Immuno-Diagnosis of Active Tuberculosis by a Combination of Cytokines/Chemokines Induced by Two Stage-Specific Mycobacterial Antigens: A Pilot Study in a Low TB Incidence Country.

Active tuberculosis (aTB) remains a major killer from infectious disease, partially due to delayed diagnosis and hence treatment. Classical microbiological methods are slow and lack sensitivity, molecular techniques are costly and often unavailable. Moreover, available immuno-diagnostic tests lack sensitivity and do not differentiate between aTB and latent TB infection (LTBI). Here, we evaluated the performance of the combined measurement of different chemokines/cytokines induced by two different stage-specific mycobacterial antigens, Early-secreted-antigenic target-6 (ESAT-6) and Heparin-binding-haemagglutinin (HBHA), after a short in vitro incubation of either peripheral blood mononuclear cells (PBMC) or whole blood (WB). Blood samples were collected from a training cohort comprising 22 aTB patients, 22 LTBI subjects and 17 non-infected controls. The concentrations of 13 cytokines were measured in the supernatants. Random forest analysis identified the best markers to differentiate M. tuberculosis-infected from non-infected subjects, and the most appropriate markers to differentiate aTB from LTBI. Logistic regression defined predictive abilities of selected combinations of cytokines, first on the training and then on a validation cohort (17 aTB, 27 LTBI, 25 controls). Combining HBHA- and ESAT-6-induced IFN-γ concentrations produced by PBMC was optimal to differentiate infected from non-infected individuals in the training cohort (100% correct classification), but 2/16 (13%) patients with aTB were misclassified in the validation cohort. ESAT-6-induced-IP-10 combined with HBHA-induced-IFN-γ concentrations was selected to differentiate aTB from LTBI, and correctly classified 82%/77% of infected subjects as aTB or LTBI in the training/validation cohorts, respectively. Results obtained on WB also selected ESAT-6- and HBHA-induced IFN-γ concentrations to provided discrimination between infected and non-infected subjects (89%/90% correct classification in the training/validation cohorts). Further identification of aTB patients among infected subjects was best achieved by combining ESAT-6-induced IP-10 with HBHA-induced IL-2 and GM-CSF. Among infected subjects, 90%/93% of the aTB patients were correctly identified in the training/validation cohorts. We therefore propose a two steps strategy performed on 1 mL WB for a rapid identification of patients with aTB. After elimination of most non-infected subjects by combining ESAT-6 and HBHA-induced IFN-γ, the combination of IP-10, IL-2 and GM-CSF released by either ESAT-6 or HBHA correctly identifies most patients with aTB.

Differential expression of maturation and activation markers on NK cells in patients with active and latent tuberculosis.

NK cells were recently suggested to be important for the initial control of M. tuberculosis infection. The phenotypes of the 3 main NK blood subsets, CD56bright , CD56dim , and CD56neg cells, were characterized by flow cytometry in a cohort of 81 prospectively enrolled subjects (21 untreated patients with active tuberculosis -aTB-, 35 latently TB infected -LTBI- subjects, and 25 non-infected controls), using 9 different mAbs added to whole blood. Compared to LTBI subjects, patients with aTB had lower proportions of total NK cells, lower proportions and numbers of CD56neg cells expressing early maturation markers (CD161, NKp30, NKp46), but higher density of NKp30 and NKp46 expression on both CD56neg and CD56dim subsets, associated with higher expression of granzymes A/B. They also had higher proportions of activated CD69pos cells within all 3 NK cell subsets and, the percentage of CD69pos CD56dim cells among CD69pos and/or NKG2Cpos NK cells was identified as a potential biomarker to discriminate aTB from LTBI. LTBI subjects were in contrast characterized by higher expression of late maturation markers (CD57, KIR molecules) on the CD56neg subset, by higher proportions of NKG2Cpos KIRpos CD56dim NK cells, and by higher in vitro IFN-γ production than patients with aTB. Thus, the in-depth phenotypic characterization of blood NK cell subsets provides new insights on possible functional modifications and the potential role of NK cells in the control of M. tuberculosis infection in humans.

Tuberculosis Risk Stratification of Psoriatic Patients Before Anti-TNF-α Treatment.

Psoriasis is a skin inflammatory condition for which significant progress has been made in its management by the use of targeted biological drugs. Detection of latent M. tuberculosis infection (LTBI) is mandatory before starting biotherapy that is associated with reactivation risk. Together with evaluation of TB risk factors and chest radiographs, tuberculin skin tests (TST) and/or blood interferon-γ-release assays (IGRA), like the QuantiFERON (QFT), are usually performed to diagnose M. tuberculosis infection. Using this approach, 14/49 psoriatic patients prospectively included in this study were identified as LTBI (14 TST+, induration size ≥ 10mm, 8 QFT+), and 7/14 received prophylactic anti-TB treatment, the other 7 reporting past-treatment. As the specificity and sensitivity of these tests were challenged, we evaluated the added value of an IGRA in response to a mycobacterial antigen associated with latency, the heparin-binding haemagglutinin (HBHA). All but one TST+ patient had a positive HBHA-IGRA, indicating higher sensitivity than the QFT. The HBHA-IGRA was also positive for 12/35 TST-QFT- patients. Measurement for 15 psoriatic patients (12 with HBHA-IGRA+) of 8 chemokines in addition to IFN-γ revealed a broad array of HBHA-induced chemokines for TST+QFT- and TST-QFT- patients, compared to a more restricted pattern for TST+QFT+ patients. This allowed us to define subgroups within psoriatic patients characterized by different immune responses to M. tuberculosis antigens that may be associated to different risk levels of reactivation of the infection. This approach may help in prioritizing patients who should receive prophylactic anti-TB treatment before starting biotherapies in order to reduce their number.

Specific Host Signatures for the Detection of Tuberculosis Infection in Children in a Low TB Incidence Country.

Diagnosis of tuberculosis (TB) in children remains challenging due to unspecific clinical presentation and low bacillary load. In low TB incidence countries, most cases are diagnosed by a contact screening strategy after exposure to an index TB case. Due to the severity of TB in young children, the priority is to determine whether a child is infected or not, whereas differential diagnosis between active TB (aTB) and latent TB constitutes a second step. In Belgium, a low TB incidence country, we prospectively included 47 children with a defined M. tuberculosis infection status (12 children with aTB, 18 with latent TB, and 17 uninfected) (exploratory cohort), and determined the optimal combinations of cytokines secreted by their peripheral blood mononuclear cells in response to a 5-days in vitro stimulation with four different mycobacterial antigens, in an attempt to classify the children according to their infectious status. Correct identification of all infected children was obtained by several combinations of two purified protein derivative (PPD)-induced cytokines (IFN-γ and either GM-CSF, MIP-1α, sCD40L or TNF-α), or by combining PPD-induced IFN-γ with culture-filtrate protein-10 (CFP-10)-induced TNF-α. Alternatively, combining CFP-10-induced TNF-α and IP-10 with heparin-binding haemagglutinin (HBHA)-induced-IFN-γ was more effective in testing recently BCG-vaccinated children or those suspected to be infected with non-tuberculous mycobacteria, providing a correct classification of 97% of the M. tuberculosis-infected children. This combination also correctly classified 98% of the children from a validation cohort comprising 40 M. tuberculosis infected children and 20 non-infected children. Further differentiation between aTB and children with latent TB was more difficult. Combining ESAT-6-induced MIP1-α and IP-10, CFP-10-induced MIG, and HBHA-induced MIG provided a correct classification of 77% of the children from the exploratory cohort but only of 57.5% of those from the validation cohort. We conclude that combining the measurement of 2-4 cytokines induced by three different mycobacterial antigens allows an excellent identification of M. tuberculosis-infected children, whereas differentiating children with aTB from those with latent TB remains far from perfect.

Early diagnosis of miliary tuberculosis in a hemodialysis patient by combining two interferon-γ-release assays: a case report.

BACKGROUND: Patients with end-stage renal disease undergoing chronic hemodialysis (HD) are at high risk to develop tuberculosis (TB) associated with a high mortality rate. TB diagnosis is often delayed due to non-specific symptoms, frequent extra-pulmonary manifestations, and rare microbiological confirmation. This case report illustrates the clear added value of combined interferon-γ -release assays (IGRA) in response to different mycobacterial antigens for an early diagnosis of TB in HD patients. CASE PRESENTATION: We report the case of an Egyptian patient under chronic HD treatment, who presented with recurrent episodes of fever and myalgia of unknown origin, associated with an important inflammatory syndrome. These episodes resolved partially or completely within less than 1 month without any treatment but recurred 10 times within 3 years. Chest Computed Tomography and 18F-fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (18FDG PET-CT) demonstrated several active mediastinal lymphadenopathies. TB was the first suspected diagnosis but cultures and polymerase chain reaction (PCR) remained negative on a mediastinal lymph node aspiration. In contrast, the results from two different IGRA performed on blood were highly suggestive of TB disease. Several granulomas, some of them with central non-caseating necrosis, were demonstrated on a pulmonary nodule obtained by thoracoscopic resection, but PCR and culture remained negative for M. tuberculosis. Three years after the initial symptoms, a new PET-CT revealed a retro-clavicular lymphadenopathy in addition to the mediastinal lymphadenopathies, and the M. tuberculosis culture performed on the resected lymphadenopathy was positive. Antibiotic treatment for TB was started and resulted in a clear improvement of the patient's clinical condition, allowing him to successfully receive a renal graft. CONCLUSIONS: In view of the high frequency of TB in patients undergoing chronic HD and of the limitations of the classical diagnosis procedures, nephrologists have to diagnose TB mostly on clinical suspicion. We demonstrate here that the use of a combined IGRA to two different mycobacterial antigens may significantly raise the index of suspicion and help clinicians to decide starting anti-TB treatment in HD patients.

Tuberculosis Transmission in a Primary School and a Private Language School. An Estimation of Infectivity.

Introduction: Belgium is a country with low incidence of tuberculosis (TB) and a very low number of TB cases in children. Children in contact with an adult smear-positive TB case are at high risk of transmission. Early diagnosis is important as young children have a significant predisposition of developing TB disease. In this paper, we describe two outbreaks after exposure to, respectively, two teachers with smear-positive pulmonary TB: one in a primary school, a nursery teacher, and another in a private language school. Methods: An exposure investigation was carried out in both index cases household and school, according to the stone-in-the-pond principle. The tuberculin skin test (TST) was used a screening tool. The time elapsed between TB diagnosis in the index case and contact investigation was, respectively, 1 and 3 weeks. If this initial test was negative, it was repeated after a "window period" of ≥8 weeks. Results: Index cases showed a transmission rate of, respectively, 13 and 40% in their classes at school, defined as casual contacts. The proximity of contact increased the risk of infection. TB disease was observed in, respectively, 4 and 11% of all the casual contacts; all of them were children younger than 5 years old. TB-infected and children with active TB disease had good compliance with recommended treatment. Uptake of chemoprophylaxis during the "window period" was poor, respectively, only 32-42%, in children under 5 years with an initially negative TST. Discussion: The World Health Organization recommends to screen all young children (<5 years old) who have close contact with a person affected by pulmonary TB and to initiate Latent tuberculosis infection treatment even before infection can be demonstrated, after ruling out active TB disease. Despite this knowledge, a small percentage of the children younger than 5 years with no proof of infection was treated with the proposed chemoprophylactic treatment, in both cases. Conclusion: This exposure investigation of two teachers detects high transmission among family contacts and school casual contacts. Recommendations for chemoprophylactic treatment in children <5 years showed low compliance, reflecting the difficulty of communication to staff, parents, and children in a school outbreak. It is essential to develop a new approach for this vulnerable group of patients. This approach could be improved, applied, and evaluated by National TB Control Programs, involving public and private health services. Public health authorities play a role in raising public awareness about the risks of TB for young children.

Identification of Mycobacterium tuberculosis Infection in Infants and Children With Partial Discrimination Between Active Disease and Asymptomatic Infection.

Background: Improved diagnostic tests are needed for the early identification of Mycobacterium tuberculosis-infected young children exposed to an active TB (aTB) index case. We aimed to compare the diagnostic accuracy of new blood-based tests to that of the tuberculin skin test (TST) for the identification of all infected children and for a potential differentiation between aTB and latent TB infection (LTBI). Methods: 144 children exposed to a patient with aTB were included, and those who met all inclusion criteria (130/144) were classified in three groups based on results from classical investigations: non-infected (NI: n = 69, 53%, median age 10 months), LTBI (n = 28, 22%, median age 96 months), aTB disease (n = 33, 25%, median age 24 months). The first whole blood assay consisted of a 7-days in vitro stimulation of blood with four different mycobacterial antigens (40 µl/condition), followed by flow cytometric measurement of the proportions of blast cells appearing among lymphocytes as a result of their specific activation. Thresholds of positivity were determined by Receiver Operating Characteristic (ROC) curve analysis (results of NI children vs. children with LTBI/aTB) in order to identify infected children in a first stage. Other cut-offs were determined to discriminate subgroups of infected children in a second step (results from children with aTB/LTBI). Analysis of blood monocytes and dendritic cell subsets was performed on 100 µl of blood for 25 of these children as a second test in a pilot study. Results: Combining the results of the blast-induced CD3+ T lymphocytes by Heparin-Binding Haemagglutinin and by Culture Filtrate Protein-10 identified all but one infected children (sensitivity 98.2% and specificity 86.9%, compared to 93.4 and 100% for the TST). Further identification among infected children of those with aTB was best achieved by the results of blast-induced CD8+ T lymphocytes by purified protein derivative (sensitivity for localized aTB: 61.9%, specificity 96.3%), whereas high proportions of blood type 2 myeloid dendritic cells (mDC) were a hallmark of LTBI. Conclusions: New blood-based tests requiring a very small volume allow the accurate identification of M. tuberculosis-infected young children among exposed children and are promising to guide the clinical classification of children with aTB or LTBI.

Assessment of humoral and cell-mediated immune responses to pertussis vaccination: a systematic review protocol.

INTRODUCTION: Globally, some studies show a resurgence of pertussis. The risks and benefits of using whole-cell pertussis (wP) or acellular pertussis (aP) vaccines in the control of the disease have been widely debated. Better control of pertussis will require improved understanding of the immune response to pertussis vaccines. Improved understanding and assessment of the immunity induced by pertussis vaccines is thus imperative. Several studies have documented different immunological outcomes to pertussis vaccination from an array of assays. We propose to conduct a systematic review of the different immunological assays and outcomes used in the assessment of the humoraland cell-mediated immune response following pertussis vaccination. METHODS AND ANALYSIS: The primary outcomes for consideration are quality and quantity of immune responses (humoral and cell-mediated) post-pertussis vaccination. Of interest as secondary outcomes are types of immunoassays used in assessing immune responses post-pertussis vaccination, types of biological samples used in assessing immune responses post-pertussis vaccination, as well as the types of antigens used to stimulate these samples during post-pertussis vaccination immune response assessments. Different electronic databases (including PubMed, Cochrane, EBSCO Host, Scopus and Web of Science) will be accessed for peer-reviewed published and grey literature evaluating immune responses to pertussis vaccines between 1990 and 2019. The quality of included articles will be assessed using standardised risk and quality assessment tools specific to the study design used in each article. Data extraction will be done using a data extraction form. The extracted data will be analysed using STATA V.14.0 and RevMan V.5.3 software. A subgroup analysis will be conducted based on the study population, type of vaccine (wP or aP) and type of immune response (cell-mediated or humoral). Guidelines for reporting systematic reviews in the revised 2009 Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement will be used in this study. ETHICS AND DISSEMINATION: Ethics approval is not required for this study as it is a systematic review. We will only make use of data already available in the public space. Findings will be reported via publication in a peer-reviewed journal and presented at scientific meetings and workshops. TRIAL REGISTRATION NUMBER: CRD42018102455.

Proportions of interferon-γ-producing ascites lymphocytes in response to mycobacterial antigens: A help for early diagnosis of peritoneal tuberculosis in a low TB incidence country.

BACKGROUND: Peritoneal tuberculosis (TB) remains difficult to diagnose because of its non-specific clinical features and the lack of efficient microbiological tests. As delayed diagnosis is associated with high mortality rates, new diagnostic tools are needed. METHODS AND FINDINGS: We investigated for 24 patients prospectively enrolled with a possible diagnosis of peritoneal TB, the diagnostic value of the analysis of IFN-γ production by peritoneal fluid lymphocytes in response to a short in vitro stimulation with mycobacterial antigens. The patients were classified in two groups: non-TB and confirmed or highly probable TB. Diagnosis of TB was based on microbiological and histopathological criteria and/or a favorable response to anti-TB treatment. The IFN-γ production by peritoneal CD4+ T lymphocytes was analyzed by flow cytometry after an overnight in vitro stimulation with three different mycobacterial antigens, purified protein derivative (PPD), heparin-binding haemagglutinin (HBHA) or early-secreted-antigen-target-6 (ESAT-6). The percentages of PPD-, HBHA- or ESAT-6-induced IFN-γ-producing peritoneal fluid CD4+ T lymphocytes were higher in the TB group than in the non-TB group (p = 0.0007, p = 0.0004, and p = 0.0002 respectively). Based on cut-off values determined by ROC curve analysis of the results from TB and highly probable TB compared to those of non-TB patients, the sensitivity of these three tests was 100% with a specificity of 92%. CONCLUSIONS: The analysis of mycobacterial-induced IFN-γ production by peritoneal lymphocytes is a promising tool to reliably and rapidly diagnose peritoneal TB. Further studies should be performed on larger cohorts of patients in high-TB-incidence countries to confirm the clinical value of this new diagnostic approach for peritoneal TB.

2018 Belgian guidelines for the screening for latent tuberculosis in HIV-infected patients.

Objectives: To review the current knowledge on screening for latent tuberculosis infection (LTBI) in HIV-infected adults and provide specific guidelines for Belgium. Focus is given to who to test, which testing method to use, timing of screening and choice of LTBI treatment. Methods: Expert review by the members of the Belgian LTBI group, in consultancy with the ARC College. Results: Target population, timing of screening, testing method, active TB exclusion, treatment of LTBI and guideline implementation are all reviewed. Conclusions: The principal changes include a selective approach to screen for LTBI (screening only of the HIV-infected patients at highest risk of active TB) as well as the timing of screening (testing for LTBI performed only after immune-restauration by antiretroviral therapy).

Granzyme B induced by Rv0140 antigen discriminates latently infected from active tuberculosis individuals.

Nearly two billion people are latently infected with Mtb (LTBI). Detection of LTBI with high risk to develop active tuberculosis (aTB) is considered the cornerstone to control the disease. The current challenge is to identify markers that better classify LTBI versus aTB. It has been previously shown that Rv0140, a reactivation-associated antigen of Mtb, induces significantly higher IFN-γ production in LTBI individuals as compared to aTB patients. Herein, we show that Rv0140 induces high granzyme B level by PBMCs derived from LTBI (n = 34) as compared to aTB (n = 18). Receiving operator characteristic (ROC) curves were used to evaluate the capacity of Rv0140 to discriminate between LTBI and aTB by measuring IFN-γ and granzyme B secretion. Our results show that, in response to Rv0140, granzyme B seems to allow better discrimination of LTBI from aTB with areas under the curve (AUC) of 0.88 (95% CI 0.79-0.98) as compared to IFN-γ with AUC of 0.85 (95% CI 0.74-0.96) even though CI overlap. Intracellular staining (ICS) experiments and the use of anti-MHC I antibody showed that granzyme B is mainly produced by CD8+ T cells in response to Rv0140. Thus, we propose granzyme B as a host marker to help identify LTBI individuals.

HBHA-Induced Polycytotoxic CD4+ T Lymphocytes Are Associated with the Control of Mycobacterium tuberculosis Infection in Humans.

Heparin-binding hemagglutinin (HBHA), a surface protein of Mycobacterium tuberculosis, is an attractive vaccine candidate and marker of protective immunity against tuberculosis, although the mechanisms underlying this protective immunity are not fully understood. Comparisons of the immune responses of latently M. tuberculosis-infected (LTBI) subjects to those of patients with active tuberculosis (aTB) may help to identify surrogate markers of protection, as LTBI subjects are most often lifelong protected against the disease. HBHA was shown to induce strong Th1 responses and cytotoxic CD8+ responses in LTBI subjects, but additional mechanisms of control of M. tuberculosis infection remain to be identified. In this study, using HBHA-induced blast formation as a readout of specific T lymphocyte activation, we report the presence in M. tuberculosis-infected subjects of HBHA-induced CD4+ T cell blasts that degranulate, as measured by surface capture of CD107a. This suggests the induction by HBHA of a CD4+ T cell subset with cytolytic function, and as nearly half of these cells also contained IFN-γ, they had both Th1 and cytotoxic characteristics. We further identified a CD4+ T lymphocyte subset producing IFN-γ together with a combination of mediators of cytotoxicity, i.e., perforin, granzymes, and granulysin, and we called them polycytotoxic CD4+ T lymphocytes. Interestingly, whereas purified protein derivative induced such cells in both LTBI subjects and patients with aTB, HBHA-specific polycytotoxic CD4+ T lymphocytes were detected in LTBI subjects and not in patients with pulmonary aTB. To our knowledge, we thus identified a new HBHA-induced CD4+ T cell subset that may contribute to the control of M. tuberculosis infection.

Blood tolerogenic monocytes and low proportions of dendritic cell subpopulations are hallmarks of human tuberculosis.

BACKGROUND: The immune mechanisms underlying the pathogenesis of tuberculosis (TB) need better understanding to improve TB management, as the disease still causes more than 1.5 million deaths annually. This study tested the hypothesis that a modulation of the proportions or activation status of APC during Mycobacterium tuberculosis infection may impact on the course of the disease. PROCEDURE: Proportions of circulating APC subsets and the expression of stimulatory (CD86), inhibitory (ILT-3, ILT-4, ILT-7), or apoptosis-inducing (PDL-1, PDL-2) molecules were analyzed in 2 independent cohorts, on blood monocytes and dendritic cell (DC) subsets from patients with active or latent TB infection (aTB /LTBI) and from uninfected subjects. RESULTS: Higher proportions of classical CD14+ CD16- and intermediate CD14+ CD16+ monocytes, and lower proportions of plasmacytoid DC (pDC) and type 2 myeloid DC were observed in the blood from untreated patients with aTB compared with those with LTBI and with healthy subjects, with an early normalization of the proportions of pDC during treatment. In addition, monocytes from M. tuberculosis-infected subjects expressed higher levels of ILT-3, ILT-4, and PDL-1 compared with healthy controls, these differences being more important for patients with aTB than for those with LTBI. CONCLUSIONS: These results confirm the hypothesis of a modulation of the proportions and activation status of APC during M. tuberculosis infection and suggest that these cells could play a role in driving the course of M. tuberculosis infection.

Age-Stratified T Cell Responses in Children Infected with Mycobacterium tuberculosis.

Tuberculosis (TB) in young children differs from adult TB in that the risk of rapid progression to active TB (aTB) is higher in children than in adults. The reasons for this increased risk are not fully understood. Early differentiation remains difficult between children at risk to develop aTB from those who will remain healthy and develop a latent TB infection (LTBI). Biomarkers to differentiate aTB from LTBI in children, especially in very young children, are urgently needed. To identify M. tuberculosis-specific functional T cell subsets related to clinical manifestations in children, we enrolled 87 children exposed to M. tuberculosis. After standard clinical assessment, the children were classified as aTB, LTBI, or uninfected. Their CD4+ T cell cytokine profiles (IFN-γ, TNF-α, IL-2, IL-17) were analyzed at the single-cell level by flow cytometry after stimulation with three mycobacterial antigens, purified protein derivative (PPD), early-secreted-antigenic target-6 (ESAT-6), or heparin-binding hemagglutinin (HBHA). This approach identified age-related discriminative markers between aTB and LTBI. Whereas among the 3- to 15-year-old children, an excellent discrimination between aTB and LTBI was provided by comparing the ratio between the proportions of ESAT-6-induced IFN-γsingle+ and ESAT-6-induced TNF-αsingle+CD4+ T lymphocytes, this was not the case for children younger than 3 years. By contrast, in this group (<3years), the analysis of HBHA-induced IL-17single+CD4+ T lymphocytes allowed us to identify children with LTBI by the high proportion of this cellular lymphocyte subset, whereas this was not the case for children with aTB. The analysis at the single-cell level of T cell immune responses induced by mycobacterial antigens are, thus, different in infected children younger or older than 3 years of age. HBHA-induced IL-17 production by CD4+ T lymphocytes was associated with protection only in children under 3 years who are at high risk for rapid progression to aTB. This suggests that the HBHA-induced IL-17 production by CD4+ T lymphocytes is a potential new correlate of protection against M. tuberculosis in humans, and that the distinction between children with LTBI and those with aTB is possible based on age-related diagnostic markers.

MCL-1 Is a Key Antiapoptotic Protein in Human and Rodent Pancreatic ß-Cells.

Induction of endoplasmic reticulum stress and activation of the intrinsic apoptotic pathway is widely believed to contribute to ß-cell death in type 1 diabetes (T1D). MCL-1 is an antiapoptotic member of the BCL-2 protein family, whose depletion causes apoptosis in rodent ß-cells in vitro. Importantly, decreased MCL-1 expression was observed in islets from patients with T1D. We report here that MCL-1 downregulation is associated with cytokine-mediated killing of human ß-cells, a process partially prevented by MCL-1 overexpression. By generating a ß-cell-specific Mcl-1 knockout mouse strain (ßMcl-1KO), we observed that, surprisingly, MCL-1 ablation does not affect islet development and function. ß-Cells from ßMcl-1KO mice were, however, more susceptible to cytokine-induced apoptosis. Moreover, ßMcl-1KO mice displayed higher hyperglycemia and lower pancreatic insulin content after multiple low-dose streptozotocin treatment. We found that the kinase GSK3ß, the E3 ligases MULE and ßTrCP, and the deubiquitinase USP9x regulate cytokine-mediated MCL-1 protein turnover in rodent ß-cells. Our results identify MCL-1 as a critical prosurvival protein for preventing ß-cell death and clarify the mechanisms behind its downregulation by proinflammatory cytokines. Development of strategies to prevent MCL-1 loss in the early stages of T1D may enhance ß-cell survival and thereby delay or prevent disease progression.

Immune Activation by Mycobacterium tuberculosis in HIV-Infected and -Uninfected Subjects.

INTRODUCTION: This study investigates the influence of Mycobacterium tuberculosis infection on immune activation biomarkers, both in HIV-infected and -uninfected subjects. METHODS: Forty-eight treatment-naive HIV-infected patients and 74 HIV-uninfected subjects were recruited and divided into groups according to their M. tuberculosis infection status: latent tuberculosis infection (LTBI), active tuberculosis (TB), and no evidence of M. tuberculosis infection. The expression of cellular markers CD38 and HLA-DR on circulating CD8 T lymphocytes and the plasmatic levels of soluble markers interleukin-6, sCD14, and D-Dimer were measured and compared between groups. The HIV-infected patients with no evidence of M. tuberculosis or with LTBI who initiated antiretroviral treatment were tested again for these biomarkers once viral suppression was reached. RESULTS: In both HIV-infected and -uninfected groups, patients with TB had higher levels of immune activation markers than subjects with LTBI and with no evidence of M. tuberculosis. Among the HIV-uninfected subjects, no significant difference in biomarker level was found between those presenting LTBI and those with no evidence of M. tuberculosis. The effect of LTBI on activation biomarkers in the HIV-infected groups was inconclusive because of the small number of individuals in the HIV+/LTBI group. sCD14 and D-Dimer levels were significantly higher in the TB-only group than in the HIV-only group. DISCUSSION: Although TB is associated with an increase in biomarkers of immune activation, the effect of LTBI is less evident. Further investigation is warranted, and according to our results, soluble markers may offer greater sensitivity for the evaluation of M. tuberculosis-associated immune activation than cellular markers.

Added Value of Long-Term Cytokine Release Assays to Detect Mycobacterium tuberculosis Infection in HIV-Infected Subjects in Uganda.

OBJECTIVES: To investigate whether mycobacterial antigen-induced cytokine secretions are helpful in detecting Mycobacterium tuberculosis (Mtb) infection in a cohort of HIV-infected patients living in a country with a high burden of Mtb and HIV infections, and to determine their predictive value for the development of tuberculosis (TB)-associated immune reconstitution inflammatory syndrome. DESIGN: A total of 352 HIV-infected patients (186 with active TB) were prospectively enrolled when initiating antiretroviral therapy (ART). Sequential blood samples were collected during the first 6 months of ART. Eighty-three HIV-uninfected subjects (39 with active TB) were enrolled as controls. METHODS: The concentrations of 13 cytokines were measured in supernatants from blood mononuclear cells in vitro stimulated with purified protein derivative (PPD), heparin-binding hemagglutinin (HBHA) or early secreted antigen-6 (ESAT-6) and culture filtrate protein-10 (CFP-10), and results were compared with those of tuberculin skin tests (TST). RESULTS: The best detection of Mtb infection was achieved by ESAT-6/CFP-10-induced interferon-γ concentrations, but results were often negative for patients with CD4 T-cell counts <50 per cubic millimeters. Patients with active TB were identified by high ESAT-6/CFP-10-induced interleukin-6. Conversions of interferon-γ-release assays (IGRA) and TST occurred under ART, and combined TB and antiretroviral treatments of coinfected patients resulted in a decrease of ESAT-6/CFP-10-induced and an increase of HBHA-induced interferon-γ responses. No Mtb antigen-induced cytokines allowed us to predict TB-immune reconstitution inflammatory syndrome or ART-associated TB. CONCLUSIONS: In Uganda, ESAT-6/CFP-10-IGRA is better in detecting Mtb infection than TST and, when combined with an HBHA-IGRA, could help to evaluate anti-TB treatment success.

Contribution of a heparin-binding haemagglutinin interferon-gamma release assay to the detection of Mycobacterium tuberculosis infection in HIV-infected patients: comparison with the tuberculin skin test and the QuantiFERON-TB Gold In-tube.

BACKGROUND: The screening and treatment of latent tuberculosis (TB) infection reduces the risk of progression to active disease and is currently recommended for HIV-infected patients. The aim of this study is to evaluate, in a low TB incidence setting, the potential contribution of an interferon-gamma release assay in response to the mycobacterial latency antigen Heparin-Binding Haemagglutinin (HBHA-IGRA), to the detection of Mycobacterium tuberculosis infection in HIV-infected patients. METHODS: Treatment-naïve HIV-infected adults were recruited from 4 Brussels-based hospitals. Subjects underwent screening for latent TB using the HBHA-IGRA in parallel to a classical method consisting of medical history, chest X-ray, tuberculin skin test (TST) and QuantiFERON-TB Gold In-Tube (QFT-GIT). Prospective clinical and biological follow-up ensued, with repeated testing with HBHA-IGRA. A group of HIV-infected patients with clinical suspicion of active TB was also recruited and tested with the HBHA-IGRA. Multiplex analysis was performed on the culture supernatants of this in-house assay to identify test read-outs alternative to interferon-gamma that could increase the sensitivity of the test. RESULTS: Among 48 candidates enrolled for screening, 9 were identified with latent TB by TST and/or QFT-GIT results. Four of these 9 patients and an additional 3 screened positive with the HBHA-IGRA. This in-house assay identified all the patients that were positive for the TST and showed the best concordance with the presence of a M. tuberculosis exposure risk. During follow-up (median 14 months) no case of active TB was reported and HBHA-IGRA results remained globally constant. Fourteen HIV-infected patients with clinical suspicion of active TB were recruited. Active TB was confirmed for 6 of them among which 3 were HBHA-IGRA positive, each with very high interferon-gamma concentrations. All patients for whom active TB was finally excluded, including 2 non-tubercular mycobacterial infections, had negative HBHA-IGRA results. Multiplex analysis confirmed interferon-gamma as the best read-out. CONCLUSIONS: The HBHA-IGRA appears complementary to the QuantiFERON-TB Gold In-Tube for the screening of latent TB in HIV-infected patients. Large-scale studies are necessary to determine whether this combination offers sufficient sensitivity to dismiss TST, as suggested by our results. Furthermore, HBHA-IGRA may help in the diagnosis work-up of clinical suspicions of active TB.

Human dendritic cell maturation and cytokine secretion upon stimulation with Bordetella pertussis filamentous haemagglutinin.

In addition to antibodies, Th1-type T cell responses are also important for long-lasting protection against pertussis. However, upon immunization with the current acellular vaccines, many children fail to induce Th1-type responses, potentially due to immunomodulatory effects of some vaccine antigens, such as filamentous haemagglutinin (FHA). We therefore analysed the ability of FHA to modulate immune functions of human monocyte-derived dendritic cells (MDDC). FHA was purified from pertussis toxin (PTX)-deficient or from PTX- and adenylate cyclase-deficient Bordetella pertussis strains, and residual endotoxin was neutralized with polymyxin B. FHA from both strains induced phenotypic maturation of human MDDC and cytokine secretion (IL-10, IL-12p40, IL-12p70, IL-23 and IL-6). To identify the FHA domains responsible for MDDC immunomodulation, MDDC were stimulated with FHA containing a Gly→Ala substitution at its RGD site (FHA-RAD) or with an 80-kDa N-terminal moiety of FHA (Fha44), containing its heparin-binding site. Whereas FHA-RAD induced maturation and cytokine production comparable to those of FHA, Fha44 did not induce IL-10 production, but maturated MDDC at least partially. Nevertheless, Fha44 induced the secretion of IL-12p40, IL-12p70, IL-23 and IL-6 by MDDC, albeit at lower levels than FHA. Thus, FHA can modulate MDDC responses in multiple ways, and IL-10 induction can be dissociated from the induction of other cytokines.

Key role of effector memory CD4+ T lymphocytes in a short-incubation heparin-binding hemagglutinin gamma interferon release assay for the detection of latent tuberculosis.

The treatment of latent tuberculosis infection (LTBI) in target populations is one of the current WHO strategies for preventing active tuberculosis (TB) infection and reducing the Mycobacterium tuberculosis reservoir. Therefore, powerful LTBI screening tools are indispensable. A gamma interferon release assay (IGRA) in response to the stimulation of peripheral blood mononuclear cells by the latency antigen native heparin-binding hemagglutinin (nHBHA-IGRA) has proven its potential for this purpose. We have evaluated its possible optimization through a reduction of incubation time from 96 to 24 h, while compensating for this by adding interleukin 7 (IL-7) to the medium. We have also investigated the phenotypes of the gamma interferon (IFN-γ)-producing cells after both short and long incubation times. One hundred thirty-one nonimmunocompromised patients were recruited from 3 Brussels-based university hospitals. They were divided into 1 of 4 subgroups according to their M. tuberculosis infection status (LTBI, TB infection, undetermined M. tuberculosis infection status, and noninfected controls). The novel 24-h nHBHA-IGRA was performed for all subjects, and a simultaneous 96-h classical HBHA-IGRA was performed for 79 individuals. The results showed a good correlation between the two tests, and the novel 24-h nHBHA-IGRA maintained the principal advantages of the classical test, namely, a high specificity for LTBI diagnosis, an absence of interference of Mycobacterium bovis BCG vaccination during infancy, and a relative discrimination between LTBI and TB infection. Whereas the commercialized IGRAs show a greater sensitivity for recent than for remote M. tuberculosis infections, the 24-h nHBHA-IGRA appears to have comparable diagnostic powers for recent and remote LTBI. The IFN-γ detected by the 24-h nHBHA-IGRA was mainly secreted by effector memory CD4(+) T lymphocytes, a finding suggestive of continuous HBHA presentation during latency.