P4 and P5) induced significant IFN-gamma production in the infected group. In humans, only the CFA-induced IFN-gamma production and cell proliferation in the group of patients with actinomycetoma. There was no stimulation in tuberculosis patients nor healthy controls. These results suggest that some culture-filtrate antigens are recognized by patients with active actinomycetoma and do not cross-react with M. tuberculosis antigens, being therefore potential candidates to develop a diagnostic test.

Regulatory Role of Promoter and 3′ UTR Variants of Vitamin D Receptor Gene on Cytokine Response in Pulmonary Tuberculosis

Vitamin D receptor (VDR) gene variants are shown to regulate immune response in tuberculosis. We studied the influence of VDR promoter (Cdx-2 and A1012G), 3' untranslated region (Apa I, Bsm I, and Taq I) and start codon (Fok I) polymorphisms on 1,25(OH)(2)D(3)-modulated IL-12p40, IFN-gamma, IL-10, and IL-5 response to live Mycobacterium tuberculosis and its culture filtrate antigen (CFA) in 60 normal healthy subjects and 51 pulmonary tuberculosis patients. In peripheral blood mononuclear cell cultures with CFA and 1,25(OH)(2)D(3), IL-12p40, and IFN-gamma levels were significantly decreased (p < 0.05) and IL-10 levels were significantly increased (p < 0.05) in patients with GG genotype. The extended genotype bbaaTT (baT haplotype) was associated with decreased IL-12p40 and IFN-gamma levels and significantly increased IL-10 levels (p < 0.05). The Cdx-2 GG genotype and baT haplotype are associated with a suppressed Th1 and increased IL-10 response, which suggests that 1,25(OH)(2)D(3) probably through the VDR polymorphic variants augments the anti-inflammatory response at the site of M. tuberculosis infection.

Cytokine Profiles of Macrophages by Mycobacterium abscessus Conditional Morphotype Variants and Comparison of Their Extracellular-Protein Expressions

Mycobacterium abscessus has been identified as an emerging pulmonary pathogen in humans. Previously, it was documented that a spontaneously formed rough variant of M. abscessus causes persistent and invasive infection in mice, while a smooth isogenic variant does not. However, little is known for immune responses elicited by M. abscessus variants artificially induced by culture conditions and their culture filtrate antigens. Thus, morphological variants of M. abscessus type strain (ATCC19977T) were generated by an acidic and low oxygen culture conditions. Overall comparison between the variant and its original smooth strain showed that the rough variant was less virulent than original smooth strain in murine bone-marrow derived macrophage. To understand the basis for the difference, the protein expression pattern in the culture filtrates of each strain was analyzed by 1-dimensional electrophoresis. Generally, the protein expressions were more influenced by pH conditions than oxygen pressures. Interestingly, several proteins, mainly lower than 30 kDa molecular weight, were uniquely expressed in normal culture conditions. In contrast, several high molecular weight proteins (＞55 kDa) were induced by acidic and low oxygen culture conditions. These findings not only provide new insights of association between morphological change and the virulence, but may also be useful in the design of immunological diagnosis and vaccines for M. abscessus infection.

Roles of SigB and SigF in theMycobacterium tuberculosisSigma Factor Network

To characterize the roles of SigB and SigF in sigma factor regulation in Mycobacterium tuberculosis, we used chemically inducible recombinant strains to conditionally overexpress sigB and sigF. Using whole genomic microarray analysis and quantitative reverse transcription-PCR, we investigated the resulting global transcriptional changes after sigB induction, and we specifically tested the relative expression of other sigma factor genes after knock-in expression of sigB and sigF. Overexpression of sigB resulted in significant upregulation of genes encoding several early culture filtrate antigens (ESAT-6-like proteins), ribosomal proteins, PE-PGRS proteins, the keto-acyl synthase, KasA, and the regulatory proteins WhiB2 and IdeR. Of note, the induction of sigB did not alter the expression of other sigma factor genes, indicating that SigB is likely to serve as an end regulator for at least one branch of the M. tuberculosis sigma factor regulatory cascade. Analysis of the 5'-untranslated region (UTR) of SigB-dependent transcripts revealed a putative consensus sequence of NGTGG-N(14-18)-NNGNNG. This sequence appeared upstream of both sigB (Rv2710) and the gene following it, ideR (Rv2711), and in vitro transcription analysis with recombinant SigB-reconstituted RNA polymerase confirmed SigB-dependent transcription from each of these promoters. Knock-in expression of sigF revealed that only the sigC gene was significantly upregulated 6 and 12 h after sigF induction. The previously identified SigF promoter consensus sequence AGTTTG-N(15)-GGGTTT was identified in the 5' UTR of the sigC gene, and SigF-dependent in vitro transcription of the promoter upstream of sigC was confirmed by using recombinant SigF-reconstituted RNA polymerase. These two knock-in recombinant strains were tested in a macrophage model of infection which showed that overexpression of sigB and sigF resulted in reduced rates of M. tuberculosis intracellular growth. These results define the SigB promoter consensus recognition sequence and members of the SigB regulon. Moreover, the data suggest that, in addition to serving as an end regulator in a sigma factor cascade, SigB may auto-amplify its own expression under certain conditions.

1, 25 Dihydroxyvitamin D 3 modulated cytokine response in pulmonary tuberculosis

1, 25 Dihydroxyvitamin D 3 (1, 25(OH) 2 D 3) has gained significant importance in tuberculosis with regard to its immunoregulatory activities. Our aim was to evaluate the effect of 1, 25(OH) 2 D 3 on cytokine response to Mycobacterium tuberculosis antigens in pulmonary tuberculosis. Peripheral blood mononuclear cells obtained from 60 healthy controls and 52 pulmonary tuberculosis patients were cultured with culture filtrate antigen (CFA) of M. tuberculosis and live M. tuberculosis with and without 1, 25(OH) 2 D 3 (10 −9, 10 −8 and 10 −7 M concentrations). The culture supernatants were used to estimate IL-8, IL-6, TGF-β, IL-10, IFN-γ, IL-12p40, IL-2, IL-4 and IL-5 levels by ELISA. 1, 25 Dihydroxyvitamin D 3 significantly suppressed IL-12p40 and IFN-γ production in response to CFA and live M. tuberculosis with a maximum suppression at 10 −7 M concentration ( p < 0.0001). In CFA stimulated cultures, addition of 1, 25(OH) 2 D 3 significantly suppressed IL-8, IL-6 and IL-10 whereas the IL-2 levels were significantly increased in controls. It variably influenced the Th2 cytokines, showing an increased trend for IL-4 and suppressed IL-5 levels. We report that 1, 25(OH) 2 D 3 differentially modulates production of cytokines in response to M. tuberculosis antigens by predominantly suppressing IL-12p40 and IFN-γ production in a dose dependent manner. Our results suggest a role for vitamin D in restricting acquired immune response against tuberculosis by regulating cytokine production.

Influence of HLA-DRB1 alleles on Th1 and Th2 cytokine response to Mycobacterium tuberculosis antigens in pulmonary tuberculosis

The influence of human leukocyte antigens (HLA) on the immune response is well established. We investigated the regulatory role of HLA-DRB1 alleles on cytokine response to live M. tuberculosis and its culture filtrate antigen (CFA) in normal healthy subjects (NHS) and pulmonary tuberculosis (PTB) patients. Th1 (IFN-gamma and IL-12p40), Th2 (IL-4 and IL-5), pro-inflammatory (IL-6 and IL-8) and anti-inflammatory (TGF-beta and IL-10) cytokines were measured by ELISA in 72-h-old peripheral blood mononuclear cell culture supernatants from 58 NHS and 48 PTB patients. HLA-DRB1 genotyping was carried out by polymerase chain reaction and dot-blot hybridization with biotinylated sequence-specific oligonucleotide probes and detection by chemiluminescence. In response to live M. tuberculosis and CFA, significantly increased levels of IL-6, IL-8 and TGF-beta and decreased IFN-gamma, IL-12p40 and IL-10 were seen in PTB patients compared to NHS. We observed a significantly increased IFN-gamma response in HLA-DRB1*03-positive NHS (p=0.03) and decreased IFN-gamma response in HLA-DRB1*15-positive patients (p=0.04) than respective allele-negative individuals. An increased level of IL-12p40 in DRB1*10 (p=0.02) and IL-10 in DRB1*12- (p=0.03) positive NHS and an increased level of IL-6 in DRB1*04- (p=0.02) positive patients were observed. The study suggests that HLA-DRB1 alleles differentially modulate the various cytokine responses to M. tuberculosis antigens, which may influence the cellular and humoral immune responses to M. tuberculosis infection in a susceptible host.

Associations between cytokine gene expression and pathology in Mycobacterium bovis infected cattle

An impediment to the development of efficacious vaccines for bovine tuberculosis has been the failure to demonstrate strong associations between immune function and protective immunity. Cytokine gene expression in response to Mycobacterium bovis ( M. bovis) infection was evaluated to identify correlates of immunity. Ten Holstein calves were infected with M. bovis by intratonsillar inoculation. Five uninfected animals served as controls. At 15, 30, 60 and 85 days post-infection (dpi) peripheral blood mononuclear cells (PBMC) were isolated and stimulated with either purified protein derivative of M. bovis (PPD), a recombinant fusion protein comprised of 6 kDa early secretory antigenic target and 10 kDa culture filtrate antigen (rESAT6:CFP10), or PBS. After a 16 h incubation period, total leukocyte RNA was isolated and gene expression evaluated using reverse transcriptase real-time PCR. In addition, gene expression adjacent to gross lesion in the retropharyngeal lymph node (LN) was analyzed. Pathology was evaluated at necropsy. Expression of IFN-γ, TNF-α, iNOS and IL-4 by PBMC increased in response to infection, whereas, IL-10 expression decreased. Differences in gene expression between PBMC from infected and uninfected animals was greatest at 30 dpi. Infected animals were divided into two groups based on pathology. Animals in the low pathology group had lesions primarily in LN of the head; whereas, animals in the high pathology group also had lesions in the lungs and lung associated LN. Gene expression in PBMC and LN was compared between animals in the high and low pathology groups. Cells from animals in the high pathology group expressed more IFN-γ, TNF-α, iNOS and IL-4 than did animals in the low pathology group at early time points. IL-10 gene expression decreased with time in PBMC from animals in the high pathology group. At 85 dpi, animals in the high pathology group expressed twofold less IL-10 mRNA than did animals in the low pathology group and the uninfected controls. IFN-γ and iNOS gene expression were significantly greater in tissues from infected animals compared to tissues from uninfected animals. The pathological outcome of M. bovis infection of cattle may be established early after infection since expression of both the TH1 and TH2 cytokines were differentially expressed by animals in the high and low pathology groups at early time points. In addition, more robust immunological responses were associated with increased pathology. These results suggest that early immune responses play a critical role in establishing the pathological outcome.