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Introduction: Pleural tuberculosis (TB) is the archetype of extrapulmonary TB (EPTB), which mainly affects the pleural space and leads to exudative pleural effusion. Diagnosis of pleural TB is a difficult task predominantly due to atypical clinical presentations and sparse bacillary load in clinical specimens., Area Covered: We reviewed the current literature on the globally existing conventional/latest modalities for diagnosing pleural TB. Bacteriological examination (smear/culture), tuberculin skin testing/interferon-γ release assays, biochemical testing, imaging and histopathological/cytological examination are the main modalities. Moreover, nucleic acid amplification tests (NAATs), i.e . loop-mediated isothermal amplification, PCR/multiplex-PCR, nested-PCR, real-time PCR and GeneXpert ® MTB/RIF are being utilized. Currently, GeneXpert Ultra, Truenat MTB TM , detection of circulating Mycobacterium tuberculosis ( Mtb ) cell-free DNA by NAATs, aptamer-linked immobilized sorbent assay and immuno-PCR (I-PCR) have also been exploited., Expert Opinion: Routine tests are not adequate for effective pleural TB diagnosis. The latest molecular/immunological tests as discussed above, and the other tools, i.e . real-time I-PCR/nanoparticle-based I-PCR and identification of Mtb biomarkers within urinary/serum extracellular vesicles being utilized for pulmonary TB and other EPTB types may also be explored to diagnose pleural TB. Reliable diagnosis and early therapy would reduce the serious complications associated with pleural TB, i.e . TB empyema, pleural fibrosis, etc.

Mycobacterial diseases are important health issues in farmed deer. The single intradermal tuberculin test is the standard test for tuberculosis testing in deer. We studied two factors which might influence the response of deer to skin testing: the inoculation site and the injection device. Deer included in this study were 2.5 years old farmed red deer (Cervus elaphus) hinds (n = 80). Two areas of 3 cm × 3 cm were shaved at the left side of the neck. Site A (SA) was situated about 10 cm caudal to the head, while site B (SB) was 10 cm caudal to SA. All hinds received at the same time two 0.1 ml inoculations of Mycobacterium avium derived purified protein derivative (aPPD). One inoculation was made by syringe and the other one with the needle-free syringe Dermojet. To test the inoculation site effect, half of the animals were inoculated by Dermojet in SA and by syringe in SB to compare with the inoculation in SA by syringe and Dermojet in SB in the other half. No differences were recorded for the injection device nor for the inoculation site. Ten hinds had a skinfold increase larger than 30 tenths of mm by any injection device and inoculation site. Seven (9%) and 6 (8%) hinds were classified as positive by syringe and Dermojet, and at the anterior or posterior inoculation site, respectively. The distribution of skinfold thickness increases did not differ by injection device. Our findings support the needle-free Dermojet syringe as a suitable tool for skin-testing in red deer and suggest no relevant effect of the position of the inoculation site along the neck in red deer., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Treatment monitoring is an essential aspect for tuberculosis (TB) disease management. Sputum smear microscopy is the only available tool for monitoring, but it suffers from demerits. Therefore, we sought to evaluate markers and cellular subsets of T regulatory (Treg) cells and T helper (Th) 17 cells in pulmonary TB patients (PTB) for TB treatment monitoring. Peripheral blood mononuclear cells (PBMCs) were stimulated in vitro (with purified protein derivative (PPD)) overnight which was followed by a polychromatic flow cytometry approach to study Treg and Th17 markers and cellular subsets in PTB ( n  = 12) undergoing antituberculous treatment (ATT). The baseline levels of these markers and cellular subsets were evaluated in normal healthy subjects (NHS). We observed a significant decrease in the expression of CD25 ( p <0.01) marker and percentage of T-cell subsets like CD4 + CD25 + ( p <0.001) and CD4 + CD25 + CD39 + ( p <0.05) at the end of intensive phase (IP) as well as in the continuation phase (CP) of ATT. A decrease in CD25 marker expression and percentage of CD4 + CD25 + T cell subset showed a positive correlation to sputum conversion both in high and low sputum positive PTB. In eight PTB with cavitary lesions, only CD4 + CD25 + FoxP3 Treg subset manifested a significant decrease at the end of CP. Thus, results of this study show that CD25 marker and CD4 + CD25 + T cells can serve as better markers for monitoring TB treatment efficacy. The Treg subset CD4 + CD25 + FoxP3 may be useful for prediction of favorable response in PTB with extensive lung lesions. However, these findings have to be evaluated in a larger patient cohort.

Currently Mycobacterium avium subsp. paratuberculosis (MAP) infection is diagnosed through indirect tests based on the immune response induced by the infection. The antigens commonly used in IFN-γ release assays (IGRA) are purified protein derivative tuberculins (PPD). However, PPDs, lack both specificity (Sp) and sensitivity (Se) in the early phase of infection. This study investigated the potential of 16 MAP recombinant proteins and five lipids to elicit the release of IFN-γ in goats from herds with or without a history of paratuberculosis. Ten recombinant proteins were selected as potential candidates for the detection of MAP infection in young goats. They were found to detect 25 to 75% of infected shedder (IS) and infected non-shedder (INS) kids younger than 10months of age. In comparison, PPD was shown to detect only 10% of INS and no IS kids. For seven antigens, Se (21-33%) and Sp (≥90%) of IGRA were shown to be comparable with PPD at 20months old. Only three antigens were suitable candidates to detect IS adult goats, although Se was lower than that obtained with PPD. In paratuberculosis-free herds, IGRA results were negative in 97% of indoor goats and 86% of outdoor goats using the 10 antigens. However, 22 to 44% of one-year-old outdoor goats were positive suggesting that they may be infected. In conclusion, this study showed that ten MAP recombinant proteins are potential candidates for early detection of MAP infected goats. Combining these antigens could form a possible set of MAP antigens to optimize the Se of caprine IGRA., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A functional genomics approach was used to examine the immune response for transcriptional profiling of PBMC M. bovis infected cattle and healthy control cattle to stimulation with bovine tuberculin (purified protein derivative PPD-b). Total cellular RNA was extracted from non-challenged control and M. bovis challenged MDM for all animals at intervals of 6 h post-challenge, in response to in-vitro challenge with M. bovis (multiplicity of infection 2:1) and prepared for global gene expression analysis using the Agilent Bovine (V2) Gene Expression Microarray, 8 × 60 K. The pattern of expression of these genes in PPD bovine stimulated PBMC provides the first description of an M. bovis specific signature of infection that may provide insights into the molecular basis of the host response to infection. Analysis of these mapped reads showed 2450 genes (1291 up regulated and 1158 down regulated) 462 putative natural antisense transcripts (354 up-regulated and 108 down regulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). The results provided enrichment for genes involved top ten up regulated and down regulated panel of genes, including transcription factors proliferation of T and B lymphocytes. The highest differentially-expressed genes were associated to immune and inflammatory responses, immunity, differentiation, cell growth, apoptosis, cellular trafficking and regulation of lipolysis and thermogenesis. Microarray results were confirmed in infected cattle by RT qPCR to identify potential biomarkers TLR2, CD80, NFKB1, IL8, CXCL6 and ADORA3 of bovine tuberculosis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: To compare the tuberculin reaction of 2 tuberculin unit (TU) with 5TU purified protein derivative (PPD) (both calibrated against RT 23) in healthy children., Methods: This was a cross sectional study done in the pediatric outpatient department of a tertiary care teaching hospital. Seventy healthy siblings of the children attending pediatric outpatient department in the age group of 1 to 12 y were enrolled. The exclusion criteria included previously diagnosed tuberculosis patients, malnutrition diagnosed according to the WHO classification, history of drug intake like steroids, recent history of measles, any skin lesions over forearm, history of fever, contact with tuberculosis and previous mantoux testing. The study was conducted wherein each child was subjected to simultaneous testing with 2TU and 5TU by standard technique. The reactions to both the tests was read at 48-72 h. Children with induration ≥10 mm were evaluated for tuberculosis by taking chest x-ray, gastric lavage or sputum smear examination for acid fast bacilli (AFB)., Results: Considering ≥10 mm induration as positive, subjects positive with 5TU were 7 (10%) and 2TU was 1(p value = 0.031); thus, there is no agreement between the two methods (McNemar's test). Comparing the mean diameter of induration of 2TU and 5TU (p < 0.001, Wilcoxon test), signified no agreement between the two strengths. Bland-Altman plot and kappa statistic showed no agreement between the two strengths., Conclusions: Cutaneous hypersensitivity to 2TU PPD is not comparable to that of 5TU PPD.

The measurement of bovine interferon-gamma (IFN-γ) forms the basis of a diagnostic test for bovine tuberculosis where Mycobacterium bovis sensitised effector T cells produce IFN-γ following in vitro stimulation with tuberculin antigens. In cattle infected with M. bovis it is also known that the anti-inflammatory IL-10 cytokine can inhibit in vitro production of IFN-γ leading to a reduced response in the IFN-γ diagnostic test. In order to investigate this in greater detail, whole blood samples from tuberculin skin test positive and negative cattle were stimulated with bovine and avian tuberculin antigens and in parallel with a neutralising anti-IL-10 monoclonal antibody. The results showed that IFN-γ protein levels increased when IL-10 activity was suppressed by Anti - IL-10. By using a standard diagnostic interpretation, the elevated levels of IFN-γ were shown to change the level of agreement between the performance of the single intradermal comparative tuberculin test (SICTT) and IFN-γ assay, depending on the tuberculin treatment. A transcriptomic analysis using RT-qPCR investigated the influence of IL-10 activity on expression of a suite of cytokine genes (IFNG, IL12B, IL10 and CXCL10) associated with antigen-stimulated production of IFN-γ. The IFNG and IL12B genes both experienced significant increases in expression in the presence of Anti-IL-10, while the expression of IL10 and CXCL10 remained unaffected., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Up to now, there has been no universal consensus on the agreement between the tuberculin skin test (TST) and the QuantiFERON-TB-Gold test (QFT) in the detection of latent tuberculosis infection (LTBI) among high-risk populations., Aims: To estimate the agreement between TST and QFT among health care workers (HCWs)., Methods: A meta-analysis in which all major electronic databases, including Medline, Scopus, Web of Sciences and Ovid, were searched until June 2014. All cross-sectional and cohort studies addressing the agreement between TST and the QFT were included. The extracted data were analysed and the results were reported using random effect models., Results: The overall kappa statistic between TST and the QFT was 0.27 [95% confidence interval (CI) 0.22, 0.32] and the adjusted kappa statistic for prevalence and bias was 0.41 (95% CI 0.32, 0.50). The kappa for subjects with and without bacillus Calmette-Guérin (BCG) vaccination was 0.27 (95% CI 0.18, 0.36) and 0.31 (95% CI 0.15, 0.46) respectively. The figures were 0.30 (95% CI 0.16, 0.43) and 0.82 (95% CI 0.74, 0.90) for prevalence-adjusted and bias-adjusted kappa, respectively., Conclusions: The overall agreement between TST and QFT in the detection of LTBI among HCWs was poor. After adjusting for the prevalence and bias indices, kappa statistics reached fair agreement. The utility of each of these two tests is dependent on the prevalence and burden of tuberculosis as well as the BCG vaccination status., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Occupational Medicine. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The most widely used ante-mortem diagnostic tests for tuberculosis in cattle are the tuberculin skin test and the interferon-gamma (IFN-γ) release assay, both of which measure cell-mediated immune responses to Mycobacterium bovis infection. However, limitations in the performance of these tests results in a failure to identify all infected animals. In attempting to increase the range of diagnostic tests for tuberculosis, measurement of the cytokine IP-10 in antigen-stimulated blood has previously been shown to improve the detection of M. tuberculosis and M. bovis infection, in humans and African buffaloes (Syncerus caffer), respectively. In the present study, 60 cattle were identified by the single intradermal comparative tuberculin test as tuberculosis reactors (n = 24) or non-reactors (n = 36) and the release of IFN-γ and IP-10 in antigen-stimulated whole blood from these animals was measured using bovine specific ELISAs. There was a strong correlation between IP-10 and IFN-γ production in these samples. Moreover, measurement of the differential release of IP-10 in response to stimulation with M. bovis purified protein derivative (PPD) and M. avium PPD distinguished between reactor and non-reactor cattle with a sensitivity of 100% (95% CI, 86%-100%) and a specificity of 97% (95% CI, 85%-100%). These results suggest that IP-10 might prove valuable as a diagnostic biomarker of M. bovis infection in cattle.

Autoimmune polyendocrinopathy - candidiasis - ectodermal dystrophy (APECED) is caused by mutations in the Autoimmune regulator (AIRE) gene and is associated with neutralizing anti-cytokine autoantibodies. We have used an in vivo challenge model to analyze antigen-specific CD4(+) T cell responses. Bacille Calmette-Guérin (BCG)-vaccinated patients and controls were injected tuberculin intradermally, skin blisters were induced by suction on the indurations and on unexposed skin, and the infiltrating cells harvested. The patients had a quantitatively normal CD4(+) T cell response and no significant abnormalities in the expression of T helper type (Th) 1- or Th2-related genes. The expression of interleukin (IL)-22, in contrast, was lower in the patients. Two patients, both with a pre-existing ocular keratopathy, experienced a relapse of keratoconjunctivitis, suggesting a possible immunological basis for this APECED component. Our in vivo data are compatible with a selective IL-22 defect in the activated CD4(+) T cells of APECED patients, affecting also unexposed skin in steady-state conditions.

There is an urgent need to identify additional diagnostic biomarkers for bovine TB to complement existing read-out systems such as interferon-gamma and for predictive markers of vaccine efficacy to accelerate vaccine development. To evaluate the potential of miRNAs as such biomarkers, we have analysed their expression in bovine PPD stimulated PBMC isolated from unvaccinated and BCG vaccinated cattle before and following Mycobacterium bovis (M. bovis) infection. Using a bovine microRNA microarray, miR-155 was found to show a significant up-regulation in expression in early (week 2) and late (week 11) M. bovis post-infection samples from unvaccinated cattle, while in BCG vaccinated cattle up-regulation was observed only in late post-infection samples. No differential expression of miR-155 was observed in pre-infection samples from unvaccinated and vaccinated cattle. These observations suggest that miR-155 could be exploited as a marker distinguishing vaccinated from infected animals (DIVA). Analysis by TaqMan RT-PCR, verified the up-regulation of miR-155 in unvaccinated cattle post-infection. Significant correlation was found between the degree of pathology and miR-155 induction in the experimentally infected cattle, suggesting miR-155 is a biomarker of disease development and/or severity. Induction of miR155 expression in cattle sourced from farms with confirmed bTB that tested positive in the tuberculin skin or interferon-gamma blood test was found to be significantly higher in cattle presenting with more advanced pathology (defined by the presence of visible TB lesions) compared to infected cattle without visible pathology and thus likely to be of lower infectivity than those with more advanced disease. In conclusion, our data indicate that miR-155 has potential both as a diagnostic and prognostic biomarker that could be used to identify animals with advanced pathology and as a DIVA test read-out. Its role in the immune biology of bovine TB will also be discussed., (Copyright © 2014. Published by Elsevier Ltd.)

Bovine tuberculosis (TB), mainly caused by Mycobacterium bovis, is a zoonotic disease with implications for Public Health and having an economic impact due to decreased production and limitations to the trade. Bovine TB is subjected to official eradication campaigns mainly based on a test and slaughter policy using diagnostic assays based on the cell-mediated immune response as the intradermal tuberculin test and the gamma-interferon (IFN-γ) assay. Moreover, several diagnostic assays based on the detection of specific antibodies (Abs) have been developed in the last few years with the aim of complementing the current diagnostic techniques in the near future. This review provides an overview of the current ante-mortem diagnostic tools for diagnosis of bovine TB regarding historical background, methodologies and sensitivity (Se) and specificity (Sp) obtained in previous studies under different epidemiological situations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Infection with Cytomegalovirus is associated with accelerated immunosenescence. Expansions of CMV-specific T cell responses have previously been demonstrated to affect the ability of T cells to respond to other infections. Most people above 60years of age display M. tuberculosis-specific immunity because of vaccination, exposure, or both. T-cell responses can be assessed by measuring intracellular IFN-γ in vitro after tuberculin stimulation. Here we investigated tuberculin-specific CD4 T-cell responses in independently living healthy older people in the South of England using flow-cytometry. Individuals were investigated for tuberculin and CMV-specific T-cell immunity using in vitro antigen stimulation followed by intracellular staining for IFN-γ, TNF-α, IL2, as well as degranulation and CD154 upregulation. We also examined a control group of younger individuals (20-35years of age). There was no significant difference between older and young people in regards to tuberculin responsiveness of CD4 T-cells; however, older people seemed to show more outliers. Increased responsiveness to tuberculin was significantly correlated to CMV responsiveness but not age. In older donors, the memory phenotype of tuberculin-induced T-cells was significantly skewed towards a more terminal differentiation phenotype in CMV-infected compared to uninfected individuals and the degree of skewing correlated quantitatively with the size of the CMV-specific CD4 T-cell response. This is a fundamental advance over previous reports of changes of the tuberculin-specific CD4 T-cell response with CMV serostatus. Our results show that how the immune system responds to CMV has a fundamental impact on the phenotype and function of the immune response to mycobacterial antigens in older life., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Macrophages are cellular targets for infection by bacteria and viruses. The fate of infected macrophages plays a key role in determining the outcome of the host immune response. Apoptotic cell death of macrophages is considered to be a protective host defense that eliminates pathogens and infected cells. In this study, we investigated the involvement of Notch signaling in regulating apoptosis in macrophages treated with tuberculin purified protein derivative (PPD). Murine bone marrow-derived macrophages (BMMs) treated with PPD or infected with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) induced upregulation of Notch1. This upregulation correlated well with the upregulation of the anti-apoptotic gene mcl-1 both at the transcriptional and translational levels. Decreased levels of Notch1 and Mcl-1 were observed in BMM treated with PPD when a gamma secretase inhibitor (GSI), which inhibits the processing of Notch receptors, was used. Moreover, silencing Notch1 in the macrophage-like cell line RAW264.7 decreased Mcl-1 protein expression, suggesting that Notch1 is critical for Mcl-1 expression in macrophages. A significant increase in apoptotic cells was observed upon treatment of BMM with PPD in the presence of GSI compared to the vehicle-control treated cells. Finally, analysis of the mcl-1 promoter in humans and mice revealed a conserved potential CSL/RBP-Jκ binding site. The association of Notch1 with the mcl-1 promoter was confirmed by chromatin immunoprecipitation. Taken together, these results indicate that Notch1 inhibits apoptosis of macrophages stimulated with PPD by directly controlling the mcl-1 promoter.

Purpose: The purpose of this study was to evaluate Interferon (IFN)-γ and Interleukin(IL)-17 profiles in patients with different clinical presentations of pulmonary tuberculosis (TB) and to compare them with those of tuberculin-negative and tuberculin-reactive healthy controls, Methods: Peripheral blood mononuclear cells (PBMCss), isolated from patients (n=52) and controls (n=30), were stimulated ex vivo with purified protein derivative (PPD) and IFN-γ and IL-17 levels in the supernatant were measured., Results: At baseline, PBMCss from patients with TB released a significantly lower amount of IL-17 (p=0.043) than PBMCss from healthy controls, whereas IFN-γ levels were similar in the two groups. After PPD stimulation, a significant rise in IL-17 levels was found only among healthy controls (p=0.02). This rise in IL-17 levels was similar between tuberculin-reactive and tuberculin-negative subjects. After PPD stimulation, patients with infiltrative TB secreted higher levels of IL-17 and IFN-γ than those affected with chronic, miliary and cavitary TB (p < 0.01). IFN-γ production from patients with infiltrative TB was even higher than for healthy controls (p < 0.01). PBMCss from tuberculin-reactive patients released higher levels of IFN-γ than tuberculin-negative subjects after PPD stimulation (p < 0.01)., Conclusion: Ex vivo PPD stimulation of PBMCs from patients with pulmonary TB does not significantly stimulate IL-17 release; however, higher IL-17 and IFN-γ levels are found in patients with infiltrative disease, in comparison with those affected with miliary, cavitary and chronic TB.

Previously, we reported that ultraviolet B-radiation (UVR) suppressed Bacillus Calmette-Guérin (BCG) vaccine-induced resistance to Mycobacterium tuberculosis in guinea pigs (GP). Herein, we investigated the cellular and molecular changes within the irradiated GP epidermis and the in vivo effect of supernatants from UV-irradiated (200 J m(-2)) epidermal cells (UV-sup) on M. bovis BCG vaccination. UVR increased the number of nucleated keratinocytes in the skin, but caused a decrease in the proportions of CD25(+)T cells. In the spleen, UVR resulted in a decrease in the proportions of T-cell subsets including CD25(+)T cells, and major histocompatibility complex (MHC) class II(+) and CD14(+) cells. Similarly, significant up-regulation of several cytokine mRNAs including IL-10 was also observed. Furthermore, UV-sup significantly reduced the MHC class II expression in peritoneal cells and reduced T-cell proliferation to ConA. The proliferation to purified protein derivative (PPD) was restored to normal levels by anti-IL-10 antibody. The UV-sup when injected into BCG-vaccinated GP significantly diminished the skin test response and T-cell proliferation to PPD and up-regulated the expression of IL-10, IL-4, IL-1β and Foxp3 mRNAs in the lymph node or spleen. Thus, whole body UVR induces profound cellular and molecular changes and injection of UV-sup from epidermal cells mimics the effect of whole body UVR in BCG-vaccinated GP., (© 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.)

Objectives: To evaluate the cytokine response pattern in Inuit in Greenland in relation to age, gender, body mass index (BMI), Mycobacterium tuberculosis infection (MTI), and otitis media (OM) to assess whether Inuit may have signs of impaired immune responsiveness to infection., Methods: A cross-sectional health assessment was conducted among inhabitants of Maniitsoq, West Greenland, in 2009, and several health outcomes were measured. The prevalence of MTI, overweight, and obesity was assessed among 263 school children and 137 adults, and OM was assessed among the children. Cytokine responses were measured in whole blood cultures after stimulation with phytohemagglutinin or purified protein derivative (PPD). Associations between cytokine concentrations, age, gender, BMI, MTI, and OM were estimated by linear regression., Results: Adults had generally higher cytokine concentrations than children. Children with MTI had 2.7 times higher interleukin (IL)-10 concentrations than those without (P = 0.01), and girls had 80% higher IL-10 than boys (P < 0.01) after phytohemagglutinin stimulation. Interferon (IFN)γ and tumor necrosis factor (TNF) concentrations were strongly elevated among children (P(IFNγ) < 0.001 and P(TNF) < 0.001) and adults (P(IFNγ) < 0.001 and P(TNF) <0.01) with MTI compared to those without after PPD stimulation. Adult women had significantly lower IFNγ (P = 0.03) and TNF (P = 0.04) concentrations than men. TNF was positively correlated with BMI in children (P = 0.01), and IL-10 was positively correlated with BMI in adults (P = 0.0004) after PPD stimulation., Conclusion: We found cytokine patterns similar to those reported from other immune competent study populations. Therefore, the study does not support the suggestion that Inuit may have impaired immune reactivity to infection., (Copyright © 2012 Wiley Periodicals, Inc.)

Mycobacterium tuberculosis (MTB)-specific cytokine responses in the peripheral blood and at the site of infection may differ significantly within the same individual, but the under-lying T-cell subset changes are largely unknown. Here, we measured effector and memory T-cell markers on CD4⁺ T cells (CD45RO, cysteine chemokine receptor (CCR)7, and CD27) in peripheral blood and at the site of active tuberculosis (TB). Additionally, T cells were stimulated overnight with purified protein derivative (PPD) and early secretory antigenic target (ESAT)-6 to determine which T-cell subset produces MTB-specific interferon (IFN)-γ. A striking decrease in CCR7 and CD27 expression on T cells was noted at the site of active TB. Likewise, IFN-γ expressing, ESAT-6 specific CD4⁺CD45RO⁺CD27⁻ T cells were dramatically increased at the site of infection but were not detectable in peripheral blood. An antigen-specific expansion of differentiated T cells at the site of active TB infection was poorly reflected in peripheral blood. Insight in these changes in MTB-specific effector T cells in different compartments of the body could lead to new approaches for immune-based diagnosis and interventions., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Recent publications have suggested that altered proportions of functional CD4 T-cell subsets correlate with active pulmonary TB. Also, CD27-expression on tuberculin-activated IFN-γ(+) CD4 T-cells is known to differ significantly between patients with active pulmonary TB and healthy TB-unexposed BCG vaccinees. Here, we explore links between CD4 T-cell phenotype, multiple functional subsets, and control of TB., Methods: We examined ex-vivo overnight tuberculin activated CD4 T-cells in regards to CD27-expression and the activation markers, CD154 upregulation, IFN-γ, TNF-α, IL-2, and degranulation in 44 individuals, including cases of clinically active pulmonary TB, and hospital staff with prolonged TB exposure, some of whom had latent TB., Results: Active pulmonary TB generally showed an excess of TNF-α(+) subsets over IFN-γ(+) subsets, paralleled by decreased CD27 expression on activated IFN-γ(+) or CD154(+) CD4 T-cells. The single subset distinguishing best between active pulmonary TB and high TB exposure was CD154(+) /TNF-α(+) / IFN-γ(-) /IL-2(-) /degranulation(-) (AUROC 0.90). The ratio between the frequencies of TNF-α(+) /IFN-γ(+) CD4 T-cells was an effective alternative parameter (AUROC 0.87)., Conclusions: Functional subsets and phenotype of tuberculin induced CD4 T-cells differ between stages of TB infection. Predominance of TNF-α(+) CD4 T-cells in active infection suggests an increased effort of the immune system to contain disease., (Copyright © 2012 International Clinical Cytometry Society.)

The high prevalence of Mycobacterium tuberculosis (MTB) despite widely available Bacille Calmette-Guerin (BCG) vaccination may be associated with Mycobacterium avium (M. avium), which may influence the host response to MTB. In this study, we demonstrate that pretreatment of murine macrophages with low-dose Mycobacterium avium-derived lipids (MALs), but not Escherichia coli-derived lipids (ELs), attenuates the clearance of intracellular Mycobacterium bovis BCG (M. bovis BCG) and the production of TNF-α, IL-6, IL-12 and nitric oxide (NO) following stimulation with purified protein derivatives (PPD) or heat-inactivated M. bovis BCG in vitro. Furthermore, a significant decrease in NF-κB activity was observed in MALs-pretreated RAW264.7 cells that were co-transfected with pSV-β-galactosidase and pGL4.32[luc2P/NF-κB-RE/Hygro] prior to stimulation with PPD or heat-inactivated M. bovis BCG. In contrast, IRAK-M, an inhibitor of NF-κB activation, was increased in these cells. This observed hyporesponsiveness is not related to the expression of Toll-like receptor 2 (TLR2). In conclusion, pretreatment with low-dose MALs can induce hyporesponsiveness to MTB components in murine macrophages.

Introduction: Guidelines recommend storing tuberculin purified protein derivative (PPD) refrigerated. However, especially in developing countries, maintaining the product refrigerated under field conditions can be difficult, limiting its use. Here we determine the effect of prolonged exposure to high temperatures on the potency of human, bovine and avian tuberculin PPD., Methodology: Human, bovine and avian tuberculin PPD were stored for several weeks exposed to temperatures ranging from 37º to 100ºC. The potency was evaluated in vivo, in sensitized or naturally infected animals., Results: Most test situations didn't affect the biological activity of the tuberculin PPDs and only very long and extreme incubations (several days at 100 °C) compromised the potency., Conclusions: Tuberculin PPD is very stable and can be stored or transported for long periods without refrigeration.

Objective: We have previously epidemiologically shown that type 1 diabetes is inversely associated with contact allergy. This finding is intriguing as type 1 diabetes and contact allergy are two completely different diseases, although T cells are involved in both diseases. The objective of this study was therefore to experimentally study the effect of contact allergens on the development of diabetes in non-obese diabetic mice., Methods: Non-obese diabetic mice 4 weeks of age were separated into seven groups. One group was exposed to tapped water every 14th day, whereas the remaining six groups were split into sensitizations groups or elicitation groups (exposure every 14th day). These groups were then treated with one of the selected contact allergens (PPD or DNCB) or vehicle (AOO). All groups received the sensitizing treatment regime, and hereafter only the elicitation groups were further treated. If the blood glucose reached 14 mM, the mice were considered diabetic and euthanized. Cardiac heart blood was drawn at euthanization, and a Luminex analysis was done on the serum., Results: We showed that repeated application of a low dose of PPD reduced the incidence of diabetes compared to application with water (47% versus 93%, p=0.004). The rest of the groups developed diabetes with a cumulative incidence rate above 80%. The Luminex cytokine analysis revealed no differences between the groups, and no elevated cytokine level suggested a systemic response. Dermatitis was not noticeable by visual inspection, a histological examination, however, revealed a slight infiltration in the ears in the elicitation groups exposed to contact allergens., Conclusion: This study showed that repeated topical application on the ears with a contact allergen could prevent the development of diabetes in non-obese diabetic mice. The contact allergens gave a non-visible, subclinical dermatitis on the application site. Activation of NKT cells to the ear lymph nodes seems to be involved., (Copyright © 2011 John Wiley & Sons, Ltd.)

Our objective was to evaluate the effect of stress-related dose of dopamine (DA) on the in vitro proliferation and cytokine production in polyclonally-activated T cells from healthy individuals or individuals with generalized anxiety disorder (GAD). Our results demonstrated that cell cultures from GAD group proliferated less following T cell activation, as compared with control group. The addition of DA reduced the proliferative response in cell cultures from healthy but not from GAD individuals. The cytokine profile in GAD individuals revealed Th1 and Th2 deficiencies associated with a dominant Th17 phenotype, which was enhanced by DA. A similar DA-induced immunomodulation was also observed in PPD-activated cell cultures from GAD individuals. Unlike the control, DA-enhanced Th17 cytokine production in GAD individuals was not affected by glucocorticoid. In conclusion, our results show that the T cell functional dysregulation in GAD individuals is significantly amplified by DA. These immune abnormalities can have impact in increasing the susceptibility of individuals with anxiety disorders to infectious diseases and inflammatory/autoimmune disorders., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Different cytokines have been suggested to be involved in the pathogenesis of pulmonary tuberculosis (TB). The frequencies of Mycobacterium tuberculosis (MTB) specific CD4(+) and CD8(+) T cells, CD4(+)CD25(+) Forkhead Box Protein (FoxP)3(+) T cells, interleukin (IL)-6, interferon (IFN)-γ, Tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β and IL-10 were assessed in HIV-negative, pulmonary tuberculosis (TB) patients (n=30) and in healthy controls (n=23) in Gabon. Peripheral blood mononuclear cells (PBMC) were stimulated with purified protein derivative (PPD) and early secretory antigenic target-6 (ESAT-6). In patients, a pronounced pro-inflammatory cytokine response with highly significant increased levels of IL-6 and TNF-α accompanied by increased TGF-β was detectable. Differences in IFN-γ responses between patients and healthy individuals were less pronounced than expected. FoxP3 expression did not differ between groups. A distinct cytokine pattern is associated with active pulmonary TB in patients from Central Africa., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The control of mycobacterial infection by the host depends on cell-mediated immunity (CMI), involving activated macrophages, T cells, and type 1 cytokines (Th1). Here we evaluated the capacity of antigen-induced proliferation by peripheral blood mononuclear cells (PBMCs) and the production of proinflammatory tumor necrosis factor-alpha (TNF-α) and interleukin 12 (IL-12p40). The Th1 cytokine interferon-gamma (IFN-γ) and Th2 cytokines interleukin 4 (IL-4) and interleukin 5 (IL-5) in 62 pulmonary tuberculosis (TB) patients (40 Warao indigenous patients [WP] and 22 Creole non-indigenous patients [CP]) and 24 healthy controls (12 Warao indigenous controls [WC] and 12 Creole non-indigenous controls [CC]) at 24 and 48 hours in response to purified protein derivative (PPD) from Mycobacterium tuberculosis. The overall results revealed that testing of CP and CC' PBMCs for TNF-α, IFN-γ, and IL-12p40 production was higher compared with WP and WC' PBMCs after stimulating for 24 and 48 hours (p < 0.0001), within the WP group, the lower productions of IL-12p40 and IFN-γ significantly correlated (r(2) = 0.91, p < 0.01). Although in general there was interindividual variability in the observed responses of Th2 cytokines, especially with IL-4, there was a trend to produce higher PPD-induced IL-5 by WP' PBMCs compared with WC' PBMCs and CP' PBMCs at 24 and 48 hours, respectively. High IL-5 production correlated inversely with low IFN-γ production (r(2) = -0.97, p < 0.002). In conclusion, our results suggest that PPD-induced responses observed in patients from both populations can be divided into two groups: one group that activates Creole' PBMCs to preferentially secrete TNF-α, IL-12p40 and IFN-γ and another group that activates preferential secretion of IL-5 in Warao' PBMCs., (Copyright © 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

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-gamma) secretion and 5 fractions induced increased proliferative response, when compared to unfractionated CFP. In the 11 fractions which showed increased IFN-gamma 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., (Copyright 2009 Elsevier Inc. All rights reserved.)

Seventy-two patients aged 4 to years, who had first detected X-ray changes in the lung or intrathoracic lymph nodes and had received no antituberculous drugs before study, were examined to estimate the sensitivity and specificity of the new skin test Diaskintest® (recombinant CFR-10--ESAT-6 protein produced by Escherichia coli BL21(DE3)/pCFP--ESAT). According to the results of examination and treatment, the patients were divided into 3 groups: 1) 52 patients with active respiratory tuberculosis; 2) 7 patients with residual changes after spontaneously cured tuberculosis; 3) 13 with nontuberculous diseases. By comparing the results of the skin tuberculin test (Mantoux test with 2 TE PPD-L) and Diaskintest®, the mean size of an infiltrate and the percentage of hyperergic, negative and doubtful reactions were estimated in each group, which allowed evaluation of the sensitivity and specificity of the new skin test Diaskintest® versus the conventional skin tuberculin test. The sensitivity of Diaskintest® (Group 1) was 96.2%; false-negative responses with doubtful results being kept in mind were 3.8% (2 patients with limited tuberculous lung lesion). Diaskintest® was the most sensitive in patients with infiltrative pulmonary tuberculosis, in those with disseminated processes or lung tissue decay, as the hyperergic reaction to Diaskintest® just in these cases was more than 80.0%. Its specificity (Group 3) was 84.6%; false-positive results were seen in 15.4%. Determining the activity of first detected posttuberculosis changes--Ghon's focus (Group 2) indicated that Diaskintest® was of more informative value than the Mantoux test with 2 TE PPD-L since its speci- ficity in this case (a negative result) was 100.0%, and that of the Mantoux tuberculin test was 14.3%. The high sensitivity (96.2%) and specificity (84.6%) of the new skin test Diaskintest® permit its use in the comprehensive examination of patients with clinical and radiological signs in a specialized hospital, contributing to the shorter duration of differential diagnosis in difficult diagnostic cases. Diaskintest® enables one to rule out the activity of the process with a high assurance in patients with first detected post-tuberculosis changes.

In Russia, an intradermal Diaskintest® drug has been designed, which is a recombinant tuberculosis allergen based on M. tuberculosis-- specific proteins: ESAT-6 and CFP-10 produced by a genetically modified Escherichia coli culture. Diaskintest® test and Mantoux test with 2TE PPD-L were concurrently carried out in 300 children and adolescents with tuberculosis and followed up in risk groups at a tuberculosis dispensary to determine the sensitivity of the new skin test in active tuberculosis infection. Diaskintest® showed a high sensitivity not only in active tuberculosis, but also in occult, the so-called latent, tuberculosis infection. This is suggested by the following evidence. The high percentage (83.8%) of positive responses to Diaskintest® is noted in children and adolescents with tuberculosis, receiving an intensive course of chemotherapy. Negative tests were observed only in minor forms at the resolution stage. In the children who had completed treatment, positive tests were seen in 78.3%, moreover in those with prior tuberculosis of intrathoracic lymph nodes; negative tests were observed not earlier than 18 months after start of treatment. The highest sensitivity of Diaskintest® was shown in children with early primary tuberculosis infection and through family contact with bacteria-excreting subjects (91.7%). These children may be judged with the highest assurance to have latent tuberculosis infection, the population of which is in an active state at the moment of the study. The children with early primary tuberculosis infection, but in no family contact with bacteria-excreting individuals, showed a lower percentage of positive responses to Diaskintest® both before (37.5%) and after (10%) treatment, which suggests that there must be a lower bacterial burden in the child. A high percentage of positive responses to Diaskintest® (76.2%) were found in subjects with hyperergic reactions to tuberculin. These were in only 16.7% in the group of patients receiving preventive therapy. In children and adolescents with a persistent positive Mantoux test (for more than 3 years), the response to Diaskintest® was negative in most cases since in early infection when mycobacteria propagated, the reaction to the drug was positive, but as 3 years pass the probability of the infection transition to the persistence stage is high--at that time the response to Diaskintest® becomes negative. Diaskintest® induces no delayed hypersensitivity associated with BCG vaccination, suggesting its high specificity. There were no positive reactions in patients with nonspecific lung diseases.

Background: The incidence of allergic diseases is increasing in industrialized countries and the immunological mechanisms leading to tolerance or allergy are poorly understood. Cytokines with suppressive abilities and CD4(+)CD25(+) regulatory T cells have been suggested to play a central role in allergen-specific responses. The aim was to determine whether major grass allergens induce production of suppressive cytokines in allergic and healthy subjects and to examine the inhibitory effect of these cytokines on allergic responses., Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy and grass-allergic donors and stimulated with the major grass allergens Phl p 1 or Phl p 5. The effects of endogenous IL-10 and/or TGF-beta on proliferation and cytokine production were determined by use of blocking antibodies. In addition, the number of CD4(+)CD25(+) T cells and their expression of chemokine receptors were investigated by flow cytometry., Results: Phl p 1 and Phl p 5 induced IL-10 production, which down-regulated proliferation and cytokine production, in PBMC cultures from atopic but not from non-atopic donors. Comparable frequencies of CD4(+)CD25(+) T cells were present in PBMCs in the two groups, but fewer cells from atopic donors were CD4(+)CD25(+)CCR4(+) and more cells were CD4(+)CD25(+)CLA(+) compared to healthy donors., Conclusion: Allergen-specific responses of grass allergic patients but not in non-atopic subjects are influenced by regulatory cytokines produced in response to the important allergens. Differences in CD4(+)CD25(+) T cell expression of chemokine receptors in allergic compared to non-atopic donors could suggest that the homing of CD4(+)CD25(+) T cells is important for the regulation of allergen-specific responses., (2010 S. Karger AG, Basel.)

Approximately 20% of persons living in areas hyperendemic for tuberculosis (TB) display persistent lack of tuberculin skin test (TST) reactivity and appear to be naturally resistant to infection by Mycobacterium tuberculosis. Among those with a positive response, the intensity of TST reactivity varies greatly. The genetic basis of TST reactivity is not known. We report on a genome-wide linkage search for loci that have an impact on TST reactivity, which is defined either as zero versus nonzero (TST-BINa) or as extent of TST in millimeters (TST-quantitative trait locus [QTL]) in a panel of 128 families, including 350 siblings, from an area of South Africa hyperendemic for TB. We detected a major locus (TST1) on chromosomal region 11p14 (P = 1.4 x 10(-5)), which controls TST-BINa, with a lack of responsiveness indicating T cell-independent resistance to M. tuberculosis. We also detected a second major locus (TST2) on chromosomal region 5p15 (P < 10(-5)), which controls TST-QTL or the intensity of T cell-mediated delayed type hypersensitivity (DTH) to tuberculin. Fine mapping of this region identified SLC6A3, encoding the dopamine transporter DAT1, as a promising gene for further studies. Our results pave the way for the understanding of the molecular mechanisms involved in resistance to M. tuberculosis infection in endemic areas (TST1) and for the identification of critical regulators of T cell-dependent DTH to tuberculin (TST2).

Tuberculosis (TB) represents one of the leading killers among all infectious disease. Protection against TB depends on the activation of T-helper type I (Th1) immune response. Carbon nanotubes (CNTs) have attracted considerable attention because of their potential applications as new nanovehicle. In the current study, tuberculin purified protein derivative (PPD) was conjugated to carboxylated single-walled carbon nanotubes (SWCNTs). Cytotoxicity of the carboxylated SWCNT and SWCNT-PPD conjugate was analyzed with MTT assay and by reactive oxygen species (ROS) and nitric oxide (NO) generation. Male BALB/c mice were immunized with BCG, PPD, SWCNT-PPD conjugate and PPD in complete Freund's adjuvant (CFA). Induction of cellular immune response was analyzed by measuring the levels of Th1 cytokines (IFN-gamma and IL-12) and Th2 cytokines (IL-10 and IL-5). Immunization with non-conjugated PPD or PPD in Freund's adjuvant induced a Th2 cytokine response while immunization with BCG resulted to a mixed Th1/Th2 cytokine response. In contrast, PPD in conjugation with SWCNT generated preferentially a Th1-type cytokine response in the absence of potential cytotoxic effects.

Despite its use widely as a therapeutic agent, and proposed use as vaccine adjuvant, the effect of IFNalpha on T cell function is poorly understood. As a pleiotropic innate cytokine produced rapidly in response to pathogens, it is well placed to impinge on specific immune responses. The aim of this study was to examine the impact of IFNalpha on the function of human memory CD4(+) T cells using the recall Ags purified protein derivative, tetanus toxoid, and hemagglutinin. IFNalpha administered either in vivo or added exogenously in vitro tended to enhance proliferative responses of purified protein derivative-specific T cells in marked contrast to the other cognate populations whose responses were often diminished. Purifying the memory CD4(+)CD45RO(+) T cells confirmed IFNalpha acted directly on these cells and not via an intermediate. The T cells could be divided into two broad categories depending on how IFNalpha effected their responses to cognate Ag: 1) enhanced proliferation and a striking increase in IFNgamma-production compared with smaller increases in IL-10 (increased ratio of IFNgamma:IL-10), and 2) neutral or diminished proliferation coupled with a smaller increase in IFNgamma relative to the increase in IL-10 (reduced IFNgamma:IL-10 ratio). IFNalpha has a role in modifying memory T cell responses when they are exposed to cognate Ag and may be important in vaccination strategies designed to augment particular Th memory responses.

Cytokine messenger RNA (mRNA) expression was investigated in the spleen and lung digest cells of bacillus Calmette-Guérin (BCG)-vaccinated and non-vaccinated guinea pigs following low-dose, pulmonary exposure to virulent Mycobacterium tuberculosis. After purified protein derivative (PPD) stimulation, the levels of lung cell interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and spleen cell interleukin-12 (IL-12) p40 mRNAs were significantly increased in the non-vaccinated M. tuberculosis-infected guinea pigs compared to the BCG-vaccinated guinea pigs. In contrast, the expression of anti-inflammatory transforming growth factor-beta and IL-10 mRNAs was significantly enhanced in the spleens of BCG-vaccinated animals. Despite the presence of protective cytokine mRNA expression, the non-vaccinated guinea pigs had significantly higher lung and spleen bacterial burdens. In contrast, BCG-vaccinated guinea pigs controlled the bacterial multiplication in their lungs and spleens, indicating that both protective as well as anti-inflammatory cytokine responses are associated with a reduction in bacteria. In addition, lung digest cells from non-vaccinated guinea pigs contained a significantly higher percentage of neutrophils, CD3(+) and CD8(+) T cells, while the percentage of macrophages was increased in the BCG-vaccinated animals. Total and purified lung digest T cells co-cultured with lung macrophages (LMøs) proliferated poorly after PPD stimulation in both non-vaccinated and BCG-vaccinated animals while robust proliferation to PPD was observed when T cells were co-cultured with peritoneal macrophages (PMøs). Macrophages within the lung compartment appear to regulate the response of T cells irrespective of the vaccination status in guinea pigs. Taken together, our results suggest that type I cytokine mRNA expression is not associated with vaccine-induced protection in the low-dose guinea pig model of tuberculosis.

Background: The gradual aging of population has increased the number of elderly patients receiving kidney transplants. In elderly transplant recipients, careful immunosuppression has to be maintained to avoid both rejection and adverse effects. Clinical protocols after kidney transplantation include use of the calcium channel antagonist diltiazem to ameliorate the hypertensive effect and nephrotoxicity of the immunosuppressant agent ciclosporin (cyclosporine)., Objective: Since immune response can be impaired by senescence, we evaluated the influence of diltiazem on lymphocyte proliferation both alone and in the presence of ciclosporin in younger versus older subjects., Methods: Peripheral blood mononuclear cells (PBMC) from younger healthy donors (aged 19-24 years) and older subjects (aged 59-65 years) were isolated and stimulated with mitogens, recombinant human interleukin-2 (IL-2), purified protein derivative (PPD) antigen from Mycobacteriumtuberculosis, and anti-CD3 monoclonal antibody (alphaCD3 moAb) in the presence or absence of 10(-4), 10(-5), 10(-6), 10(-7) mol/L concentrations of diltiazem. In some experiments, lymphocytes from younger and older subjects were used as responder cells in an allogeneic mixed lymphocyte reaction (MLR) in the presence of different concentrations of diltiazem and 10 ng/mL of ciclosporin., Results: We found that PBMC from older subjects were more susceptible to immunosuppression induced by low concentrations of diltiazem when mitogens were used to stimulate cells. In particular, when pokeweed mitogen was used, diltiazem 10(-7) mol/L was associated with statistically significant immunosuppression in older subjects compared with younger subjects. This effect was not observed when IL-2, PPD antigen and alphaCD3 moAb were used as stimulators. Moreover, in the allogeneic MLR, we found no differences between younger and older subjects when the 10(-)(5), 10(-)(6) and 10(-7) mol/L concentrations of diltiazem were used alone or in the presence of ciclosporin. Only addition of the supratherapeutic 10(-4) mol/L concentration of diltiazem to ciclosporin was associated with statistically significant immunosuppression in older versus younger subjects., Discussion: Our results show that PBMC from older subjects are no more susceptible than PBMC from younger subjects to therapeutic doses of diltiazem when T-cell receptors are directly or indirectly involved. On the contrary, when PBMC activation was not mediated by T-cell receptor involvement, as in the case of pokeweed mitogen, susceptibility to a therapeutic concentration of diltiazem in older subjects was enhanced. Moreover, co-administration of therapeutic doses of diltiazem and ciclosporin in an MLR showed no significant differences between younger and older subjects in an in vitro model of lymphocyte response to allogeneic transplantation., Conclusion: Since we found no variations in immunosuppression between older and younger subjects when therapeutic doses of diltiazem were added to ciclosporin, our data do not discourage the use of diltiazem in older kidney transplant recipients receiving ciclosporin therapy.

We pursued a breeding strategy intended to generate disease-resistant mice with exclusive expression of the H-2(u)-restricted myelin basic protein (MBP) 1-11 peptide-specific transgenic (Tg) T-cell receptor (TCR) on the T-cell-deficient RAG1KO (H-2(b)) background. Utilizing specific screening assays for the offspring, analyses of the F1 intercross and subsequent crosses revealed that the TgTCR-associated clonotypic marker detected by the 3H12 mAb could be found only in association with the H-2(b) homozygous background in offspring possessing a functional rag1 gene. Moreover, expression of the MBP-specific TgTCR could not be found in H-2(b) homozygous offspring that were RAG1 deficient (rag1(-/-)). PCR analysis of genomic DNA from these 3H12-negative offspring verified the presence of the TCR transgenes. Thus, the presence of a functional rag1 gene was required for the expression of the MBP-specific TgTCR on the H-2(b) background. Given the role for RAG1, the results have important implications for T-cell repertoire development., (2008 Wiley-Liss, Inc.)

Background: Bovine tuberculosis (BTB) caused by Mycobacterium bovis continues to cause substantial losses to global agriculture and has significant repercussions for human health. The advent of high throughput genomics has facilitated large scale gene expression analyses that present a novel opportunity for revealing the molecular mechanisms underlying mycobacterial infection. Using this approach, we have previously shown that innate immune genes in peripheral blood mononuclear cells (PBMC) from BTB-infected animals are repressed in vivo in the absence of exogenous antigen stimulation. In the present study, we hypothesized that the PBMC from BTB-infected cattle would display a distinct gene expression program resulting from exposure to M. bovis. A functional genomics approach was used to examine the immune response of BTB-infected (n = 6) and healthy control (n = 6) cattle to stimulation with bovine tuberculin (purified protein derivative - PPD-b) in vitro. PBMC were harvested before, and at 3 h and 12 h post in vitro stimulation with bovine tuberculin. Gene expression changes were catalogued within each group using a reference hybridization design and a targeted immunospecific cDNA microarray platform (BOTL-5) with 4,800 spot features representing 1,391 genes., Results: 250 gene spot features were significantly differentially expressed in BTB-infected animals at 3 h post-stimulation contrasting with only 88 gene spot features in the non-infected control animals (P < or = 0.05). At 12 h post-stimulation, 56 and 80 gene spot features were differentially expressed in both groups respectively. The results provided evidence of a proinflammatory gene expression profile in PBMC from BTB-infected animals in response to antigen stimulation. Furthermore, a common panel of eighteen genes, including transcription factors were significantly expressed in opposite directions in both groups. Real-time quantitative reverse transcription PCR (qRT-PCR) demonstrated that many innate immune genes, including components of the TLR pathway and cytokines were differentially expressed in BTB-infected (n = 8) versus control animals (n = 8) after stimulation with bovine tuberculin., Conclusion: The PBMC from BTB-infected animals exhibit different transcriptional profiles compared with PBMC from healthy control animals in response to M. bovis antigen stimulation, providing evidence of a novel gene expression program due to M. bovis exposure.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a live vaccine that has been used in routine vaccination against tuberculosis for nearly 80 years. However, its efficacy is controversial. The failure of BCG vaccination may be at least partially explained by the induction of poor or inappropriate host responses. Dendritic cells (DCs) are likely to play a key role in the induction of immune response to mycobacteria by polarizing the reactivity of T lymphocytes toward a Th1 profile, contributing to the generation of protective cellular immunity against mycobacteria. In this study we aimed to investigate the production of Th1 and Th2 cytokines by naive CD4+ T cells to mycobacterial antigen-pulsed DCs in the group of young, healthy BCG vaccinated volunteers. The response of naive helper T cells was compared with the response of total blood lymphocytes. Our present results clearly showed that circulating naive CD45RA+CD4+ lymphocytes from BCG-vaccinated subjects can become effector helper cells producing IFN-gamma and IL-5 under the stimulation by autologous dendritic cells presenting mycobacterial protein antigen-PPD or infected with live M. bovis BCG bacilli.

Dendritic cells (DCs) are the most powerful antigen presenting cells, capable of inducing T-dependent immune responses even in naive T cells. DCs are of special interest as cellular adjuvants for immunity induction in clinical settings and several methods for their generation and maturation are recently under investigation. The present study was set out to define the effects of PPD (Purified Protein Derivative), a mycobacterial extract used in the tuberculin skin test, on in vitro differentiation and maturation of human monocyte derived dendritic cells. Immature DCs were prepared from the peripheral blood monocytes of healthy volunteers by culturing in a medium supplemented with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). The resultant cells were then stimulated with PPD extract and their properties such as cell morphology and the expression of key surface molecules were compared with tumor necrosis factor-alpha (TNF-alpha) stimulated immature DCs. Our results suggest that mycobacterial purified extract is as potent as TNF-alpha, a well-established DC stimulator, in induction of maturation in human monocyte derived DCs. We also ruled out the contribution of lipopolysaccharide (LPS) and beta-glucan contamination in maturation effect of PPD preparations. So, PPD as an examined safe material for in vivo consumption could be used to stimulate DC maturation in DC based immunotherapy protocols.

Macrophages and dendritic cells are involved in the immune response to Mycobacterium tuberculosis (Mtb). Such a response, although extensively studied using animal models and cells from human blood, has not been characterized in cells from pulmonary hilar lymph nodes (PHLN). We characterized populations of myeloid APC from PHLN and determined their expression of CCR2, CCR5, CCR7, CD40, CD54, CD80, and CD86 as well as the cytokine/chemokine microenvironment before and after purified protein derivative (PPD) and mannosilated lipoarabinomannan (ManLAM) stimulation. Results show that there are at least three APC populations in PHLN, defined as CD14highHLA-DRlow/-, CD14dimHLA-DRdim, and CD14-HLA-DRhigh/dendritic cells (DC), with the largest number represented by CD14dimHLA-DRdim cells (where dim indicates intermediate levels). CD14-HLA-DRhigh/DC expressed higher levels of costimulatory molecules and lower levels of CCR2 and CCR5, but all cell populations showed similar CCR7 levels. PPD and ManLAM specifically down-regulated CCR2 expression but not that of CCR5 and CCR7, and such down-regulation was observed on all APC populations. Mtb Ag did not affect the expression of costimulatory molecules. PPD but not ManLAM specifically induced MCP-1/CCL2 production, which was likely associated with the induction of IFN-gamma because this cytokine was highly induced by PPD. We characterized, for the first time, different APC from human PHLN and show that Mtb Ag exert fine and specific regulation of molecules closely associated with the immune response to Mtb infection. Because knowledge of this response in secondary lymphoid tissues is still poorly understood in humans, such studies are necessary and important for a better understanding of lymphoid cell microenvironment and migrating capacities and their role in the immunopathogenesis of tuberculosis.

Background: Gammadelta T cells have been described as one of immune regulators in patients with infection, malignancy, and allergy., Objective: To elucidate the ability of gammadelta T cells as an allergen immunotherapy candidate, the effectiveness of human gammadelta T cells in allergen-specific T-helper type 2 (Th2)-type T cells was evaluated in vitro., Methods: House dust mite-specific Th2-type T cell clones, Bacillus Calmette-Guerin (BCG)-specific Th1-type T cell clones, and gammadelta T cell lines were established from the peripheral blood mononuclear cells of two patients with allergic rhinitis. The effectiveness of gammadelta T cells and BCG-specific Th1-type T cell clones in the modulation of allergen-specific Th2 cells in terms of their cytokine productions was evaluated., Results: In response to cognate antigens, the gammadelta T cell lines demonstrated a proliferation and production of IFN-gamma that exceeded that of BCG-specific Th1-type T cell clones (mean stimulation index: 14.5 vs. 2.8, mean IFN-gamma: 130.5 vs. 10.0 pg/mL). When the gammadelta T cell lines and mite-allergen-specific Th2 clones were co-cultured with each other, only the levels of IL-4 (mean, -87%) decreased, but not the levels of IL-5 and IL-13, with an increasing concentration of gammadelta T cell antigen and IFN-gamma production (mean, +730%)., Conclusion: These results demonstrated that gammadelta T cells derived from allergic patients might thus have a partial ability to modulate allergen-specific Th2-skewed immunity.

Objective: Children <5 years old are at increased risk of miliary/meningeal tuberculosis, but the immunologic factors that place them at risk are unknown. BCG vaccine protects against miliary/meningeal tuberculosis, but the mechanism of protection is unknown. We assessed for abnormalities in immune response associated with miliary/meningeal or pulmonary tuberculosis in young children., Patients and Methods: We conducted a case-control study among HIV-seronegative Brazilian children who were <5 years old. Case subjects had previous culture-confirmed or clinical miliary/meningeal tuberculosis. There were 2 sets of control subjects: those with culture-confirmed pulmonary tuberculosis and purified protein derivative-positive household contacts. All of the children had completed treatment. Peripheral blood mononuclear cells were stimulated (phytohemagglutinin, phytohemagglutinin + interleukin 12, lipopolysaccharide, lipopolysaccharide + interferon-gamma, and purified protein derivative), and cytokine responses (interleukin 1beta, interleukin-4, interleukin-6, interleukin-8, interleukin 10, interleukin 12, interferon-gamma, tumor necrosis factor-alpha, and monocyte chemoattractant protein 1) were quantified by bead-based assay. Median cytokine responses were compared by the Kruskal-Wallis test. Multivariate analysis of variance accounted for multiple comparisons., Results: There were 18 case subjects with miliary/meningeal tuberculosis, 28 pulmonary control subjects, and 29 purified protein derivative-positive control subjects. The median age was 4.2 years. There was no difference in case and control subjects by age, gender, race, BMI, or median CD4 count. Twelve (67%) of 18 case subjects, 26 (93%) of 28 pulmonary control subjects, and 28 (97%) of 29 purified protein derivative-positive subjects had received BCG vaccine. No cytokine defects were identified in case subjects with miliary/meningeal tuberculosis compared with either set of control subjects. Pulmonary control subjects had uniformly higher monocyte chemoattractant protein 1 levels than case subjects with miliary/meningeal tuberculosis and purified protein derivative-positive control subjects, both at rest and with lipopolysaccharide, lipopolysaccharide + interferon-gamma, and purified protein derivative stimulation. Pulmonary control subjects did not have a higher frequency of allele G in the -2518 monocyte chemoattractant protein 1 promoter polymorphism. Case subjects with miliary/meningeal tuberculosis who had received BCG vaccine (n = 12) had lower stimulated interleukin 8 production than children who did not receive BCG vaccine (n = 6)., Conclusions: Children with previous miliary/meningeal tuberculosis did not have a major defect in the cytokine pathways studied. Increased monocyte chemoattractant protein 1 levels were associated with pulmonary disease, occurred despite BCG vaccination, and were not associated with a polymorphism in the monocyte chemoattractant protein 1 promoter.

The pathogenesis of tuberculosis-causing Mycobacterium bovis is largely due to its ability to enter and survive in alveolar macrophages. Its mechanism of entry, mediated by proteins encoded by mammalian cell entry (mce) genes, is important for its pathogenesis. Here we focussed on the role of the Mce4A protein in the pathogenesis of M. bovis in cattle. Cell livability decreased in a dosage-dependent manner when Mce4A proteins were used to stimulate alveolar macrophages, which suggested that the recombinant Mce4A protein significantly inhibited alveolar macrophage activity. To test whether Mce4A modulates the gene expression profile of alveolar macrophages, alveolar macrophages were stimulated by Mce4A protein and other proteins/ligands (such as MtbPPD, MbPPD, and BCG), followed by real-time RT-PCR assay for the mRNA expression level of TNF-alpha, iNOS, IL-6, and IL-12. The results showed that the expression of TNF-alpha, iNOS, and IL-6 in alveolar macrophages was up-regulated by stimulation with the recombinant Mce4A protein of M. bovis; in contrast, expression of IL-12 was unaffected. MbPPD and BCG up-regulated the mRNA expression of TNF-alpha, iNOS, IL-6, and IL-12 (P < 0.05), whereas MtbPPD stimulated the mRNA expression of TNF-alpha, IL-6, and IL-12 with no effect on iNOS. This study suggests that Mce4A proteins may induce the body's inflammation response to M. bovis and therefore may play an important role in the immune response.

Buruli ulcer (BU), caused by Mycobacterium ulcerans, is the third most common mycobacterial infection in immunocompetent humans besides tuberculosis and leprosy. We have compared by ex vivo enzyme-linked immunospot analysis interferon-gamma (IFN-gamma) responses in peripheral blood mononuclear cells (PBMC) from BU patients, household contacts, and individuals living in an adjacent M. ulcerans nonendemic region. PBMC were stimulated with purified protein derivative (PPD) and nonmycobacterial antigens such as reconstituted influenza virus particles and isopentenyl-pyrophosphate. With all three antigens, the number of IFN-gamma spot-forming units was reduced significantly in BU patients compared with the controls from a nonendemic area. This demonstrates for the first time that M. ulcerans infection-associated systemic reduction in IFN-gamma responses is not confined to stimulation with live or dead mycobacteria and their products but extends to other antigens. Interleukin (IL)-12 secretion by PPD-stimulated PBMC was not reduced in BU patients, indicating that reduction in IFN-gamma responses was not caused by diminished IL-12 production. Several months after surgical excision of BU lesions, IFN-gamma responses of BU patients against all antigens used for stimulation recovered significantly, indicating that the measured systemic immunosuppression was not the consequence of a genetic defect in T cell function predisposing for BU but is rather related to the presence of M. ulcerans bacteria.

Responses to tuberculin in Zebu cattle of the transhumant pastoral farming system in Karamoja region and Nakasongola district in the north-eastern and mid-central regions in Uganda, respectively, were investigated using a comparative intradermal tuberculin skin test. Of the 1864 cattle tested from 30 large units (superherds) in Karamoja and 7 herds in Nakasongola, a total of 28 animals from 19 herds (51.4%) tested positive. Inter-district tuberculin reactor prevalence variations seemed to be influenced by climate, with impact on both the management patterns and transmissibility of agent. High herd tuberculin reactor prevalence (51.4%) was attributed to widespread contacts and mixing of animals between herds. Low individual animal tuberculin test positivity (mean = 1.4%) was attributed to low transmissibility of the agent under the Karamoja climate, which is semi-arid, and to increased resistance due to non-specific response to environmental mycobacteria and natural selection, since there was no active control against bovine tuberculosis. Owing to similarities in management practices in Karamoja and widespread risk factors, it was difficult to identify which were more important, but variations in sources of drinking water pointed to provision of lake and borehole water during dry season as reducing the risk. Positive bovine tuberculin reactor prevalence and skin reactor status were related to age.

The German medical bacteriologist Robert Koch is commonly considered one of the founding fathers of medical bacteriology. His investigations into the aetiology of tuberculosis uncovered the pathogen of this condition, the tubercle bacillus today known as Mycobacterium tuberculosis, in 1882. This work can be seen as a cornerstone of contemporary medical bacteriology, its technologies and methods. It has often been asked how such successful research connected to the tuberculin episode of 1890/91, when Koch produced a medicine for that disease, which spectacularly failed when applied in practice. The analysis concentrates on the path of mostly experimental investigations which Koch followed between 1882 and 1890. From Koch's laboratory notes it becomes clear that tuberculin therapy did in fact work in Koch's laboratory, even though it failed to do so almost anywhere else. The clue to this contradictory picture lies in the peculiar nature of Koch's understanding of tuberculosis as a disease e.g. his reliance an animal experiments, which essentially differed from what many of his contemporaries held as essentials of that condition.

IFN-gamma responses to Mycobacterium tuberculosis antigens ESAT-6 and CFP-10 have been proposed as specific markers of M. tuberculosis infection. Monokine induced by gamma interferon (MIG/CXCL9) has been shown to be expressed by IFN-gamma stimulated mononuclear cells and to attract activated T-cells through the chemokine receptor CXCR3. Since MIG is induced early in the response to IFN-gamma, measuring MIG may provide an interesting marker to assess downstream IFN-gamma induced responses, in contrast to assays that mainly focus on quantifying production of IFN-gamma per se. We, therefore, investigated MIG and IFN-gamma responses to a fusion protein of ESAT-6 and CFP-10, and compared responses to the conserved mycobacterial antigen 85B (Ag85B) and purified protein derivative (PPD) of M. tuberculosis, in 29 BCG vaccine controls and 24 TB patients. IFN-gamma secreting cells were determined by ELISPOT, and MIG production was measured by ELISA and flow cytometry. Production of MIG in response to ESAT-6/CFP-10, Ag85B and PPD correlated overall with increased numbers of IFN-gamma secreting cells (r=0.55, P<0.0001). A significant increase was noted among patients compared to controls in the secretion of IFN-gamma and MIG following stimulation with ESAT-6/CFP-10 or PPD (P<0.05). Moreover, MIG intracellular expression was higher in TB patients compared to BCG vaccines (P<0.05) in response to ESAT-6/CFP-10 or PPD. We conclude that MIG production correlates significantly with enhanced T-cell IFN-gamma production induced by M. tuberculosis-specific antigens ESAT-6/CFP-10. These results point to MIG as a potential novel biomarker that may be helpful in assessing downstream responses induced by IFN-gamma in TB.

Rationale and Objectives: Sarcoidosis is a granulomatous disease of unknown etiology characterized by a helper T-cell type 1-mediated process. Previously we demonstrated a role for interleukin-18 in sarcoidosis. Here we examine the regulation of interleukin-18 in this condition., Methods: Cytokine levels in sarcoid epithelial lining fluid were measured by ELISA. We examined interleukin-18 promoter activity and mRNA and protein levels in the epithelial lining fluid of individuals with active sarcoidosis, and of individuals recovered from sarcoidosis, in response to purified protein derivative of Mycobacterium tuberculosis, beryllium sulfate, zirconium sulfate, aluminum sulfate, and lipopolysaccharide. Endotoxin levels in the epithelial lining fluid of individuals with sarcoidosis, individuals recovered from sarcoidosis, and control subjects were assessed by Limulus amebocyte lysate analysis. Allele-specific polymerase chain reaction was used to genotype 94 patients with sarcoidosis and 97 control subjects for the interleukin-18 -607(A/C) polymorphism. Species-specific polymerase chain reaction identified bacterial DNA in fluid samples., Results: Epithelial lining fluid from active sarcoids contained elevated levels of interleukin-18, interferon-gamma, and interleukin-12 compared with recovered patients and also contained significantly higher levels of endotoxin. Depletion of endotoxin from this epithelial lining fluid reduced its effect on the human interleukin-18 promoter in vitro. There was a higher frequency of the -607C allele and -607(C/C) genotype in the sarcoidosis population compared with control subjects; however, this was not associated with a functional response to endotoxin treatment. Finally, bacterial 16S rRNA from Haemophilus influenzae and Moraxella catarrhalis was detected in sarcoid fluid samples., Conclusions: The pathogenesis of sarcoidosis is propagated through the actions of a helper T-cell type 1-driven response. This study shows that gram-negative bacteria may contribute to this effect by upregulating interleukin-18 expression.

Objectives: The aim of the present study was to determine the immune response profile that differentiates patients with newly diagnosed (non-treated) pulmonary tuberculosis from multidrug-resistant (MDR) ones, as well as from healthy, tuberculin positive individuals., Methods: Lymphocytes proliferative response to non-specific mitogen (PHA) and PPD were evaluated by 3H thymidine incorporation and cytokines were quantified using an ELISA assay., Results: Patients with active disease showed a diminished proliferative response to PHA and PPD, while multidrug-resistant patients showed a diminished proliferative response to PHA, but a normal response to PPD. The cytokine production of newly diagnosed patients was characterized by a diminished production of IFNgamma and normal production of transforming growth factor (TGFbeta), while MDR patients revealed a normal production of IFNgamma accompanied by an increase in TGFbeta., Conclusions: The production of significant amounts of TGFbeta in MDR patients leads to a poor immune response and may contribute to the resistance of tuberculosis patients to drugs.

The gamma interferon assay was evaluated for diagnosis of paratuberculosis in goats with special emphasis on false positive reactions. Four categories of herds were tested: (A) herds that had a history of paratuberculosis, had given positive Mycobacterium avium subsp. paratuberculosis fecal samples and were vaccinated against paratuberculosis; (B) herds that had been vaccinated but had never shown clinical signs of paratuberculosis nor given positive M. a. paratuberculosis fecal samples; and (C) non-vaccinated herds without paratuberculosis. To extend the analysis of samples from young goats free of paratuberculosis, animals less than 18 months of age from non-vaccinated herds without paratuberculosis, category D, were included. Heparinized blood was stimulated with purified protein derivate (PPD) from M. a. paratuberculosis for 24 h and plasma was assayed for the presence of gamma interferon. Results were recorded as the difference between OD values of PPD stimulated and control samples. Vaccinated animals from herds with paratuberculosis, category A, showed significant higher gamma interferon responses than animals from vaccinated herds without paratuberculosis, category B. In both these groups the responses were correlated to age with higher responses in younger animals. Some of the vaccinated animals in herds without paratuberculosis had a gamma interferon response lasting for several years, which demonstrate a long lasting interference with diagnostic testing in vaccinated goats. Only three of the 121 non-vaccinated animals free of paratuberculosis in category C had responses against PPD (corrected OD values at 0.2, 0.24 and 0.5), and none of the 255 young animals in category D had corrected OD values exceeding 0.2. This indicates that false positive reactions do not appear to the same extent in young goats as in young cattle. We conclude that the low responses of non-infected goats could make the gamma interferon assay useful in monitoring the paratuberculosis status of non-vaccinated herds. However, more information about the early gamma interferon responses of naturally infected goats and the presence of false negative samples are needed.

Lymphatic filariasis is a disease characterized by immune dysregulation involving APC and T cell populations. To assess the contribution of TLR in mediating this dysregulation, we examined the expression of TLR1, TLR2, TLR4, and TLR9 on B cells and monocytes of filaria-infected and uninfected individuals. Baseline expression of TLR was significantly lower in B cells but not in monocytes of the filaria-infected group compared with the uninfected group. Upon stimulation with filarial Ag, a diminished up-regulation of TLR was observed in both B cells and monocytes of infected individuals. Finally, stimulation of B cells and monocytes with TLR ligands resulted in decreased B cell and monocyte activation/cytokine production, indicating a state of immune tolerance. This dysregulation is associated with diminished CD4(+) T cell production of IFN-gamma and IL-5. The diminished expression and function of TLR is thus a likely consequence of chronic Ag stimulation and could serve as a novel mechanism underlying the dysfunctional immune response in filariasis.