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Purified protein derivative (PPD) or tuberculin skin testing is used to identify infected individuals with Mycobacterium tuberculosis (Mtb) and to assess cell-mediated immunity to Mtb. In the present study, we compared PBMC cultures in the presence of tuberculin or Candida antigens using cytokine bead arrays and RNA microarrays. Measurements of different cytokines and chemokines in supernatants of PMBC cultures in the presence of PPD showed increased levels of interferon (IFN)-gamma in active tuberculosis infection (ATBI) and latent TB infected (LTBI) compared to controls, and increased levels of TNF-alpha in ATBI compared with LTBI. Also, we found increase of IL-6 in cultures of PPD positive and controls but not in the cultures with Candida. We also report the molecular signature of tuberculosis infection, in ATBI patients, the following genes were found to be up-regulated and absent in LTBI individuals: two kinases (JAK3 and p38MAPK), four interleukins (IL-7, IL-2, IL-6, and IFNbeta1), a chemokine (HCC-4) a chemokine receptor (CxCR5), two interleukin receptors (IL-1R2 and IL-18R1), and three additional ones (TRAF5, Smad2, CIITA, and NOS2A). By contrast, IL-17 and IGFBP3 were significantly up-regulated in LTBI. And, STAT4, GATA3, Fra-1, and ICOS were down-regulated in ATBI but absent in LTBI. Conversely, TLR-10, IL-15, DORA, and IKK-beta were down-regulated in LTBI but not in ATBI. Interestingly, the majority of the up-regulated genes found in ATBI were found in cultures stimulated with tuberculin (PPD) or Candida antigens, suggesting that these pathogens stimulate similar immunological pathways. We believe that the molecular signature distinguishing active from latent tuberculosis infection may require using cytokine bead arrays along with RNA microarrays testing cell cultures at different times following in vitro proliferation assays using several bacterial antigens and PPD.

We have previously described that antibodies and T cells against myelin basic protein (MBP) rose under conditions to induce acute experimental autoimmune encephalomyelitis (EAE) bind other proteins present in the synaptosomal fraction, some of them identified as synapsin I. The aim of this study was to evaluate whether anti-MBP T-cell lines can be also activated by synapsin. The analysis of rat anti-MBP T-cell lines cultured with each antigen showed that these cells responded also to purified rat synapsin and to the amino terminal portion of this protein. This recognition originated a proliferative response with a concomitant pattern of cytokine secretion similar to that induced by MBP itself implicating that this recognition would be mediated by the T-cell receptor. On the other hand, anti-synapsin T-cell lines were not capable of responding to MBP stimulation. Therefore, the immunological cross-reactivity between both proteins occurs only in one direction and these cross-reactive cells would be elicited only in animals sensitized with MBP. A possible implication of immunological agents against MBP cross-reactive with extra-myelin proteins in the process of EAE is considered.

The clinical characteristics of pulmonary tuberculosis and its detection methods were studied in patients with hyperergic (Group 1, n = 84) and another (Group 2, n = 75) tuberculin susceptibility. The groups were identical in clinical, X-ray, and laboratory characteristics. The major clinical forms were infiltrative (35.2%) and thoracic lymphatic tuberculosis (34%). Mass tuberculin diagnosis revealed 64.7% of children with hyperergic susceptibility, which confirms the topicality of this technique in the early detection of tuberculosis in children. This technique was realized in adolescents with hyperergic susceptibility to a lesser degree (12.1%). Overall, 55.9% of patients with hyperergic tuberculin susceptibility (mainly adolescents) were identified by X-ray studies performed routinely and if there were clinical indications. In these cases, individual tuberculin diagnosis was used when X-ray changes were found.

In recent years, biological agents have emerged as the most popular drugs for the treatment of rheumatoid arthritis (RA). The most frightening side effects of biological agents are infections, with tuberculosis being the leader. On account of the fact that biological agents have been used widespread, a number of algorithms have been developed to search latent tuberculosis. Among these algorithms, the most popular is the purified protein derivatives (PPD) test which is based upon late sensitivity reaction. The objective of this trial is to investigate the relevance of PPD response for the disease in RA patients. A total of 149 subjects (80 patients, 69 healthy), 35 RA patients who have not been treated before, 23 RA and 22 AS patients who are candidates for biological agents and being treated with immunosuppressive drugs, and 69 healthy subjects, have been included in this trial. Swelling joints, number of tender joints, visual analog scale, erythrocyte sedimentation rate, C-reactive protein, and rheumatoid factor were recorded. PPD was performed using the Mantoux method and was measured 72 h later. Statistically significant lower PPD values were determined in untreated RA patients compared to PPD values of treated RA and AS patients and healthy subjects. No correlation was determined between disease activity score 28 activity and PPD values in untreated and treated groups. Similarly, there was no correlation between acute phase reactants and PPD. Lower PPD responses in patients not being treated with immunosuppressive are due to the disease itself, rather than to the drugs being used. It is also important to interpret PPD results in early RA patients with suspicion, when it is intended to start anti tumor necrosis factor therapy.

This study examined the performance of a tuberculosis (TB)-specific enzyme-linked immunospot assay in 48 patients awaiting liver transplantation. They were tested with T-SPOT.TB, tuberculin skin test (TST), and lymphocyte transformation test (LTT) using tuberculin as a stimulus. A questionnaire was used to gain information on TB exposure. Four patients were defined as positive by T-SPOT.TB, 6 by TST, and 28 by LTT. The patients displaying positive results to T-SPOT.TB were also positive in the TST and LTT. We considered them to have latent TB because they were repeatedly (two or three times) positive to the T-SPOT.TB and reported TB exposure. Active TB was excluded by negative multislice computed tomography, negative culture, and absence of symptoms. In 1 patient, T-cell reactivity toward TB peptides was lost 1 and 2 months posttransplantation. Another patient, however, tested 8 and 13 months posttransplantation, displayed measurable cellular TB immune responses. This finding suggests that the measurement of cellular TB immune responses shortly after transplantation may fail. If possible, patients with end-stage liver disease should be screened for TB prior to transplantation. Our data are the first evidence that T-SPOT.TB may be useful to diagnose latent TB in patients awaiting liver transplantation.

Sarcoidosis is a systemic granulomatous disease associated with local epithelioid granulomas, CD4(+) T cells, and Th1 cytokines. The tissue Ags that drive this granulomatous inflammation are uncertain. In this study, we used IFN-gamma-ELISPOT assays and flow cytometry to assess lung and blood T cell responses to the candidate pathogenic Ag, Mycobacterium tuberculosis catalase-peroxidase (mKatG) in patients with sarcoidosis from two centers. Despite differences in patient phenotypic, genetic, and prognostic characteristics, we report that T cell responses to mKatG were remarkably similar in these cohorts, with higher frequencies of mKatG-reactive, IFN-gamma-expressing T cells in the blood of sarcoidosis patients compared with nontuberculosis sensitized healthy controls, and (in a subset) in greater numbers than T cells reactive to purified protein derivative. In sarcoidosis, mKatG-reactive CD4(+) Th1 cells preferentially accumulated in the lung, indicating a compartmentalized response. Patients with or without Löfgren syndrome had similar frequencies of mKatG specific IFN-gamma-expressing blood T cells. Circulating mKatG-reactive T cells were found in chronic active sarcoidosis but not in patients with inactive disease. Together, these results demonstrate that T cell responses to mKatG in sarcoidosis fit a profile expected for a pathogenic Ag, supporting an immunotherapeutic approach to this disease.

Multi-colour flow cytometry was applied to determine T-cell-specific interferon-gamma, interleukin-2 and tumour necrosis factor-alpha expression in children with tuberculosis and non-tuberculosis mycobacterial lymphadenopathy (NTM-L). In vitro stimulation of peripheral blood mononuclear cells with purified protein derivative from Mycobacterium tuberculosis (tuberculin) and M. avium (sensitin) revealed differential recognition of tuberculin and sensitin in both study groups. Ratios of tuberculin-specific and sensitin-specific T-cell proportions in individual patients discriminated between children with tuberculosis or NTM-L. These findings have the potential to improve the differential diagnosis of mycobacterial infections.

Background: Although the biological basis for the increased susceptibility of diabetic patients to tuberculosis remains unclear, the world is undergoing a type 2 diabetes pandemic. We hypothesize that chronic hyperglycemia leads to immunocompromise that facilitates progression to active tuberculosis. To assess this possibility, we determined whether patients with tuberculosis and diabetes (particularly those with chronic hyperglycemia), compared with patients with tuberculosis who did not have diabetes, presented altered cytokine responses to a mycobacterial antigen., Methods: Samples of whole blood from patients with tuberculosis and diabetes and from patients with tuberculosis who did not have diabetes was stimulated in vitro with purified protein derivative from Mycobacterium tuberculosis. We then determined whether there was an association between the levels of innate and adaptive cytokines secreted in response to the antigen and diabetes status, or diabetes with chronic hyperglycemia (measured by glycosylated hemoglobin level), after controlling for possible confounders., Results: Innate and type 1 cytokine responses were significantly higher in patients with tuberculosis who had diabetes than in nondiabetic control subjects. The effect was consistently and significantly more marked in diabetic patients with chronic hyperglycemia., Conclusions: These data provide preliminary evidence that type 2 diabetes, especially type 2 diabetes involving chronic hyperglycemia, is associated with an altered immune response to M. tuberculosis. More-detailed knowledge of the underlying mechanisms should focus on the effect of chronic hyperglycemia on the immune response to help in understanding the enhanced susceptibility of diabetic patients to tuberculosis.

We investigated the effect of 25 wk of exercise training on in vivo immune measures that depend on T helper 1 (Th1) and T helper 2 (Th2) immune responses in the elderly as a substudy of a randomized controlled trial to investigate health benefits of regular exercise training for the elderly. Sixty-five healthy elderly volunteers were randomly assigned to either an exercise training group (n = 32) or a sedentary control group (n = 33). The area of skin reaction to purified protein derivative (PPD) of tuberculin that depends on Th1 activation and the concentrations of serum IgG subclasses and IgE were evaluated before and after 25-wk intervention. All participants completed 25 wk of training. Thirty-one participants of the exercise group and all control group underwent immunological analyses, but only 30 from the exercise group and 21 from the control group had the PPD skin reaction assessment. Repeated-measures ANOVA revealed a significant interaction between time and exercise intervention, which appeared as an enhanced skin reaction to tuberculin PPD (P < 0.05) and a reduced serum IgG4 concentration, the production of which depends on Th2-dependent class switching (P < 0.05), in the exercise group after 25 wk. No immune variables changed in the control group. These result supports the hypothesis that exercise training favors in vivo Th1 immune response in elderly persons.

RUTI is a vaccine consisting of Mycobacterium tuberculosis bacilli grown in stress conditions that is fragmented, detoxified and liposomed. RUTI was designed to shorten the treatment of latent tuberculosis infection (LTBI) with isoniazid from 9 months to just 1 month, by additional treatment with two inoculations of RUTI 4 weeks apart. During the validation process for monitoring the immunogenicity of administration of RUTI in a Phase I clinical trial, the question arose whether to introduce the tuberculin skin test (TST) in the screening of non-LTBI volunteers. This study was designed to evaluate the effect of TST on subsequent different T-cell interferon-gamma release assay (TIGRA) responses, using a spectrum of M. tuberculosis-related antigens (ESAT-6, CFP-10, 16 kDa, 19 kDa, MPT64, Ag 85B, 38 kDa, hsp65, PPD and BCG). The results showed an increase in post-TST response even in non-LTBI subjects for most antigens tested, as measured both by whole blood assay (WBA) and ELISPOT. Increased ELISPOT response decreased toward pre-TST levels within 1 month whereas the WBA response did not. Taking into account that there is no definitive correlation between TST and TIGRA tests to diagnose LTBI and the feasibility that TST might alter the immune monitoring included in clinical trials, these data suggest that TST determination should be carefully planned to avoid any interference with TIGRA.

Accurate diagnosis of tuberculosis (TB) infection is critical for the treatment, prevention, and control of TB. Conventional diagnostic tests based on purified protein derivative (PPD) do not achieve the required diagnostic sensitivity. Therefore, in this study, we have evaluated the immunogenic properties of Rv1168c, a member of the PPE family, in comparison with PPD, which is routinely used in the tuberculin test, and Hsp60 and ESAT-6, well-known immunodominant antigens of Mycobacterium tuberculosis. In a conventional enzyme immunoassay, the recombinant Rv1168c protein displayed stronger immunoreactivity against the sera obtained from patients with clinically active TB than did PPD, Hsp60, or ESAT-6 and could distinguish TB patients from Mycobacterium bovis BCG-vaccinated controls. Interestingly, Rv1168c antigen permits diagnosis of smear-negative pulmonary TB as well as extrapulmonary TB cases, which are often difficult to diagnose by conventional tests. The immunodominant nature of Rv1168c makes it a promising candidate to use in serodiagnosis of TB. In addition, our studies also show that Rv1168c is a potent T-cell antigen which elicits a strong gamma interferon response in sensitized peripheral blood mononuclear cells obtained from TB patients.

CD45 is a haemopoietic tyrosine phosphatase, crucial for lymphocyte signalling. Two polymorphisms (C77G and A138G), which alter CD45 isoform expression, are associated with autoimmune and infectious diseases. Using HapMap data, we show that there is substantial linkage disequilibrium across the CD45 gene (PTPRC), with similar patterns in different populations. Employing a set of single nucleotide polymorphisms, correlated with a substantial proportion of variation across this gene, we tested for association with type 1 diabetes, Graves' disease in a Japanese population, hepatitis C in UK population and tuberculin response in a Chinese population. A limited number of common haplotypes was found. Most 138G alleles are present on only one haplotype, which is associated with Graves' disease, supporting previous data that A138G is a functionally important CD45 polymorphism.

In this paper we report the development of a sensitive and specific assay for the detection of tuberculosis (TB) in European badgers (Meles meles), based on the stimulation of lymphocytes in whole-blood culture and the subsequent detection of gamma-interferon (IFNgamma) by sandwich ELISA. The comparative levels of IFNgamma produced to bovine and avian tuberculin (B-A) was used as the basis of determining the TB status of badgers, resulting in a more sensitive test than that based on the defined Mycobacterium bovis antigens ESAT6 and CFP10. The assay was evaluated using 235 badgers. The IFNgamma EIA (enzyme immunoassay) based on a monoclonal pair (mEIA) was more sensitive than one using a rabbit polyclonal antiserum (pEIA). At a specificity of 93.6%, the mEIA was 80.9% sensitive, compared to a sensitivity of 74.5% for the pEIA. At the same specificity as the EIA, the current serological ELISA test for TB in badgers (Brock test) had a sensitivity of 48.9%. Only one of the culture positive badgers missed by the mEIA was correctly diagnosed by the Brock test, suggesting that the combination of both a T-cell and serological test has little diagnostic advantage.

Background: Simple biomarkers are required to identify TB in both HIV(-)TB(+) and HIV(+)TB(+) patients. Earlier studies have identified the M. tuberculosis Malate Synthase (MS) and MPT51 as immunodominant antigens in TB patients. One goal of these investigations was to evaluate the sensitivity and specificity of anti-MS and -MPT51 antibodies as biomarkers for TB in HIV(-)TB(+) and HIV(+)TB(+) patients from a TB-endemic setting. Earlier studies also demonstrated the presence of these biomarkers during incipient subclinical TB. If these biomarkers correlate with incipient TB, their prevalence should be higher in asymptomatic HIV(+) subjects who are at a high-risk for TB. The second goal was to compare the prevalence of these biomarkers in asymptomatic, CD4(+) T cell-matched HIV(+)TB(-) subjects from India who are at high-risk for TB with similar subjects from US who are at low-risk for TB., Methods and Results: Anti-MS and -MPT51 antibodies were assessed in sera from 480 subjects including PPD(+) or PPD(-) healthy subjects, healthy community members, and HIV(-)TB(+) and HIV(+)TB(+) patients from India. Results demonstrate high sensitivity (approximately 80%) of detection of smear-positive HIV(-)TB(+) and HIV(+)TB(+) patients, and high specificity (>97%) with PPD(+) subjects and endemic controls. While approximately 45% of the asymptomatic HIV(+)TB(-) patients at high-risk for TB tested biomarker-positive, >97% of the HIV(+)TB(-) subjects at low risk for TB tested negative. Although the current studies are hampered by lack of knowledge of the outcome, these results provide strong support for the potential of these biomarkers to detect incipient, subclinical TB in HIV(+) subjects., Conclusions: These biomarkers provide high sensitivity and specificity for TB diagnosis in a TB endemic setting. Their performance is not compromised by concurrent HIV infection, site of TB and absence of pulmonary manifestations in HIV(+)TB(+) patients. Results also demonstrate the potential of these biomarkers for identifying incipient subclinical TB in HIV(+)TB(-) subjects at high-risk for TB.

Background: Targeted preventive chemotherapy of individuals with progressive subclinical (incipient) disease before it becomes contagious would break the chain of tuberculosis transmission in high endemic regions. We have studied the ability of a skin test response to ESAT-6 and CFP10 (E6/C10) to predict later development of tuberculosis disease in the guinea pig model., Methods and Findings: Guinea pigs, either vaccinated with BCG or unvaccinated, were infected with a low dose of Mycobacterium tuberculosis by the aerosol route and the development of delayed type hypersensitivity responses to E6/C10 and to purified protein derivative (PPD) were followed until the onset of clinical disease. We demonstrated a negative correlation between the size of the skin test response and the time to the onset of clinical disease; a large E6/C10 skin test response correlated to a shorter survival time post skin testing, while a small E6/C10 skin test reaction correlated with a longer survival time (r = -0.6 and P<0.0001). No correlation was found using PPD., Conclusions: Our data suggest that it may be possible to develop a prognostic skin test based on E6/C10 that will allow the identification of individuals with incipient disease, who have the highest risk of developing active tuberculosis in the near future.

European badgers (Meles meles) are considered to be an important reservoir of infection for Mycobacterium bovis and are implicated in the transmission of tuberculosis to cattle in Ireland and Great Britain. Accurate tests are required for tuberculosis surveillance in badger populations and to provide a basis for the development of strategies, including vaccination, to reduce the incidence of the infection. In this study, we have developed an endobronchial M. bovis infection model in badgers in which we measured cell-mediated immune and serological responses for up to 24 weeks post-infection. Groups of badgers were subjected to necropsy at 6-week intervals and the gross lesion severity status compared with immune responses measured in blood samples taken throughout the course of the study. The panel of antigens included bovine and avian tuberculins (PPD) as well as single antigens, ESAT-6, CFP-10, MPB70, Rv3019c, Rv3873, Rv3878 and Rv3879, all known to be recognised by the immune system in other animal models of tuberculosis infection. Our results demonstrated that M. bovis infected badgers responded to specific antigens as early as 6 weeks post-infection, consistent with the presence of visible lesions. The data also revealed unique patterns of antigen recognition with high levels of PBMC proliferation in the presence of CFP-10 but low proliferation levels with ESAT-6. Using a multi-antigen print immunoassay (MAPIA), we were able to confirm that MPB83 is the dominant antigen recognised by serum antibodies in infected badgers.

The roles of intracellular reactive oxygen species (ROS) and related signalling pathways in mycobacterial infection are largely unknown. Here we show that tuberculin purified protein derivative (PPD)/Toll-like receptor (TLR) 2/ROS signalling through activation of apoptosis-regulating signal kinase (ASK) 1 and p47phox pathways is responsible for the induction of proinflammatory responses during tuberculosis (TB) infection. Tuberculin PPD stimulation resulted in rapid activation of mitogen-activated protein kinases (MAPKs) and an early burst of ROS in monocytes/macrophages in a TLR2-dependent manner. PPD-induced ROS production led to robust activation of ASK1 upstream of p38 MAPK, via TLR2. Interestingly, phosphorylation of the cytosolic NADPH oxidase subunit p47phox and ASK1 activation are mutually dependent on PPD/TLR2-mediated signalling. Furthermore, active pulmonary TB patients showed upregulated ROS generation, as well as enhanced activation of ASK1/p38/p47phox pathways in their primary monocytes compared with healthy controls, which suggests a systemic primed status during TB. Taken together, these results indicate that activation of the ASK1/p38 MAPK/p47phox cascade plays a central role in PPD/TLR2-induced ROS generation and suggests the existence of a 'ROS/ASK1' inflammatory amplification feedback loop in monocytes/macrophages. The altered regulation of this axis with an increasing free-radical burden may contribute to the immunopathogenesis of human TB.

Micronutrient deficiencies are frequently associated with tuberculosis (TB) worldwide. We tested the effect of exogenous vitamin E on proliferation and cytokine production of peripheral blood mononuclear cells (PBMC) from TB patients and healthy purified protein derivative (PPD)+ volunteers. Proliferation was stimulated with mycobacterial antigen (PPD) and evaluated by the incorporation of tritiated thymidine in PBMC cultured with or without 50 microm-vitamin E for 6 d. Cytokine production (IL-2 and interferon (IFN)-gamma) was determined by intracellular cytokine staining and by ELISA in the supernatant of PBMC stimulated for 24 h with phytohaemagglutinin or PPD. Our results show that culture with vitamin E increased (P < or = 0.05 ) the antigen-induced proliferation of PBMC in TB patients but not in healthy PPD+ volunteers. No significant changes in the number of cytokine-producing cells or in the production of IFN-gamma were observed with vitamin E treatment. These results indicate that vitamin E may enhance the antigen-specific in vitro response of PBMC from TB patients.

Alternative activation of macrophages (Mphi) during helminth infection is a characteristic feature of the host immune response. Alternatively activated macrophages (AAMphi) are distinguished from others by high arginase 1 (Arg-1) activity, low nitric oxide (NO), and high interleukin 10 (IL-10) production. In murine models, these cells have been shown to possess anti-inflammatory properties. They have also been implicated in exacerbating a subsequent infection with a secondary pathogen. In this study we used cattle experimentally infected with Fasciola hepatica to monitor the kinetics of IL-4 and IL-10 over the course of infection. Using naïve Mphi in vitro, we examined the effects of exposure to F. hepatica excretory/secretory products (FhepES) alone or in combination with IL-4. Our results suggest that FhepES may work in combination with IL-4 to produce AAMphi. The effects of FhepES on the subsequent responses to lipopolysaccharide (LPS) and purified protein derivative from Mycobacterium bovis (PPD-B), which are bovine Toll-like receptor 4 (TLR4) and TLR2 antagonists, respectively, were also examined. We found that Mphi stimulated with FhepES together with LPS or PPD-B have reduced NO or gamma interferon production, respectively. The ability of FhepES to produce AAMphi was found to be heat labile and partially dependent on glycan residues. A possible role for TLR recognition is discussed.

Antimycobacterial T cell reactivity at different stages of HIV infection was investigated. Subjects were screened with purified protein derivative (PPD), early secreted antigenic target (ESAT)-6, and culture filtrate protein (CFP)-10 antigens for interferon (IFN)-gamma-producing effector T cell responses by direct ex vivo enzyme-linked immunospot (ELISpot) assay. The proportion of responders to PPD tuberculin decreased with a reduction in CD4 T cell count, whereas the proportion of responder to ESAT-6 and CFP-10 did not. The main sources of IFN-gamma secretion were CD4 cells, and the relative responses to ESAT-6 and CFP-10 significantly increased in HIV-infected patients with decreasing CD4 cell count. This may reflect early signs of reactivation, reinfection, or a restricted, inefficient immune response to Mycobacterium tuberculosis.

In recent years, there have been reports on the reduced diagnostic value of a tuberculin test with 2 TE PPD-L. Tuberculin diagnosis is a ground for suspecting tuberculosis only in 25.9 +/- 0.06% of cases in senior pupils and in 10.7 +/- 0.03% amongst adolescents. This is associated with the higher proportion tuberculin-positive individuals and it is very difficult to make the diagnosis in children with monotonous tuberculin sensitivity. A contact with a bacterium-discharging person undoubtedly increases a risk for the disease, but fails to affect the true outcome of delayed hypersensitivity. According to the date of this study, comorbidity and helminthic invasion have no significant impact on tuberculin sensitivity. Reactivation impairs immune homeostasis towards inadequate immune system performance.

Setting: A chest disease centre in Istanbul, Turkey., Objective: The diagnostic accuracy of interferon-gamma-based assays for Mycobacterium tuberculosis infection may be improved by using lower cut-off values for the tuberculin skin testing (TST), QuantiFERON-TB Gold (QFT) and T-SPOT.TB (T-SPOT) assays., Design: Three assays, TST, QFT and T-SPOT, were evaluated for their diagnostic performance with respect to different cut-off values. This evaluation was carried out in a comparative study involving 100 patients with untreated culture-confirmed cavitary pulmonary tuberculosis (TB) and 47 healthy subjects., Results: The sensitivities of the assays were: TST 70%, QFT 78% and T-SPOT 83.5%, while their specificities were TST 35%, QFT 89.4% and T-SPOT 84.8%. Both QFT and T-SPOT were significantly more specific than TST (both P < 0.001), but were similiar to each other (P = 0.5). Receiver operating characteristic analysis revealed that a cut-off value of 0.818 IU/ml for QFT maximises specificity without significant loss of test sensitivity. Using lower cut-off values for T-SPOT and TST, however, also increased the sensitivity of the assay but resulted in a significant decrease in specificity., Conclusion: Lower cut-off values for TST, QFT and T-SPOT increased the sensitivity of each assay, but only with a lower cut-off value for QFT could specificity be maintained.

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.

The diagnosis of tuberculosis remains among public health concerns due to shortcomings of the purified protein derivative (PPD). Recombinant truncated 38 kDa protein (rTPA38) of Mycobacterium tuberculosis was evaluated to screen new tuberculosis-specific tuberculin. 539 patients, 1133 healthy controls, and 55 guinea pigs were recruited to assess their sensitivity and specificity to rTPA38; 221 healthy controls, with negative responses to rTPA38 and PPD, were vaccinated with M. bovis BCG to determine their cross-reactions with M. bovis BCG. The Mantoux technique was adopted to perform skin tests. No difference in the sensitivity of skin tests was detected between rTPA38 and PPD (78.2% vs 83.4%), but there was a significant difference in the specificity of skin tests between rTPA38 and PPD (75.2% vs 47.0%). Compared to PPD, rTPA38 elicited low positive responses for those recruitments vaccinated with M. bovis BCG. The rTPA38 had significant skin reactions in M. tuberculosis-sensitized guinea pigs, and the opposite was true for both M. fortuitum- and M. kansasii-sensitized guinea pigs. These findings indicate that rTPA38 may have potential as a tuberculosis-specific skin test antigen.

Tuberculosis (TB) still remains the leading cause of mortality due to bacterial pathogen. The only currently available vaccine against TB, Bacille Calmette-Guérin (BCG) is at best credited with a 50% overall protective efficacy. Skin testing with purified protein derivative (PPD) from Mycobacterium tuberculosis is the method of detecting BCG-induced cell mediated immunity, in vivo. In the previous study we found that approximately 60% young volunteers with no history of TB, who had been subjected to neonatal BCG vaccination and revaccination(s) at school age, developed delayed type hypersensitivity (DTH) to tuberculin. The remaining volunteers were persistently tuberculin negative. Moreover, we found a significant association between BCG driven development of DTH to PPD and the polymorphism within the CD14 C/T(-159) gene for macrophage receptor recognising mycobacterial compounds. It has suggested that the CD14 gene variants may play a role in the appearance and persistence of DTH to PPD in BCG vaccinated subjects. In order to extend our study on a possible role of CD14 in BCG driven DTH response to PPD, we measured the expression of mCD14 on macrophages, stimulated or not stimulated with mycobacterial antigens, and the serum levels of sCD14. Considering the importance of CD14 - TLR2/TLR4 interactions in macrophage signalling, we determined the polymorphism of TLR2 and TLR4 genes as well as macrophage expression of TLR2 for the volunteers with and without skin reactivity to PPD. We observed a subtle but significant decrease in CD14 density on adherent monocytes from tuberculin positive versus tuberculin negative volunteers. However, we found no difference in CD14 density on monocytes enriched in CD14+ cells using anti-CD14 mAb coupled to magnetic beads. A significant increase in CD14 density was observed on macrophages stimulated with PPD and LPS but not with live BCG bacilli. However, this increase as well as serum levels of soluble sCD14 were similar in the volunteers with and without skin reactions to PPD. Thus, our suggestion on the role of CD14 in the generation of DTH to tuberculin in BCG vaccinated subjects should be further explored. The most important CD14 co-receptors are Toll-like receptors (TLRs) which activate nuclear factors for the production of inflammatory cytokines. However, we could see no association between the polymorphisms of TLR4 (Asp299Gly and Thr399Ile) and TLR2 genes (Arg753Gln and Arg677Trp) and skin responses to PPD. Also, the TLR2 density was similar on monocytes from tuberculin negative and tuberculin positive volunteers.

Reactions to tuberculin in cows from a healthy herd were ascertained to be caused by Mycobacterium tuberculosis as latent tuberculous infection was detected in 10.4% of cattle-breeders, including milkmaids looking after infected cows and having a family contact with patients with open-type tuberculosis. In tuberculin-positive cows, the level of antibodies to M. tuberculosis was higher than that of antibodies to M. bovis; mycobacterial antigens and their complexes with antibodies circulated in blood; the investigators isolated mycobacteria with the greatly changed morphology and cultural properties, which were referred to as an M. tuberculosis-M. bovis complex in their antigenic composition and specific DNA sites. Alongside with the traditional diagnostic methods for tuberculosis, the authors underline the need for using enzyme immunoassay, polymerase chain reaction, blood and sputum tests using the VKC medium.

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.

Studies of sarcoidosis immunology have noted oligoclonal T cell populations, suggesting cell-mediated immunity that is antigen-specific. Sarcoidosis immunology and pathology are most similar to mycobacterial infections. Mycobacterium tuberculosis infection in mice and humans reflects T helper 1 (Th1) immune responses to multiple cell wall and secreted antigens. We investigated if the oligoclonal immune response in individual sarcoidosis subjects could be elicited by multiple secreted mycobacterial antigens by performing ex vivo enzyme-linked immunospot assay (ELISPOT) on peripheral blood mononuclear cells (PBMC) from 30 sarcoidosis, 26 purified protein derivative negative (PPD-) control and 10 latent tuberculosis subjects (PPD+) to assess Th1 responses to mycobacterial superoxide dismutase A (sodA), catalase-peroxidase (katG) and early secreted antigenic target protein (ESAT-6). A significant difference was noted among the sarcoidosis and PPD- control subjects to ESAT-6 [12 of 30 versus one of 26 (P = 0.0014)], katG [nine of 30 versus none of 26 (P = 0.002)] and sodA [12 of 30 versus none of 26 (P = 0.002)]. There was no significant difference between sarcoidosis and PPD+ subjects. Twelve sarcoidosis subjects recognized two or more mycobacterial proteins, as well as multiple distinct epitopes within individual proteins. One sarcoidosis subject on whom we collected bronchoalveolar lavage (BAL) fluid and PBMC had no recognition of mycobacterial antigens using PBMC, but BAL fluid demonstrated strong Th1 immune responses to ESAT-6 and katG. Individual sarcoidosis subjects recognized not only multiple mycobacterial proteins, but multiple distinct peptides within a specific protein, thus demonstrating that multiple mycobacterial epitopes elicit the Th1 immune response observed. Immune responses by sarcoidosis T cells to mycobacterial proteins may have an important role in sarcoidosis pathogenesis.

Buruli ulcer disease (BUD) is an emerging predominantly tropical disease caused by Mycobacterium ulcerans. The initial pre-ulcerative skin lesion often breaks down into an ulcer with undermined edges. Healing is common but may require considerable time, and scarring often results in functional limitations. Considerable evidence has now emerged that patients with early BUD cannot mount a sufficient protective T helper 1 (Th1) cell response to M. ulcerans, but uncertainty remains as to whether immune protection is restored over time. This study investigates the Th1 cell response of patients with various stages of BUD on mycobacterial antigens. We measured interferon (IFN)-gamma levels after ex vivo whole blood stimulation with tuberculin purified protein derivative (PPD), and compared the Th1 cell response of individuals with pre-ulcerative, ulcerative and healed BUD as well as healthy controls. Moreover, the systemic Th1 cell response was related to histopathological features in the various stages of surgically resected BUD lesions. We show that patients with ulcerative and healed BUD produce significantly higher IFN-gamma levels after mycobacterial ex vivo whole blood stimulation than healthy controls, and that patients with a granulomatous tissue response produce higher IFN-gamma levels than individuals without. We therefore suggest that the mounted Th1 cell response in ulcerative BUD patients might be related to their histopathological tissue response.

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.

Dendritic cells (DC) are powerful inducers of primary T-cell responses, but their role in secondary responses has not been extensively analysed. Here, we address the role of two DC subsets derived from human CD16(+) (16(+) mDC) or CD16(-) (16(-) mDC) monocytes on the reactivation of memory responses. CD4(+) CD45RA(-) memory T cells were obtained from adult blood donors, and central (T(CM)) and effector (T(EM)) memory T cells were isolated by fluorescence-activated cell sorting with anti-CCR7 antibodies. The 16(+) mDC and 16(-) mDC were cocultured with autologous lymphocytes, either unpulsed or loaded with purified protein derivatives of Mycobacterium tuberculosis (PPD) or tetanus toxoid (TT), and were analysed for up to 8 days. Over a range of doses, 16(+) mDC drove stronger T-cell proliferative responses against both antigens. Overall, antigen-specific memory cells tended to acquire a phenotype of T(EM) at later time-points in the culture, whereas cells that had completed fewer cycles of division were similar to T(CM). The 16(+) mDC induced higher rates of proliferation on both T(CM) and T(EM) lymphocytes than 16(-) mDC. This phenomenon was not related to the ability of both DC to induce CD25 expression on T cells, to lower secretion of interleukin-2, or to raise production of interleukin-10 during T-cell/16(-) mDC cocultures. The induction of T(CM) effector capacity in terms of interferon-gamma production was faster and more pronounced with 16(+) mDC, whereas both DC had similar abilities with T(EM). In conclusion, these data might reveal new potentials in vaccination protocols with 16(+) mDC aimed at inducing strong responses on central memory T cells.

Factors that relate to medium-term outcome in patients with pulmonary tuberculosis (PTB) who have completed the 2-month intensive phase of treatment are incompletely understood. The relationship between in vitro production of interferon-gamma (IFN-gamma), interleukins (ILs)-5 and -10 and drug levels determined after 2 months of drug therapy, to outcome at 24 months was studied prospectively. Cytokine concentrations were determined from culture supernatants after stimulation of whole blood with purified protein derivative (PPD) of Mycobacterium tuberculosis. Plasma concentrations of rifampin, isoniazid, pyrazinamide and ethambutol were determined by high-performance liquid chromatography. The treatment failure and relapse free survival probability was 0.54 (95% CI: 0.40-0.67) at 24 months. In multivariate analysis of parameters at 2 months the strongest positive associations with disease free survival were IFN-gamma response to PPD (p=0.002) and serum creatinine (p=0.001). Drug concentrations were not associated with outcome although rifampin exposure correlated with IFN-gamma response to PPD (p=0.0132). These data suggest that the ability to mount a recall immune response to M. tuberculosis may influence treatment outcome. The data support the idea to identify persons at risk of a poor treatment outcome by monitoring of the in vitro response to tuberculosis antigens.

Type 1 (Th1) granulomas can be studied in mice sensitized with mycobacterium antigens followed by challenge of agarose beads covalently coupled to purified protein derivative. TLR9 is known to play a role in the regulation of Th1 responses; thus, we investigated the role of TLR9 in granuloma formation during challenge with mycobacterium antigens and demonstrated that mice deficient in TLR9 had increased granuloma formation, but a dramatically altered cytokine phenotype. Th1 cytokine levels of IFN-gamma and IL-12 in the lungs were decreased in TLR9(-/-) mice when compared to wild-type mice. In contrast, Th2 cytokine levels of IL-4, IL-5, and IL-13 were increased in TLR9(-/-) mice. The migration of CD4(+) T cells in the granuloma was impaired, while the number of F4/80(+) macrophages was increased in TLR9(-/-) mice. Macrophages in the lungs of the TLR9-deficient animals with developing granulomas expressed significantly lower levels of the classically activated macrophage marker, nitric oxide synthase, but higher levels of the alternatively activated macrophage markers such as 'found in inflammatory zone-1' antigen and Arginase-1. These results suggest that TLR9 plays an important role in maintaining the appropriate phenotype in a Th1 granulomatous response.

Background: Vitamin A supplementation (VAS) at birth has been associated with decreased mortality in Asia. Bacille Calmette-Guérin (BCG) vaccine is given at birth in tuberculosis-endemic countries. Previous studies suggest that VAS may influence the immune response to vaccines., Objective: Our objective was to examine whether VAS influences the immune response to simultaneously administered BCG vaccine., Design: Within a randomized trial of 50,000 IU vitamin A or placebo given with BCG vaccine at birth in Guinea-Bissau, 2710 infants were examined for BCG scar formation and delayed-type hypersensitivity (DTH) to purified protein derivative of Mycobacterium tuberculosis (PPD) at 2 and 6 mo of age. The ex vivo cytokine response to PPD was measured in 607 infants., Results: At 2 mo of age, 39% (43% of the boys and 34% of the girls) responded to PPD. The prevalence ratio of a measurable PPD reaction for VAS compared with placebo recipients was 0.90 (95% CI: 0.80, 1.02) for all infants, 0.81 (95% CI: 0.69, 0.95) for boys, and 1.04 (95% CI: 0.86, 1.26) for girls. At 6 mo of age, 42% of the infants responded to PPD. No difference was observed between VAS and placebo recipients. The prevalence of BCG scar was not affected by VAS. The ex vivo interferon-gamma response to PPD was increased by VAS (means ratio: 1.40; 95% CI: 1.03, 1.91)., Conclusions: VAS with BCG vaccination does not appear to interfere with the long-term immune response to BCG. However, VAS temporarily altered the DTH reaction to PPD in boys at 2 mo of age, suggesting sex differences in the immunologic response to VAS given with BCG. This trial was registered at www.clinicaltrials.gov as #NCT00168597.

Serum responses against Mycobacterium tuberculosis HSP16 were determined for children with tuberculosis (TB) and for healthy purified protein derivative (PPD)-positive and PPD-negative children. Immunoglobulin G (IgG) and IgM responses were higher for TB patients than for other groups. After chemotherapy, IgM and IgG responses decreased for TB patients and PPD-positive subjects. Monitoring of anti-M. tuberculosis HSP16 responses could assist in the management of pediatric TB.

Background: Tuberculin-specific T-cell responses have low diagnostic specificity in BCG vaccinated populations. While subunit-antigen (e.g. ESAT-6, CFP-10) based tests are useful for diagnosing latent tuberculosis infection, there is no reliable immunological test for active pulmonary tuberculosis. Notably, all existing immunological tuberculosis-tests are based on T-cell response size, whereas the diagnostic potential of T-cell response quality has never been explored. This includes surface marker expression and functionality of mycobacterial antigen specific T-cells., Methodology/principal Findings: Flow-cytometry was used to examine over-night antigen-stimulated T-cells from tuberculosis patients and controls. Tuberculin and/or the relatively M. tuberculosis specific ESAT-6 protein were used as stimulants. A set of classic surface markers of T-cell naïve/memory differentiation was selected and IFN-gamma production was used to identify T-cells recognizing these antigens. The percentage of tuberculin-specific T-helper-cells lacking the surface receptor CD27, a state associated with advanced differentiation, varied considerably between individuals (from less than 5% to more than 95%). Healthy BCG vaccinated individuals had significantly fewer CD27-negative tuberculin-reactive CD4 T-cells than patients with smear and/or culture positive pulmonary tuberculosis, discriminating these groups with high sensitivity and specificity, whereas individuals with latent tuberculosis infection exhibited levels in between., Conclusions/significance: Smear and/or culture positive pulmonary tuberculosis can be diagnosed by a rapid and reliable immunological test based on the distribution of CD27 expression on peripheral blood tuberculin specific T-cells. This test works very well even in a BCG vaccinated population. It is simple and will be of great utility in situations where sputum specimens are difficult to obtain or sputum-smear is negative. It will also help avoid unnecessary hospitalization and patient isolation.

The guinea pig model of low-dose pulmonary tuberculosis has been used to study the pathogenesis of infection as well as the mechanisms of bacille Calmette-Guérin (BCG) vaccine-induced resistance. We investigated the function of lung cells from naive and BCG-vaccinated guinea pigs after enzymatic digestion of lung tissue with collagenase and DNase I. The total lung digest cells proliferated poorly to purified protein derivative (PPD) but comparatively better to ConA as assessed by [(3)H]-thymidine uptake. However, the non-adherent population obtained after plastic adherence of lung digests showed an enhanced response to concanavalin A (ConA) and PPD. Therefore, proliferation to ConA and PPD of nylon wool-purified T cells co-cultured with peritoneal (PMøs), alveolar (AMøs) or lung macrophages (LMøs) was assessed. Co-cultures of lung T cells and PMøs showed maximum proliferation to PPD, whereas proliferation was suppressed significantly by the addition of AMøs or LMøs. The response of T cells to ConA was unaffected in co-cultures. Incubation of co-cultures with recombinant guinea pig interferon-gamma (rgpIFN-gamma) did not reverse the suppression. In contrast, rgpIFN-gamma-treated plastic adherent LMøs that were non-specific esterase-positive were capable of reducing the intracellular growth of Mycobacterium tuberculosis. Similarly, total, non-adherent and adherent lung digest cells from BCG-vaccinated guinea pigs showed IFN-gamma and tumour necrosis factor (TNF)-alpha mRNA expression in response to ConA, lipopolysaccharide or PPD by reverse transcription-polymerase chain reaction followed by release of TNF protein but not IFN. These studies indicate that rgp-IFN-gamma-treated lung tissue macrophages from BCG-vaccinated guinea pigs are defective for inducing antigen-specific proliferation in T cells, but control the intracellular accumulation of virulent M. tuberculosis.

Mycobacterium tuberculosis lipid antigens produce significant T cell responses in healthy tuberculin reactor [purified protein derivative (PPD-positive] individuals. In the present study, proliferation and interferon (IFN)-gamma/interleukin (IL)-4 responses were analysed to M. tuberculosis total lipid antigens in T lymphocytes from 25 patients with multi-drug-resistant tuberculosis (MDR-TB). The obtained results were compared with those of 30 asymptomatic healthy PPD-positive and 30 healthy tuberculin skin test negative (PPD-negative) subjects. Peripheral blood mononuclear cells (PBMCs) and T cells (CD4(+) and CD8(+)) were stimulated using autologous immature dendritic cells. Proliferation responses were assessed using 3-{4,5-dimethylthiazol-2-yl}-2,5 diphenyl tetrazolium bromide (MTT). IFN-gamma/IL-4 concentrations in the supernatant of the CD4(+) and CD8(+)T cells were measured by enzyme-linked immunosorbent assay. Proliferation assay showed that the peripheral blood mononuclear cells and CD4(+) T cells from the MDR-TB patients responded significantly less to the M. tuberculosis total lipid antigens than to the CD4(+) T cells in the PPD-positive subjects. Total lipid antigen-specific proliferative responses in the CD8(+) T cells from the MDR-TB patients were minimally detected and the responses were similar to those of the PPD-positive subjects. IFN-gamma production by the CD4(+) T cells stimulated by total lipid antigens from the MDR-TB patients was decreased significantly compared with the PPD-positive individuals, whereas IL-4 production in the patients was elevated. IFN-gamma and IL-4 production in the CD8(+) T cells of the MDR-TB patients was similar to those of the PPD-positive subjects. In conclusion, it is suggested that stimulated CD4(+) T cells by M. tuberculosis total lipid antigens may be shifted to T helper 2 responses in MDR-TB patients.

Objective: To assess the effect of maternal cells or cellular components on neonatal immune responses to intracellular pathogens in calves., Animals: 15 Holstein calves., Procedures: Calves were fed whole colostrum, frozen colostrum, or cell-free colostrum within 4 hours after birth. Leukocytes were obtained from calves before feeding colostrum and 1, 2, 7, 14, 21, and 28 days after ingestion. Proliferative responses against bovine viral diarrhea virus (BVDV) and mycobacterial purified protein derivatives were evaluated. Dams received a vaccine containing inactivated BVDV, but were not vaccinated against mycobacterial antigens., Results: All calves had essentially no IgG in circulation at birth, but comparable and substantial concentrations by day 1. Calves that received whole colostrum had enhanced responses to BVDV antigen 1 and 2 days after ingestion of colostrum. In contrast, calves that received frozen colostrum or cell-free colostrum did not respond to BVDV. No differences were identified among the 3 groups in response to mycobacterial antigens., Conclusions and Clinical Relevance: Results indicated that transfer of live maternal cells from colostrum to neonatal calves enhanced responses to antigens against which the dams had previously responded (BVDV), but not to antigens to which the dams were naïve (mycobacterial purified protein derivatives). Results suggested that cell-mediated immune transfer to neonates can be enhanced by maternal vaccination.

Histamine not only mediates immediate allergic reactions, it also regulates cellular immune responses. H4R is the most recently identified histamine receptor (HR). In the present study, we examined the in vitro effect of histamine and H4R agonists on the responses of human T cells to purified protein derivative from Mycobacterium tuberculosis (PPD) and to Cry j1, the major allergen of Cryptomeria japonica pollen. Dimaprit, clobenpropit and clozapine, which are H4R agonists, dose-dependently blocked both PPD-induced interferon-gamma and Cry j1-induced interleukin-5 production by both peripheral blood mononuclear cells (PBMCs) and antigen-specific T-cell lines. However, the addition of thioperamide, an H3R/H4R antagonist, as well as a mixture of d-chlropheniramine, famotidine and thioperamide, did not reverse the inhibition. Pretreatment of PBMCs with SQ22536 and 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, had varying abilities to reverse the inhibitory effects of H4R agonists, except for clobenpropit. Moreover, the addition of H4R agonists induced annexin-V expression on PBMCs, especially in CD19(+) and CD4(+) cells. cDNA microarray analysis revealed that, among 16,600 genes tested, increased expression following treatment with clozapine was seen in 0 x 8% of the genes, whereas decreased expression was seen in 3 x 0% of the genes. These results suggest that H4R agonists inhibit antigen-specific human T-cell responses, although H4R does not appear to be important for this effect. In addition, the present study indicated that there may be orphan receptors or HR subtypes which can bind dimaprit, clobenpropit and clozapine, and that can exert an inhibitory effect on antigen-specific cellular responses via a cAMP/cAMP-dependent protein kinase-dependent, apoptotic pathway.

Objective: The purpose of this study was to explore the association between tuberculin reaction size in contacts with positive tuberculin skin test results and a variety of contact and index patient variables., Patients and Methods: We reviewed the contact investigation records for tuberculosis patients identified by the Tuberculosis Prevention and Control Center of Lleida, Spain, over a period of 7 years. Patients with pulmonary and/or extrapulmonary disease were included. Tuberculosis infection had been diagnosed using the Mantoux skin test with 2 tuberculin units of purified protein derivative RT-23, and tuberculosis disease had been detected on the basis of clinical data, chest radiographs, and cultures. We examined the association between tuberculin reaction size and contact age, close contact with index patients with positive sputum smears, and diagnosis of tuberculosis disease in contacts. Associations were analyzed using the chi(2) test for linear trend., Results: Tuberculin reaction size was 10 mm or greater in 85.9% of the 768 contacts analyzed and 15 mm or greater in 63.8%. The percentage of contacts under 15 years of age (P=.006) who had close contact with an index patient with a positive sputum smear (P=.013) and who were diagnosed with tuberculosis disease (P=.029) increased with tuberculin reaction size., Conclusions: We found that larger tuberculin reaction size in contacts with positive tuberculin skin test results was most frequently associated with recent infections in the contact, close contact with an index patient with a positive sputum smear, and diagnosis of tuberculosis disease in the contact.

TNF-alpha is a principal pro-inflammatory cytokine which contributes to the activation of innate immunity and the transition to antigen-specific adaptive immunity in tuberculosis. Using recombinant guinea pig (rgp) TNF-alpha, the effect of TNF-alpha on lymphocyte activation was examined in unvaccinated and BCG-vaccinated guinea pigs. Splenocytes were stimulated with PPD or ConA, in the presence or absence of rgp TNF-alpha for 96 h. Lymphocyte proliferation was measured using [(3)H]thymidine uptake, and IL-12 p40 and IFN-gamma mRNA were analyzed using real-time PCR. rgpTNF-alpha alone was able to stimulate a significant degree of proliferation in splenocytes. The addition of rgpTNF-alpha to PPD-stimulated cells enhanced the proliferation of splenocytes from BCG-vaccinated guinea pigs. Furthermore, enhancement of proliferation by rgpTNF-alpha was found to be correlated with upregulation of the levels of Type 1 cytokine mRNA (IL-12p40 and IFN-gamma) in splenocyte cultures. This suggests that TNF-alpha plays an important role in the regulation of Type 1 T cell-mediated immune responses in the guinea pig.

Tumor necrosis factor-alpha (TNF-alpha) plays critical and opposing roles in the pathogenesis of tuberculosis (TB). We examined the effects of Mycobacterium bovis BCG vaccination on TNF-alpha production in three distinct guinea pig leukocyte populations before and after pulmonary infection with M. tuberculosis H37Rv. Following BCG vaccination alone, and following challenge, bronchoalveolar lavage cells (BALC), resident peritoneal cells (PC), and splenocytes (SPC) were stimulated with purified protein derivative (PPD). Before virulent challenge, BCG vaccination clearly enhanced the ability of BALC, PC and SPC to produce TNF-alpha in response to PPD stimulation ex vivo. Following challenge, the TNF-alpha production of all three leukocyte populations from BCG-vaccinated animals remained relatively constant at pre-challenged levels. In sharp contrast, 5 weeks post-challenge, all three leukocyte populations from unvaccinated animals produced very high amounts of TNF-alpha in response to PPD. Three weeks post-challenge, SPC from one of the unvaccinated animals produced higher levels of TNF-alpha but the others produced lower levels of TNF-alpha than BCG-vaccinated animals. As expected, BCG vaccination reduced the levels of virulent mycobacteria in both the lungs and spleens. Thus, BCG vaccination allows guinea pigs to modulate TNF-alpha levels in conjunction with a reduction in bacillary loads in their tissues.

Background: Studies in West Africa and elsewhere have suggested that Bacillus Calmette-Guérin (BCG) vaccine given at birth is beneficial for child survival. It is possible that this effect is mediated partly through an effect on malaria, a hypothesis supported by animal studies. We investigated whether revaccination with BCG at 19 months of age reduced morbidity from malaria., Method: In the capital of Guinea-Bissau, between January and November 2003, children who had previously received BCG vaccination and who did not have a strong reaction to tuberculin were individually randomised to either receive revaccination with BCG at the age of 19 months or to be a control. Episodes of malaria were recorded during the 2003 malaria transmission season through passive case detection at health centres in the study area and at the national hospital. Cross-sectional surveys were carried out at the beginning and at the end of the rainy season., Results: Incidence rates of first episodes of malaria associated with any level of parasitaemia were 0.16 episodes per child-year among 713 revaccinated children and 0.12 among 720 control children [incidence rate ratio (IRR) = 1.37; 95% confidence intervals (CI): 0.84-2.25]. Results were similar when the diagnosis of malaria was based on the presence of parasitaemia >5000 parasites/microl (IRR = 1.30; 95% CI: 0.61-2.77). The incidence of all-cause hospitalisation was higher among BCG-revaccinated children than among controls (IRR = 2.13; 95% CI: 1.10-4.13). There were no significant differences in the prevalence of parasitaemia between the two groups of children at cross-sectional surveys., Conclusion: We found no evidence that BCG revaccination reduces morbidity from malaria.

Background: Our objective was to conduct a prevalence survey of purified protein derivative (PPD) reactions among Lebanese healthy school children to identify those with tuberculosis or latent tuberculosis and to investigate the effect of bacille Calmette-Guérin (BCG) vaccine on the interpretation of PPD reactivity., Methods: A self-administered questionnaire, including demographic characteristics, time of prior BCG vaccine and number of doses, known household contact with tuberculosis as well as parents' characteristics and living conditions was administered. PPD testing was performed on all children in diverse Lebanese regions aged 3 to 19 years. Reactivity that measured <5 mm were considered negative induration, doubtful if between 5 and 9 mm and positive if 10 mm or above. Chest radiographs were obtained as part of the evaluation for children with positive induration., Results: Of 4895 children, 4271 entered into the final data analysis. A total of 3259 children (76.3%) did not develop a reaction to PPD (0 mm), 170 (4%) had 1 to <5 mm reading, 509 (11.9%) had 5 to 9 mm and 333 (7.8%) had > or =10 mm. Approximately 62% of the vaccinated children had received BCG vaccine in first year of life. Two hundred ninety (61.8%) of 469 children < or =5 were vaccinated and 179 (38.2%) were not. Only 22 of the youngest vaccinated had positive PPD. Twelve children were diagnosed with tuberculosis, a prevalence of 280 per 100,000. However, the prevalence of latent tuberculosis was 7.51%., Conclusion: Our prevalence of tuberculosis and latent tuberculosis is a sentinel indicator of continued transmission in the community. The data support the current recommendations that children who receive BCG can and should be tested with PPD for latent tuberculosis and tuberculosis.

MPB70 (mycobacterial protein of BCG 70) as T-cell stimulator has been tried with an intradermal skin test (IST) and enzyme-linked immunosorbent assay (ELISA) of bovine tuberculosis (BTB). In this study, crude mycobacterial protein 70 (CMP70) was prepared by anion exchange chromatography from the culture supernatant of Mycobacterium bovis AN5 and CMP70 ELISA was compared with purified protein derivative (PPD) ELISA. PPD and CMP70 ELISA have shown a positive reaction to the sera of M. bovis infected cattle and IST positive reactors. One of three IST negative cattle showed the nonspecific reaction in PPD ELISA, whereas all of the IST negative cattle (n=3) were did not show the nonspecific reaction in CMP70 ELISA. When each ELISA was applied to sixty-two IST positive cattle, ELISA positive reactors were eighty four per cent to CMP70 antigen and fifty-two per cent to PPD. CMP70 has been shown to be more specific and sensitive than PPD in ELISA.

Background: The identification of mycobacteria represents the gold standard in the diagnosis of tuberculosis (TB), but it is not applicable in all patients, and immunological tests, such as the tuberculin skin test (TST), are not specific and sensitive enough., Methods: By flow cytometry, we measured the CD4 T-cell response to purified protein derivative (PPD) and early secretory antigenic target-6 (ESAT-6) protein using the intracellular cytokine staining technique on whole blood samples obtained from active TB (n = 16), latent TB (n = 17), Bacille Calmette-Guérin (BCG)-vaccinated (n = 11) and healthy (n = 10) donors. All the patients were also tested with conventional TST., Results: The identification by flow cytometry of PPD-specific T lymphocytes upon antigen stimulation of whole blood enables the discrimination of active TB, latent TB and BCG-vaccinated subjects from healthy individuals, whereas the ESAT-6 response discriminated active TB from healthy and BCG-vaccinated individuals. Moreover, this test enables identification of active TB patients who were negative on TST and to distinguish between TB and non-typical mycobacteria TB infections., Conclusions: The identification by flow cytometry of antigen-specific T lymphocytes upon antigen stimulation of whole blood has a better positive predictive value than TST, and could represent a further tool in the diagnosis of TB infection., ((c) 2007 S. Karger AG, Basel.)

Incubation by means of the growth-promoting factor VCG of autoclaved culture broth of M. bovis 8 and tuberculins on the nutrient medium VCG gave rise to microorganisms that could partially restore acid resistance. The isolates reacted with antiserum to M. bovis Vallee in the agglutination test and enzyme immunoassay, which in combination with polymerase chain reaction data verifies their genetic relationship with the pathogen of tuberculosis. The isolates did not show a significant pathogenicity, but they long persisted in guinea-pigs and, in some cases, induced delayed hypersensitivity to tuberculin. When exposed to high temperatures, the pathogen of tuberculosis is presumably to form structures that are capable to restore viability under certain conditions (for example, on the VCG medium).

The study covered 56 patients (aged 6-14 years) having hyperergic tuberculin sensitivity, including 28 patients with pulmonary tuberculosis (Group 1) and 28 patients infected with Mycobacterium tuberculosis (MBT) (Group 2). All tuberculous processes were asymptomatic and limited. Minor forms of tuberculosis, diagnosed by computed tomography, were found in 71.4% of cases. The signs of incomplete calcification were detectable in 93% of cases. In the past year, tuberculin sensitivity has progressed to hyperergic one in 75% of Group 2 patients. The patients with tuberculosis were treated with 3 drugs; those infected with M BT received 2 agents. In the patients with minor forms of tuberculosis, the level of specific IgG antibodies was equal to those in healthy MBT-infected individuals with hyperergic tuberculin sensitivity, the levels of immunoglobulins of subclasses G1 and G4 were lower, that of IgE antibodies were higher than in the MTB-infected. These data may suggest the stability of the immune system in patients with minor forms of tuberculosis, detected in the phase of calcification, and its instability in the MBT-infected with tuberculin sensitivity increasing up to hyperergic one.

Sarcoidosis is an enigmatic disease with a pathology similar to that of tuberculosis. We detected Th-1 immune responses to Mycobacterium tuberculosis ESAT-6 and KatG peptides from peripheral blood mononuclear cells from 15/26 sarcoidosis, 1/24 purified-protein-derivative-negative (PPD-) (P < 0.0001, Fisher's exact test), and 7/8 PPD-positive (PPD+) subjects (P = 0.21). This finding provides immunologic links between mycobacteria and systemic sarcoidosis.