Concordant or discordant results by the tuberculin skin test and the quantiFERON-TB test in children reflect immune biomarker profiles.

The tuberculin skin test (TST) and QuantiFERON-TB-Gold-In-tube (QFTGIT) are adjunctive tests used in the diagnosis of pediatric tuberculosis (TB). Neither test can rule out TB; however, a positive test usually triggers preventive treatment in TB contacts aged <5 years. TST and QFTGIT can give divergent results and it is unclear how discordant results should be interpreted in terms of TB risk and preventive treatment. To understand the immune processes underlying concordant or discordant TST and QFTGIT results, we analyzed immune responses in children from Palamaner Taluk in India (a TB-endemic region with routine neonatal BCG vaccination) who were referred to a TB case verification ward on suspicion of TB. Two hundred and ten children aged <3 years were classified according to their TST and QFTGIT results, and their immune responses analyzed by dual-colour-Reverse-Transcriptase-Multiple-Ligation-dependent-Probe-Amplification, using a panel of 45 genes and a 10-plex antigen-specific enzyme-linked immunosorbent assay. We show that immune biomarkers FPR1, TNFRSF1A and interferon (IFN)-γ are upregulated (all P<0.05) in concordant test-positive children, whereas BPI is downregulated (P<0.05). In contrast, SEC14L1 (P=0.034) and Interferon gamma-induced protein 10 (IP-10) (P=0.001) are differentially expressed between the TST+QFTGIT- /TST-QFTGIT+ groups. Known TB exposure was more frequent in concordant positive children and results were consistent with elevated expression of genes associated with inflammatory responses. Children with discordant test results displayed a mixed profile with activation of both pro- and anti-inflammatory markers. TST and/or QFTGIT positivity appears to reflect distinct but overlapping aspects of host immunity.

Identification of biomarkers for Mycobacterium tuberculosis infection and disease in BCG-vaccinated young children in Southern India.

Pediatric tuberculosis (TB) often goes undiagnosed because of the lack of reliable diagnostic methods. With the aim of assessing biomarker(s) that can aid in the diagnosis of TB infection and disease, we investigated 746 Indian children with suspected TB. Whole-blood mRNA from 210 children was examined by dual-color Reverse-Transcriptase Multiple Ligation-dependent Probe-Amplification for the expression of 45 genes and a Bio-Plex assay for the expression of cytokines/chemokines in QuantiFERON supernatants. The study shows that transcription of SEC14L1, GUSB, BPI, CCR7 and TGFß-1 (all P ≤ 0.05) was downregulated in TB disease compared with uninfected controls, while transcription of RAB33A was downregulated in TB disease compared with both latent TB (P < 0.05) and controls (P < 0.01). The transcription of CD4, TGFß-1 (P < 0.01) and the expression of IL-2 (P < 0.01) and IL-13 (P < 0.05) was upregulated in latent TB compared with that in controls. Using the Least Absolute Shrinkage and Selection Operator (lasso) model, RAB33A alone discriminated between TB disease and latent TB (area under the curve (AUC) 77.5%), whereas a combination of RAB33A, CXCL10, SEC14L1, FOXP3 and TNFRSF1A was effective in discriminating between TB disease and controls (AUC 91.7%). A combination of 11 biomarkers predicted latent TB with moderate discriminatory power (AUC 72.2%). In conclusion, RAB33A is a potential biomarker for TB disease, whereas CD4, TGFß-1 and IL-2, IL-13 may identify latent TB in children.

Mycobacterium tuberculosis Mce1 protein complex initiates rapid induction of transcription of genes involved in substrate trafficking.

The mammalian cell entry (Mce)1 protein complex has an important role during the initial phase of a Mycobacterium tuberculosis (M. tuberculosis) infection. Murine macrophages were infected with M. tuberculosis H37Rv or Δ-mce1 H37Rv, and total RNA was isolated from the host cells at 15, 30 and 60 min, and 4 and 10 h post-infection. With the aim of studying the role for the Mce1 protein complex on host gene expression, the RNA was hybridized onto 44 K whole-genome microarrays. Selected genes were verified by reverse-transcriptase quantitative PCR (RT-QPCR). 'Transport' was the most overrepresented biological process during the first hour post H37Rv infection. Five genes (Abca1 (21.0-fold), Slc16a10 (3.1-fold), Slc6a12 (17.9-fold), Slc6a8 (2.3-fold) and Nr1h3, (5.5-fold)) involved in substrate trafficking were verified by RT-QPCR to be upregulated by >2-fold 1 h post H37Rv infection. By 1 h post Δ-mce1 H37Rv infection, only Abca1 and Slc6a12 were upregulated by >2-fold. A number of other genes, which may be directly involved in substrate trafficking or share the same transcription, were found to have expression profiles similar to the genes involved in substrate trafficking. The Mce1 protein complex has a significant role in the transcriptional activation of genes involved in substrate trafficking during the initial phase of an M. tuberculosis infection.

CD161 DEFINES EFFECTOR T CELLS THAT EXPRESS LIGHT AND RESPOND TO TL1A-DR3 SIGNALING.

Expression of NK cell markers identifies pro-inflammatory T cell subsets in the liver and intestinal immune compartments. Specifically, CD161 is expressed on Th17 cells which play an important role in the regulation of mucosal inflammation. In this study, we characterized human peripheral blood CD161+ T cells as an effector population partially resembling a gut T cell phenotype. CD161+ CD4+ T cells express the gut-associated TNF family member, LIGHT, and respond to crosslinking of DR3, a receptor to another gut-associated cytokine, TL1A. Robust IFN-γ production in response to DR3 signaling correlated with enhanced expression of surface DR3 on CD161+ T cells and co-stimulation with IL12 and IL18. CD161+ T cell effector function was directly demonstrated by activation of responder monocytes in co-culture leading to CD40 upregulation and CD14 downregulation. CD161+ T cells reciprocally responded to activated monocytes, inducing expression of activation marker, CD69, and production of IL2 and IFN-γ, further demonstrating effective CD161+ T cell cross-talk with monocytes. Finally, CD161 defined a subset of T cells that co-express CD56, a second NK marker. Our findings implicate human CD161+ T cells in gut-associated signaling mechanisms, and suggest a monocyte mediated effector function in mucosal inflammation.

Impact of age and sex on mycobacterial immunity in an area of high tuberculosis incidence.

SETTING: The extent of immune reactivity measured by the tuberculin skin test (TST) and interferon-gamma (IFN-gamma) T-cell assays is usually not analysed. OBJECTIVE: To determine the impact of age and sex on assay positivity and on the extent of reactivity of both TST and T-cell assays in young persons in an area of South Africa with high TB transmission. RESULTS: Age had a strong impact on assay positivity for all seven immune phenotypes tested (P < 0.0007). Among positive responders, the extent of purified protein derivative (PPD) triggered IFN-gamma release (P < 0.003) was sensitive to age. ESAT-6 triggered IFN-gamma release (day 7, P = 0.03) and the frequency of PPD-specific IFN-gamma(+)CD4(+) (P = 0.03) and IFN-gamma(+)CD8(+) cells (P = 0.04) were weakly dependent on age. By contrast, the extent of TST induration was insensitive to age (P > 0.05), and sex had no significant impact on any phenotype measured (P > 0.05). The high proportion of positive responders in the 1-10 year age-group observed with long-term whole blood assays, but not with 3-day assays and TST, suggests that long-term whole blood assays may be confounded by bacille Calmette-Guérin vaccination in this age group. CONCLUSION: There is a significant impact of age, but not sex, on different assays of immune reactivity in this high TB transmission setting.

Strong purified protein derivative responses are associated with poor mycobacterium inhibition in latent TB.

The tuberculin skin test (TST) using purified protein derivative (PPD) of Mycobacterium tuberculosis is traditionally used to diagnose latent tuberculosis (TB) infection (LTBI). However, LTBI diagnosis by peripheral blood mononuclear cell (PBMC) interferon (IFN)-gamma responses to M. tuberculosis-specific antigens, early secreted antigenic target 6 kDa (ESAT-6) and culture filtrate protein (CFP)-10 has greater specificity. We investigated the difference in antimycobacterium cellular immunity in TB contacts who were strong TST reactors but nonresponsive to the ESAT-6/CFP-10 assay compared with those with concordant results. Healthy TB contacts were tested using the above two assays and mycobacterium survival was measured after co-culture of infected macrophages with their PBMCs. Whether PPD reactivity was tested by TST or by PBMC-specific IFN-gamma responses, strongly PPD-reactive TB contacts without ESAT-6/CFP-10 responsiveness showed significantly better mycobacterium inhibition activity than ESAT-6/CFP-10-responsive TB contacts with the same PPD reactivity. In the former group, stronger PPD reactivity was associated with improved mycobacterium killing, whereas ESAT-6/CFP-10 responders showed the opposite result. PPD-reactive ESAT-6/CFP-10-nonresponsive TB contacts in our population may have had protective immunity related to prior mycobacterium exposure. ESAT-6/CFP10-responsive TB contacts are more likely to have LTBI and, in this group, strong PPD reactivity may paradoxically be associated with poor mycobactericidal activity.

MHC class II tetramer guided detection of Mycobacterium tuberculosis-specific CD4+ T cells in peripheral blood from patients with pulmonary tuberculosis.

Novel diagnostic tools are needed to diagnose latent infection and to provide biologically meaningful surrogate markers to define cellular immune responses against Mycobacterium tuberculosis (MTB). Interferon gamma-based assays have recently been developed in addition to the more than 100-year-old tuberculin skin test (TST) for the immune diagnosis of MTB in blood. The advent of soluble MHC/peptide tetramer molecules allows to objectively enumerate antigen-specific T cells. We identified novel MHC class II-restricted MTB epitopes and used HLA-DR4 tetrameric complexes to visualize ex vivo CD4(+) T cells directed against the antigens Ag85B and the 19-kDa lipoprotein, shared between MTB and other Mycobacterium species, and CD4(+) T cells which recognize the MTB-associated ESAT-6 antigen. MTB-reactive CD4(+) T cells reside predominantly in the CD45RA(+) CD28(+) and CD45(-) CD28(+) T-cell subset and recognize naturally processed and presented MTB epitopes. HLA-DR4-restricted, Ag85B or ESAT-6-specific CD4(+) T cells show similar dynamics over time in peripheral blood mononuclear cells (PBMC) when compared with CD8(+) T cells directed against the corresponding HLA-A2-presented MTB epitopes in patients with pulmonary MTB infection and subsequent successful therapy. This was not found to be true for T-cell responses directed against the 19-kDa lipoprotein. The dissection of the cellular immune response in M. tuberculosis infection will enable novel strategies for monitoring MTB vaccine candidates and to gauge CD4(+) T cells directed against MTB.

Combination of the cationic surfactant dimethyl dioctadecyl ammonium bromide and synthetic mycobacterial cord factor as an efficient adjuvant for tuberculosis subunit vaccines.

Recombinant, immunodominant antigens derived from Mycobacterium tuberculosis can be used to effectively vaccinate against subsequent infection. However, the efficacy of these recombinant proteins is dependent on the adjuvant used for their delivery. This problem affects many potential vaccines, not just those for tuberculosis, so the discovery of adjuvants that can promote the development of cell-mediated immunity is of great interest. We have previously shown that the combination of the cationic surfactant dimethyl dioctadecyl ammonium bromide and the immunomodulator modified lipid A synergistically potentiates Th1 T-cell responses. Here we report a screening program for other adjuvants with reported Th1-promoting activity and identify a second novel adjuvant formulation that drives the development of Th1 responses with an extremely high efficacy. The combination of dimethyl dioctadecyl ammonium bromide and the synthetic cord factor trehalose dibehenate promotes strong protective immune responses, without overt toxicity, against M. tuberculosis infection in a vaccination model and thus appears to be a very promising candidate for the development of human vaccines.

Long-lived immune response to early secretory antigenic target 6 in individuals who had recovered from tuberculosis.

We sought to understand the persistence and relevance of the long-lived immune response to early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis in humans. ESAT-6 is recognized by memory cells involved in protection of animals against tuberculosis (TB). Recent reports also showed that ESAT-6 response can be recovered in patients with TB and in those soon after anti-TB therapy. We chose 18 individuals who had recovered from pulmonary TB (some in remission for >5 years), and 14 bacille Calmette-Guérin-vaccinated healthy individuals for this study. The results showed that peripheral blood mononuclear cells of 10 (55.6%) of 18 patients with TB remission responded to ESAT-6 with stimulation indices >3.0, whereas none of the healthy controls responded. Functional analysis showed that 13 (72.2%) of 18 patients with TB remission produced significant amounts of IFN-gamma in response to ESAT-6, whereas only 1 (7.1%) of the 14 healthy control subjects did so. It appears that responses to ESAT-6 can persist in individuals who had recovered from pulmonary TB.

Specific immune-based diagnosis of tuberculosis.

Current diagnostic tests for tuberculosis based on tuberculin have poor specificity, and both BCG vaccination and exposure to non-tuberculosis mycobacteria produce a response similar to that induced by infection with Mycobacterium tuberculosis. The identification of regions of the M. tuberculosis genome that are not present in BCG and non-tuberculous mycobacteria provides a unique opportunity to develop new highly specific diagnostic reagents. We describe the current status of attempts to exploit this information and summarise recent research that has used defined antigens for an accurate and rapid test for tuberculosis infection based on the detection of T cells sensitised to M. tuberculosis either by blood tests in vitro or skin tests in vivo.

Tuberculosis vaccines: developmental work and the future.

The last year in tuberculosis vaccine research has witnessed the initial flowering of the benefits promised by the tuberculosis genome sequencing product. Although the real benefits in terms of clinical treatments are yet to be realized, genomics is making its presence felt in the rapid identification and expression of proteins with vaccine potential from Mycobacterium tuberculosis, the definition of species-specific antigens for diagnostic use, and the construction of a variety of novel living vectors for vaccination. At the same time, the recent increase in work on animal models with more direct applicability to the situations likely to be encountered in human vaccine trials are providing the basic underpinnings needed for the assessment of these new vaccines.

Prognostic value of coronary electron-beam computed tomography for coronary heart disease events in asymptomatic populations.

The predictive ability of electron-beam computed tomography (EBCT) for coronary heart disease outcomes, particularly hard coronary outcomes (myocardial infarction or death), has been questioned in asymptomatic populations. Our objective was to synthesize data on the use of EBCT for determining cardiovascular prognosis in asymptomatic populations. Studies were identified using standard systematic review methods. The outcome of interest was relative risk for myocardial infarction or sudden death, and combined events including revascularization. Nine articles met the inclusion criteria, of which 5 were of independent studies. Using meta-analytic techniques to synthesize prognostic data, there was an increased risk (summary risk ratio 8.7, 95% confidence interval 2.7 to 28.1) of a combined outcome of nonfatal myocardial infarction or death or revascularization if the calcium score was above a median score. Similarly, there was an increased risk for hard events: myocardial infarction or death (summary risk ratio 4.2, 95% confidence interval 1.6 to 11.3). However, there was significant heterogeneity in the studies' quality and patient populations. Although EBCT appears to predict combined and hard coronary outcomes similarly in high risk, asymptomatic populations, these results should be interpreted with caution. Further study is needed on the incremental value of EBCT over conventional risk prediction before this test is used in screening asymptomatic populations.

Screening for occult coronary artery disease with radiographic detection of coronary calcification.

Coronary atherosclerosis is ubiquitous among adults, yet many afflicted persons will suffer no coronary events. Atherosclerotic plaque formation in the coronary arteries is a dynamic process, and the onset of a coronary event is often unheralded, sudden, and lethal. In addition, it is known that the amount of calcification in the coronary arteries correlates with the amount of atherosclerosis in different persons and, to a lesser degree, in segments of the coronary tree in the same person. Radiographic imaging methods, including fluoroscopy, electron-beam computed tomography, and helical computed tomography, can detect coronary calcium and seem to be able to diagnose coronary atherosclerosis. However, data on the relationship between quantity of coronary calcium and event likelihood are limited. Thus, the diagnostic value and, particularly, the prognostic value of calcium detection are controversial and may be applicable only to certain subgroups of patients.

Alcohol consumption, coronary calcium, and coronary heart disease events.

This study was performed to determine if alcohol intake was associated with reduced coronary risk in a high-risk asymptomatic population, and whether this effect was independent of coronary risk factors and coronary calcium. In 1,196 asymptomatic subjects with coronary risk factors, we assessed alcohol consumption history, performed risk factor measurements, and quantified coronary calcium with electron beam computed tomography. These subjects were then followed for a mean of 41 months, and coronary events (myocardial infarction or coronary death) were noted. Significant inverse predictors of coronary events included alcohol use and serum high-density lipoprotein cholesterol level. Direct predictors of events were history of systemic hypertension, smoking, diabetes mellitus, serum cholesterol, and coronary calcium score. Subjects with coronary calcium were 3.1 times more likely to suffer a coronary event than those without calcium (95% confidence interval [CI] limits 1.3 to 7.2). Subjects who drank alcohol had a relative risk of 0.3 (95% CI limits 0.2 to 0.6) for developing coronary events. After controlling for age, gender, and other risk factors with logistic regression, these differences in relative risk persisted (relative risk 0.58; 95% CI limits 0.41 to 0.82). Alcohol consumption is a significant inverse predictor of coronary events, comparable in magnitude to standard risk factors and to radiographically measured coronary calcium. This effect is independent of coronary risk factors and coronary calcium.

T-cell recognition of Mycobacterium tuberculosis culture filtrate fractions in tuberculosis patients and their household contacts.

We examined the immune responses of patients with active pulmonary tuberculosis (TB) and their healthy household contacts to short-term culture filtrate (ST-CF) of Mycobacterium tuberculosis or molecular mass fractions derived from it. Our goal was to identify fractions strongly recognized by donors and differences among the donor groups of possible relevance for vaccine development. The study population consisted of 65 human immunodeficiency virus-negative donors from the Hossana Regional Hospital, Hossana, Ethiopia. Peripheral blood leukocytes from the donors were stimulated with different antigens and immune responses were determined. Household contacts produced significantly higher levels of gamma interferon (IFN-gamma) than the TB patients in response to antigens present in ST-CF and the 10 narrow-molecular-mass fractions. A similar difference in leukocyte proliferative responses to the antigens between the two groups was also found. In general, while all fractions stimulated immune responses, the highest activity was seen with the low-molecular-mass fractions, which include well-defined TB antigens such as ESAT-6. Leukocytes from contacts of TB patients with severe disease produced higher levels of antigen-specific IFN-gamma than those from contacts of patients with minimal disease. Both groups of contacts exhibited higher cell-mediated responses than the patients themselves. The enhanced immune response of healthy contacts, especially those of patients with severe disease, to secreted mycobacterial antigens is suggestive of an early stage of infection by M. tuberculosis, which could in time result in overt disease or containment of the infection. This possibility is currently being investigated by follow-up studies of the household contacts.

Racial differences in the significance of coronary calcium in asymptomatic black and white subjects with coronary risk factors.

OBJECTIVES: To compare the significance of a specific feature of coronary atherosclerosis--coronary calcium--in asymptomatic black and white subjects with coronary risk factors. BACKGROUND: The natural history and clinical evolution of coronary atherosclerosis differs between blacks and whites. Differences in the underlying pathobiology of atherosclerosis may be one determinant of the ethnic variability in the clinical manifestation of coronary atherosclerosis. METHODS: In 1,375 high-risk but asymptomatic subjects (93 blacks [6.8%] and 1,282 whites [93.2%]) with at least one risk factor but no prior evidence of coronary disease, we assessed coronary risk factors, calculated Framingham risk of a coronary event and evaluated coronary calcium with digital subtraction fluoroscopy. We then followed these subjects clinically for 70 +/- 13 months, noting the occurrence of the following coronary events: death due to coronary heart disease (CHD); myocardial infarction (MI); angina pectoris; and performance of coronary bypass or angioplasty. RESULTS: Risk factor profiles were similar in black and white subjects (6-year Framingham risk 15 +/- 7% in blacks, 14 +/- 8% in whites [NS]). Coronary calcium was present in 59.9% of white subjects but only 35.5% of black subjects (p = 0.0001). Nevertheless, after 70 months of follow-up, more blacks than whites (22 blacks [23.7%] vs. 190 whites [14.8%]; p = 0.04) suffered one of the following end points: CHD death, MI, angina or revascularization. The age, gender and coronary risk-adjusted odds ratio of black race for at least one event was 2.16 (95% CI 1.34 to 3.48). CONCLUSIONS: Despite having a lowered prevalence of coronary calcium than high risk whites, high risk blacks suffer more CHD events. Coronary calcium therefore does not carry the same pathobiologic significance in blacks that it does in whites, consistent with the concept that there are specific racial differences in the natural history of CHD and its evolution into clinically manifest events.

BCL-6-deficient mice reveal an IL-4-independent, STAT6-dependent pathway that controls susceptibility to infection by Leishmania major.

The BCL-6 gene negatively regulates Th2 responses as shown by the finding that BCL-6-deficient (BCL-6-/-) mice develop a lethal Th2-type inflammatory disease. The response of inbred mouse strains to infection with Leishmania major is under genetic control; BALB/c mice are susceptible and develop a progressive parasite burden, whereas most other common laboratory strains of mice are resistant to infection. We found that BCL-6-/- mice on a resistant genetic background (C57BL/6 x 129 intercrossed mice) were highly susceptible to L. major infection; they resembled BALB/c mice in terms of lesion size, parasite load, and the production of Th2 cytokines. BCL-6-/-IL-4-/- double-mutant mice were also susceptible to L. major infection and produced 10-fold higher levels of the Th2 cytokine IL-13 than IL-4-/- littermate controls. By contrast, BCL-6-/-STAT6-/- double-mutant mice were resistant to L. major infection despite also producing elevated levels of IL-13. These results show that STAT6 is required for susceptibility to L. major infection and suggest that IL-13 signaling through STAT6 may contribute to a nonhealing, exacerbated L. major infection.

Infection of HIV-1 transgenic mice with Mycobacterium avium induces the expression of infectious virus selectively from a Mac-1-positive host cell population.

Infection of HIV-1-transgenic mice with Mycobacterium avium, a common opportunistic pathogen in AIDS patients, was shown to result in increased tissue expression of viral specific transcripts. Moreover, by coculturing splenocytes from the transgenic animals with human T cells it was possible to demonstrate that the elevation in HIV-1 mRNA triggered by M. avium infection reflects increased production of infectious virions. Viral immune activation was also shown to correlate with a marked elevation of p24 in supernatants of ex vivo-cultured tissues and, more importantly, in systemic increases in the HIV-1 protein in plasma. Interestingly, these tissue and systemic p24 responses were found to be differentially regulated. Thus, while in vitro p24 production by cultured splenocytes increased concurrently with bacterial loads during the first 6 wk of infection, levels of the Ag in plasma actually decreased. In situ localization experiments together with FACS analysis of HIV-1-expressing splenocytes indicated that virus production is restricted largely to cells of the monocyte/macrophage lineage. Indeed, in vitro p24 expression by cells from noninfected transgenic mice was up-regulated by polyclonal stimulation of macrophages but not T cells. Together these results underscore the importance of the macrophage reservoir in persistent virus expression and establish a convenient and relevant animal model for studying the factors responsible for immune activation of HIV-1 induced by mycobacterial as well as other common coinfections encountered by AIDS patients.

Pregnancy-specific glycoprotein gene expression in recurrent aborters: a potential correlation to interleukin-10 expression.

PROBLEM: The expression of the pregnancy-specific glycoprotein (PSG) genes in the uterine endometrium of women experiencing recurrent first-trimester abortions, and potential correlations to cytokine expression were examined. METHOD OF STUDY: Endometrial RNA, isolated from women with a history of either repetitive first-trimester pregnancy losses or uncomplicated pregnancies, was isolated and analyzed for PSG transcripts by the reverse transcriptase-polymerase chain reaction method. PSG genes showing different patterns of expression were expressed in baculovirus, and the purified proteins examined for their effects on cytokine expression. RESULTS: The expression of PSG11 in the endometria of recurrent aborters was significantly lower than in that of controls (P < 0.01). When tested on monocytes, PSG11 stimulated secretion of interleukin (IL)-10. CONCLUSIONS: The level of expression of the PSG11 gene in the uterine endometrium, during the peri-implantation period, correlates with the risk of pregnancy loss in some women experiencing recurrent spontaneous abortions. The ability of PSG11 to influence the secretion of IL-10 suggests that PSG11 may contribute to the local modulation of the inflammatory T helper-1 response seen in the endometrium of these women.