Subsequent AS01-adjuvanted vaccinations induce similar transcriptional responses in populations with different disease statuses.

Transcriptional responses to adjuvanted vaccines can vary substantially among populations. Interindividual diversity in levels of pathogen exposure, and thus of cell-mediated immunological memory at baseline, may be an important determinant of population differences in vaccine responses. Adjuvant System AS01 is used in licensed or candidate vaccines for several diseases and populations, yet the impact of pre-existing immunity on its adjuvanticity remains to be elucidated. In this exploratory post-hoc analysis of clinical trial samples (clinicalTrials.gov: NCT01424501), we compared gene expression patterns elicited by two immunizations with the candidate tuberculosis (TB) vaccine M72/AS01, between three groups of individuals with different levels of memory responses to TB antigens before vaccination. Analyzed were one group of TB-disease-treated individuals, and two groups of TB-disease-naïve individuals who were (based on purified protein derivative [PPD] skin-test results) stratified into PPD-positive and PPD-negative groups. Although TB-disease-treated individuals displayed slightly stronger transcriptional responses after each vaccine dose, functional gene signatures were overall not distinctly different between groups. Considering the similarities with the signatures found previously for other AS01-adjuvanted vaccines, many features of the response appeared to be adjuvant-driven. Across groups, cell proliferation-related signals at 7 days post-dose 1 were associated with increased anti-M72 antibody response magnitudes. These early signals were stronger in the TB-disease-treated group as compared to both TB-disease-naïve groups. Interindividual homogeneity in gene expression levels was also higher for TB-disease-treated individuals post-dose 1, but increased in all groups post-dose 2 to attain similar levels between the three groups. Altogether, strong cell-mediated memory responses at baseline accelerated and amplified transcriptional responses to a single dose of this AS01-adjuvanted vaccine, resulting in more homogenous gene expression levels among the highly-primed individuals as compared to the disease-naïve individuals. However, after a second vaccination, response heterogeneity decreased and was similar across groups, irrespective of the degree of immune memory acquired at baseline. This information can support the design and analysis of future clinical trials evaluating AS01-adjuvanted vaccines.

[Dynamic analysis of tuberculosis specific mononuclear cells in peripheral blood of HIV patients with tuberculosis].

Objective: To evaluate the response of peripheral blood mononuclear cells (PBMCs) in patients with human immunodeficiency virus (HIV) combined with active tuberculosis (TB) to TB-specific antigen stimulation. Methods: From January to December, 2018, individuals infected with both HIV and TB (HIV/TB group) were taken as the study subjects. Individuals infected with HIV alone (HIV group), individuals infected with TB alone (TB group) and healthy people (Health control group, HC group) were taken as the control groups. PBMCs were isolated and stimulated with purified protein derivative of bacillus calmette-guerin (BCG-PPD). The expression of surface molecules in T cells (CD3+, CD4+, CD8+) and monocytes (CD14+) and the percentages of Interferon (IFN)-γ and tumor necrosis factor (TNF)-α were detected by cell surface molecular staining, direct intracellular cytokine staining and flow cytometry (CD3- lymphocytes were mainly B lymphocytes and NK cells). Analysis of non-parametric data was used to compare the data between the two groups, and paired t-test was used to compare the data before and after PPD stimulation in each group. Results: Before PPD stimulation, the percentage of IFN-γ+ CD8+ cells in the peripheral blood of HIV/TB group(mean 0.52%) was significantly lower than that in TB group(mean 0.94%, P=0.010). The TNF-α+cell percentages in CD3+, CD4+, CD8+, or CD14+ cells in the HIV/TB group(mean 19.2%) were significantly lower than those in the HIV group(mean 31.9%, P=0.002). The percentage of TNF-α secreted by monocytes in the HIV group was significantly lower than that in the HC group. The percentages of IFN-γ+ CD8+ and IFN-γ+ CD3- cells in the peripheral blood of the TB group (mean 0.94%) were significantly higher than thoset in the HC group(mean 0.51%, P=0.020), while the percentages of TNF-α+ cells in each subsets of PBMCs were significantly lower than those in the HC group. After PPD stimulation, the percentage of IFN-γ+ CD8+ cells in the HIV/TB group was significantly lower than that in the TB group(P=0.008), and the change was more marked than that before stimulation. The percentage of IFN-γ+ CD8+ cells in the HIV group(mean 0.20%) was lower than that in the HC group (mean 0.52%, P=0.044). The percentage of IFN-γ+ CD3- in the TB group was significantly higher than in the HC group. There were no significant differences in TNF-α+ cell percentages in the 3 groups compared with the control group after PPD stimulation. The percentages of IFN-γ+ CD4+ cells in the HC and the TB groups were significantly increased after PPD stimulation in each group (P=0.002, P=0.001, respectively). However, there were no significant differences of IFN-γ+ CD4+ cell percentages in the HIV/TB group and the HIV group. The percentages of TNF-α production by monocytes were significantly increased after PPD stimulation in all groups. Conclusions: Chronic Mycobacterium tuberculosis (MTB) infection reduced the ability of PBMCs to produce TNF-α. For patients with TB infection, the production of TNF-α was reduced when combined with HIV infection. The capacity of CD8+ and CD3- lymphocytes to produce IFN-γ was increased in TB patients, while the capacity of CD8+ T cells to produce IFN-γ was decreased with co-infection of HIV. Infection of HIV weakened the immune response to MTB infection, which made the clinical diagnosis and treatment of TB more difficult.

Persistent expression of activation markers on Mycobacterium tuberculosis-specific CD4 T cells in smear negative TB patients.

BACKGROUND: T cell activation (HLA-DR, CD-38), proliferation (KI-67), and functional (IFN-γ, TNF-α) markers have recently been shown to be useful in predicting and monitoring anti-TB responses in smear positive TB, but previous research did not characterize the activation and proliferation profiles after therapy of smear negative TB. METHODOLOGY: In this study, we used polychromatic flow cytometry to assess selected PPD-specific T cell markers using fresh PBMC of smear negative and positive pulmonary tuberculosis (PTB) patients, recruited from health facilities in Addis Ababa. RESULT: Levels of activation (HLA-DR, CD38) and proliferation (Ki-67) among total unstimulated CD4 T cells decreased significantly after therapy, particularly at month 6. Similarly, levels of PPD-specific T cell activation markers (HLA-DR, CD-38) were significantly lower in smear positive PTB patients following treatment, whereas a consistent decline in these markers was less apparent among smear negative PTB patients at the sixth month. CONCLUSION: After six months of standard anti-TB therapy, persistent levels of activation of HLA-DR and CD-38 from PPD specific CD4+T cells in this study could indicate that those markers have little value in monitoring and predicting anti-TB treatment response in smear negative pulmonary TB patients in Ethiopian context.

The variable prevalence of bovine tuberculosis among dairy herds in Central Ethiopia provides opportunities for targeted intervention.

Bovine tuberculosis (bTB) is an important disease for dairy productivity, as well as having the potential for zoonotic transmission. Previous prevalence studies of bTB in the dairy sector in central Ethiopia have suggested high prevalence, however, they have been limited to relatively small scale surveys, raising concerns about their representativeness. Here we carried out a cross sectional one-stage cluster sampling survey taking the dairy herd as a cluster to estimate the prevalence of bTB in dairy farms in six areas of central Ethiopia. The survey, which to date is by far the largest in the area in terms of the number of dairy farms, study areas and risk factors explored, took place from March 2016 to May 2017. This study combined tuberculin skin testing and the collection of additional herd and animal level data by questionnaire to identify potential risk factors contributing to bTB transmission. We applied the single intradermal cervical comparative tuberculin (SICCT) test using >4mm cut-off for considering an individual animal as positive for bTB; at least one reactor animal was required for a herd to be considered bTB positive. Two hundred ninety-nine dairy herds in the six study areas were randomly selected, from which 5,675 cattle were tested. The overall prevalence of bTB after standardisation for herd-size in the population was 54.4% (95% CI 48.7-60%) at the herd level, and it was 24.5% (95% CI 23.3-25.8) at the individual animal level. A Generalized Linear Mixed Model (GLMM) with herd and area as random effect was used to explore risk factors association with bTB status. We found that herd size, age, bTB history at farm, and breed were significant risk factors for animals to be SICCT positive. Animals from large herds had 8.3 times the odds of being tuberculin reactor (OR: 8.3, p-value:0.008) as compared to animals from small herds. The effect of age was strongest for animals 8-10 years of age (the oldest category) having 8.9 times the odds of being tuberculin reactors (OR: 8.9, p-value:<0.001) compared to the youngest category. The other identified significant risk factors were bTB history at farm (OR: 5.2, p-value:0.003) and cattle breed (OR: 2.5, p-value: 0.032). Our study demonstrates a high prevalence of bTB in central Ethiopia but with a large variation in within-herd prevalence between herds, findings that lays an important foundation for the future development of control strategies.

Phenotypic Changes on Mycobacterium Tuberculosis-Specific CD4 T Cells as Surrogate Markers for Tuberculosis Treatment Efficacy.

Background: The analysis of phenotypic characteristics on Mycobacterium tuberculosis (MTB)-specific T cells is a promising approach for the diagnosis of active tuberculosis (aTB) and for monitoring treatment success. We therefore studied phenotypic changes on MTB-specific CD4 T cells upon anti-tuberculosis treatment initiation in relation to the treatment response as determined by sputum culture. Methods: Peripheral blood mononuclear cells from subjects with latent MTB infection (n = 16) and aTB (n = 39) at baseline, weeks 9, 12, and 26 (end of treatment) were analyzed after intracellular interferon gamma staining and overnight stimulation with tuberculin. Liquid sputum cultures were performed weekly until week 12 and during 4 visits until week 26. Results: T cell activation marker expression on MTB-specific CD4 T cells differed significantly between subjects with aTB and latent MTB infection with no overlap for the frequencies of CD38pos and Ki67pos cells (both p < 0.0001). At 9 weeks after anti-TB treatment initiation the frequencies of activation marker (CD38, HLA-DR, Ki67) positive MTB-specific, but not total CD4 T cells, were significantly reduced (p < 0.0001). Treatment induced phenotypic changes from baseline until week 9 and until week 12 differed substantially between individual aTB patients and correlated with an individual's time to stable sputum culture conversion for expression of CD38 and HLA-DR (both p < 0.05). In contrast, the frequencies of maturation marker CD27 positive MTB-specific CD4 T cells remained largely unchanged until week 26 and significantly differed between subjects with treated TB disease and latent MTB infection (p = 0.0003). Discussion: Phenotypic changes of MTB-specific T cells are potential surrogate markers for tuberculosis treatment efficacy and can help to discriminate between aTB (profile: CD38pos, CD27low), treated TB (CD38neg, CD27low), and latent MTB infection (CD38neg, CD27high).

Effective antigen presentation to helper T cells by human eosinophils.

Although eosinophils are inflammatory cells, there is increasing attention on their immunomodulatory roles. For example, murine eosinophils can present antigen to CD4+ T helper (Th) cells, but it remains unclear whether human eosinophils also have this ability. This study determined whether human eosinophils present a range of antigens, including allergens, to activate Th cells, and characterized their expression of MHC class II and co-stimulatory molecules required for effective presentation. Human peripheral blood eosinophils purified from non-allergic donors were pulsed with the antigens house dust mite extract (HDM), Timothy Grass extract (TG) or Mycobacterium tuberculosis purified protein derivative (PPD), before co-culture with autologous CD4+ Th cells. Proliferative and cytokine responses were measured, with eosinophil expression of HLA-DR/DP/DQ and the co-stimulatory molecules CD40, CD80 and CD86 determined by flow cytometry. Eosinophils pulsed with HDM, TG or PPD drove Th cell proliferation, with the response strength dependent on antigen concentration. The cytokine responses varied with donor and antigen, and were not biased towards any particular Th subset, often including combinations of pro- and anti-inflammatory cytokines. Eosinophils up-regulated surface expression of HLA-DR/DP/DQ, CD80, CD86 and CD40 in culture, increases that were sustained over 5 days when incubated with antigens, including HDM, or the major allergens it contains, Der p I or Der p II. Human eosinophils can, therefore, act as effective antigen-presenting cells to stimulate varied Th cell responses against a panel of antigens including HDM, TG or PPD, an ability that may help to determine the development of allergic disease.

Estudio comparativo de concordancia y costes entre la prueba de la tuberculina y QuantiFERON ®-TB Gold In-Tube en el diagnóstico de la infección latente tuberculosa en contactos de pacientes con tuberculosis pulmonar / Comparative study of concordance and costs between tuberculin skin test and QuantiFERON ®-TB Gold In-Tube in the diagnosis of latent tuberculosis infection among contacts of patients with pulmonary tuberculosis

Introducción: El diagnóstico de la infección latente tuberculosa (ILT) es posible realizarlo mediante la prueba de la tuberculina (PT) o bien a través de las denominadas técnicas de interferon-γ release assays (IGRAS, «análisis de liberación del interferón-γ»), siendo QuantiFERON®-TB Gold In-Tube (QF-G-IT) la más usada. Los IGRAS permiten evitar algunos inconvenientes de la PT, especialmente la reacción cruzada con la vacuna con bacilo de Calmette-Guérin (BCG). No obstante, también presentan algunos problemas, como son los derivados del coste de la técnica, así como el ser un método de laboratorio que precisa una infraestructura y experiencia adecuadas. No existe un claro consenso sobre cuál de las técnicas debería utilizarse de forma prioritaria para el diagnóstico de la ILT. Método: Se trata de un estudio comparativo entre la PT y la QF-G-IT en nuestra cohorte de contactos de pacientes con tuberculosis pulmonar durante el período de estudio (n = 101). Se realizó un análisis de la concordancia global y por grupos según los contactos estuvieran vacunados con BCG o no. Se realizó, además, un estudio de costes de ambas técnicas y de las estrategias diagnósticas basadas en ellas. Resultados: La concordancia entre la PT y la QF-G-IT fue aceptable en el global de la muestra, pero muy buena en el grupo de no vacunados. Se registraron muy pocos casos de valores indeterminados. El estudio de costes mostró que la PT era más económica que la QF-G-IT; sin embargo, al analizar el coste de las estrategias según cada técnica, la PT mostró un mayor coste-beneficio. Conclusión: Aconsejamos considerar QF-G-IT como la única y preferente técnica para el diagnóstico de la ILT en contactos convivientes, basados en una buena concordancia general entre ambas técnicas (más aún si eliminamos el efecto de la vacuna) y un estudio de costes favorable a QF-G-IT (AU)

Introduction: Recently diagnosis of latent tuberculosis infection (LTBI) can be made using the tuberculin skin test (TST) or by techniques known as interferon-γ release assays (IGRAS), being QuantiFERON®-TB Gold In-Tube (QF-G-IT) the most used. The IGRAS avoid some drawbacks of the TST, especially cross-reaction with bacillus Calmette-Guérin (BCG) vaccine, but also present some problems such as those arising from cost and the need of having an adequate infrastructure and experience. There is no clear consensus on which technique should be preferentially used for the diagnosis of LTBI. Methods: This is a comparative study between the TST and QT-G-IT in a cohort of contacts of patients with pulmonary tuberculosis during the study period. An analysis of global agreement and groups was performed according to whether the contacts were vaccinated with BCG or not. A study of costs of both techniques and diagnostic strategies based on these techniques was performed. Results: The agreement between TST and QF-G-IT was acceptable in the whole sample yet it was very good in the unvaccinated group. Few cases of indeterminate values were recorded. The cost study showed that TST was cheaper than QF-G-IT; however when we analyzed the cost of the strategies according to each technique, the QF-G-IT showed a better cost-benefit. Conclusion: We suggest considering QF-G-IT as the only preferred technique for the diagnosis of LTBI in household contacts, based on good overall agreement between the 2 techniques (even if we eliminate the effect of the vaccine) and a cost analysis favorable to QF-G-IT (AU)

Tuberculin reaction and BCG scar: association with infant mortality.

OBJECTIVE: To test the hypothesis that having a scar and a positive tuberculin skin test (TST) response after vaccination with Bacille Calmette-Guérin (BCG) is associated with reduced infant mortality. METHODS: We studied cohorts of 2709 normal-birthweight (NBW) and 1102 low-birthweight (LBW) infants in Guinea-Bissau. Children were enrolled in randomised trials between year 2002 and 2008 and received BCG vaccination at birth. BCG scars and TST responses were assessed at 2 and 6 months of age. The infants were followed for mortality to 12 months of age, and survival was analysed using Cox regression. RESULTS: At age 2 months, 88% of NBW children and 91% of LBW children had a BCG scar, and 36% and 17% had a TST response, respectively. The LBW infants had nearly twofold higher mortality (4.5%) than the NBW infants (2.8%) between 2 and 12 months of age. In the LBW cohort, the adjusted mortality rate ratio (MRR) comparing children with a BCG scar with those without was 0.42 (95% CI = 0.19; 0.93). There was a similar tendency for TST positivity: MRR = 0.47 (95% CI = 0.14; 1.54). For LBW children who had both a positive TST reaction and a scar, the MRR was 0.22 (95% CI = 0.05; 0.87). For NBW children, a scar and a positive TST were associated with 20% reductions in mortality, which did not reach statistical significance. CONCLUSION: We confirmed previous observations that having a scar and a TST response after BCG vaccination is associated with lower mortality risk. The possibility of revaccinating scar-negative children should be considered.

Bifunctional T-cell-derived cytokines for the diagnosis of tuberculosis and treatment monitoring.

BACKGROUND: Diagnosis and treatment monitoring of patients with tuberculosis remain challenging. OBJECTIVE: We have evaluated whether Mycobacterium-specific interferon (IFN)-γ and interleukin (IL)-2 bifunctional cytokine immune response assays improve the diagnosis of and correlate to treatment response in pulmonary tuberculosis. METHODS: Early secretory antigenic target (ESAT)6/culture filtrate protein 10 (CFP10), microsomal triglyceride transfer protein 65 (MTP65) and the purified protein derivative (PPD) tuberculin-specific immune profiles were investigated in peripheral blood mononuclear cells from 19 patients with culture-confirmed tuberculosis and 23 healthy community controls (HCCs; 82.6% with latent M. tuberculosis infection) using a novel fluorescence-based dual-colour enzyme-linked immunospot (EliSpot) technology (FluoroSpot). RESULTS: The frequency of ESAT6/CFP10-induced IFN-γ+IL-2- producing cells was elevated (p < 0.001), whereas the percentages of specific IFN-γ-IL-2+ (p = 0.002) and IFN-γ+IL-2+ double producing cells (p = 0.037) were diminished in tuberculosis patients in comparison to HCCs. A 3-host marker model using a combination of those IFN-γ and IL-2 single-cell responses showed 93.8% sensitivity and 77.8% specificity for tuberculosis. During tuberculosis treatment, the PPD-induced immune responses shifted from an IFN-γ+IL-2- dominated profile towards a balance of IFN-γ-IL-2+ and IFN-γ+IL-2+ double producing cells (all p ≤ 0.05). CONCLUSIONS: The addition of antigen-specific IL-2 production to IFN-γ responses by EliSpot in IFN-γ release assays increases diagnostic sensitivity for active tuberculosis.

Description of pediatric tuberculosis evaluated in a referral center in istanbul Turkey.

PURPOSE: Diagnosis of tuberculosis (TB) in children is more challenging than in adults. This study aimed to describe demographical, clinical and laboratory findings of children diagnosed with tuberculosis in Turkey, including the issues of contact tracing, culture positivity and forms of the disease. MATERIALS AND METHODS: Clinical and laboratory data of 51 children with a mean age of 8.0±4.6 years who were diagnosed with TB were retrospectively reviewed. Main diagnostic tools included tuberculin skin test, chest X-ray, sputum/gastric aspirate culture with sensitivity testing, and direct microscopy for acid-fast bacilli on available samples. Clinical characteristics and outcomes of the patients were examined. RESULTS: Thirty-six (70.6%) children were diagnosed with intra-thoracic and 15 (29.4%) with extra-thoracic tuberculosis. Twenty-eight of the patients had a positive Bacillus Calmette-Guérin vaccine scar (28/51, 54.9%) and 23/51 (45.1%) had a positive tuberculin skin test. An adult TB contact was identified in 27 (52.9%) of the cases. On direct microscopy, acid-fast bacilli were found in nine (17.6%) patients and positive culture for Mycobacterium tuberculosis was found in 19 (37.3%). Drug resistance to isoniazid was detected in four (7.8%). One patient with nephrotic syndrome and miliary tuberculosis died during follow-up. All other patients responded well to the treatment. CONCLUSION: Focusing on active contact tracing among all household contacts of tuberculous cases may be helpful in early identification and controlling childhood disease, even in regions with low disease prevalence. Adopting a suspicious and proactive approach in this particular age group is warranted.

Description of Pediatric Tuberculosis Evaluated in a Referral Center in Istanbul Turkey

PURPOSE: Diagnosis of tuberculosis (TB) in children is more challenging than in adults. This study aimed to describe demographical, clinical and laboratory findings of children diagnosed with tuberculosis in Turkey, including the issues of contact tracing, culture positivity and forms of the disease. MATERIALS AND METHODS: Clinical and laboratory data of 51 children with a mean age of 8.0+/-4.6 years who were diagnosed with TB were retrospectively reviewed. Main diagnostic tools included tuberculin skin test, chest X-ray, sputum/gastric aspirate culture with sensitivity testing, and direct microscopy for acid-fast bacilli on available samples. Clinical characteristics and outcomes of the patients were examined. RESULTS: Thirty-six (70.6%) children were diagnosed with intra-thoracic and 15 (29.4%) with extra-thoracic tuberculosis. Twenty-eight of the patients had a positive Bacillus Calmette-Guerin vaccine scar (28/51, 54.9%) and 23/51 (45.1%) had a positive tuberculin skin test. An adult TB contact was identified in 27 (52.9%) of the cases. On direct microscopy, acid-fast bacilli were found in nine (17.6%) patients and positive culture for Mycobacterium tuberculosis was found in 19 (37.3%). Drug resistance to isoniazid was detected in four (7.8%). One patient with nephrotic syndrome and miliary tuberculosis died during follow-up. All other patients responded well to the treatment. CONCLUSION: Focusing on active contact tracing among all household contacts of tuberculous cases may be helpful in early identification and controlling childhood disease, even in regions with low disease prevalence. Adopting a suspicious and proactive approach in this particular age group is warranted.

Progesterone prevents allodynia after experimental spinal cord injury.

UNLABELLED: Chronic pain after spinal cord injury represents a therapeutic challenge. Progesterone, a neuroprotective steroid, has been shown to modulate nociceptive thresholds, whereas its effect on neuropathic pain needs to be further explored. In this study, we evaluated whether progesterone could ameliorate pain-associated behaviors in animals subjected to a spinal cord hemisection. The development of mechanical and cold allodynia was assessed in injured male rats treated with daily injections of progesterone or vehicle. The expression of N-methyl-D-aspartate receptor (NMDAR) subunits, protein kinase C gamma (PKCγ), preprodynorphin (ppD), and kappa opioid receptor (KOR), key players in chronic pain mechanisms, was determined in the dorsal spinal cord. Twenty-eight days after injury, all vehicle-treated animals presented allodynic behaviors and a marked increase in NMDAR subunits, PKCγ, and ppD mRNA levels, with no changes in KOR mRNA levels. Progesterone prevented the development of mechanical allodynia and reduced the painful responses to cold stimulation. In correlation with the attenuation of pain behaviors, the steroid prevented NMDAR subunits and PKCγ mRNAs upregulation, did not modify the elevated ppD mRNA levels, but increased KOR expression. In conclusion, progesterone modulates neuropathic pain after spinal cord injury, creating a favorable molecular environment that may decrease spinal nociceptive signaling. PERSPECTIVE: The present study suggests that progesterone administration could represent an interesting strategy to modulate neuropathic pain circuits after spinal cord injury. Further studies are needed to investigate the potential progesterone receptors involved in these actions.

Neonatal BCG vaccination induces IL-10 production by CD4+ CD25+ T cells.

To determine the optimal time of Bacillus Calmette-Guerin (BCG) vaccination for induction of Th1 immunity, we measured the interferon (IFN)-γ and interleukin (IL)-10 secretion in purified protein derivative (PPD)-stimulated peripheral blood mononuclear cell (PBMC) cultures in newborns vaccinated at birth or 2nd month of life. Moreover, role of CD4(+) CD25(+) T cells was studied by depletion assay at 8th month. Nineteen term and healthy newborns were randomized into two groups: Group I composed of 10 newborns vaccinated with BCG at birth and the remaining 9 (group II) at 2nd month of life. PBMCs were isolated at birth, 2nd and 8th months of age, and PPD-stimulated IL-10, 5 and IFN-γ secretion were assessed. The same measurements were repeated for IL-10 and IFN-γ after the depletion of CD4(+) CD25(+) T cells at the 8th month. Children vaccinated at birth demonstrated higher PPD-stimulated IFN-γ and IL-10 levels at 2 months of age when compared to non-vaccinated ones (p = 0.038 and p = 0.022, respectively), whereas at 8 months, no significant differences were detected between the two groups. Moreover, CD4(+) CD25(+)-depleted T-cell cultures resulted in lower PPD-stimulated IL-10 levels in those vaccinated at birth when compared to non-depleted condition at the 8th month (p < 0.001). BCG at birth upregulated PPD-stimulated IFN-γ secretion at the 2nd month and remained still detectable at 8 month after the vaccination, whereas those vaccinated at the 2nd month of life lacked that increase in IFN-γ response at the same time-point. Furthermore, depletion assays suggest that CD4(+) CD25(+) T cells are involved in PPD-stimulated IL-10 secretion in response to BCG vaccination.

Characterization of CD4 and CD8 T cells producing IFN-γ in human latent and active tuberculosis.

Patients with pulmonary tuberculosis (PTB) frequently have reduced IFN-γ production in response to mycobacterial antigens, compared to individuals with latent Mycobacterium tuberculosis infection (LTBi). However, it is not clear whether this reduced responsiveness is restricted to a particular T cell subset. Herein, PBMCs from 26 PTB patients, 30 household contacts (HHCs) of PTB, and 30 tuberculin positive (TST+) healthy subjects not recently exposed to PTB, were stained with CFSE and stimulated non-specific (PPD) for 120 h, and specific (CFP-10/ESAT-6) and latency (HSpX) mycobacterial antigens for 144 h and the percentage of CD4(+) and CD8(+)IFN-γ(+) T cells responding determined by flow cytometry, in addition to their memory phenotype by the CD45RO and CD27 expression. PTB had decreased frequency of both CD4(+) and CD8(+) precursor cells, as well as decreased number of CD4(+)IFN-γ(+) cells in response to all antigens, whereas CD8(+)IFN-γ(+) cells were decreased in response to PPD and ESAT-6, but not to CFP-10 and HSpX. HHCs exhibited the highest precursor frequencies and IFN-γ responses, irrespective of the antigen employed. The CD4(+)/CD8(+) cell ratios showed that in response to PPD CD4(+) precursor and IFN-γ-producer cells are more frequent than their CD8(+) counterparts, and that PTB have a decreased CD4(+)IFN-γ(+)/CD8(+)IFN-γ(+) ratio in response to PPD, CFP-10, and ESAT-6. CD4(+)IFN-γ(+) and CD8(+)IFN-γ(+) cells exhibited a central memory phenotype (CD45RO(+)CD27(+)), irrespective of the group of subjects and the antigen used for stimulation. In conclusion, PTB patients had a decreased percentage of CD4(+) and CD8(+) precursor cells and CD4(+)IFN-γ(+). HHCs exhibited the highest frequency of CD4(+) and CD8(+) precursors and CD4(+)IFN-γ(+)-producing cells.

Potential role for IL-2 ELISpot in differentiating recent and remote infection in tuberculosis contact tracing.

Interferon (IFN)-gamma release assays (IGRA) have improved tuberculosis contact tracing, but discrimination of recent from remote Mycobacterium tuberculosis contacts is not possible by IGRA alone. We present results of a tuberculosis contact investigation with a new early-secretory-antigenic-target (ESAT)-6 and culture-filtrate-protein (CFP)-10 specific interleukin (IL)-2 ELISpot in addition to ESAT-6 and CFP-10 specific IFN-gamma ELISpot and tuberculin skin testing (TST). Results of the TST, IFN-gamma ELISpot and IL-2 ELISpot were positive in 6/172 (3.4%), 7/167 (4.2%) and 6/196 (3.1%) of contacts, respectively. Close contact (> or =100 hours) to the index case increased the risk of positive results in the IFN-gamma ELISpot, TST, and IL-2 ELISpot by 40.8, 19.3, and 2.5 times, respectively. Individuals with a positive IFN-gamma ELISpot/negative IL-2 ELISpot result had a median (IQR) duration of index case exposure of 568 hours (133_1000) compared to individuals with a positive IFN-gamma ELISpot/positive IL-2 ELISpot result (median = 24 hours; 20_130; p-value = 0.047). Combination of a M. tuberculosis specific IFN-gamma ELISpot with a M. tuberculosis specific IL-2 ELISpot significantly improved the identification of individuals with the highest risk of recent M. tuberculosis infection and is a promising method that should be explored to target tuberculosis preventive chemotherapy.

CTLA-4 is expressed by human monocyte-derived dendritic cells and regulates their functions.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is the major negative regulator of T-cell responses, although growing evidence supports its wider role as an immune attenuator that may also act in other cell lineages. Here, we have analyzed the expression of CTLA-4 in human monocytes and monocyte-derived dendritic cells (DCs), and the effect of its engagement on cytokine production and T-cell stimulatory activity by mature DCs. CTLA-4 was highly expressed on freshly isolated monocytes, then down-modulated upon differentiation toward immature DCs (iDCs) and it was markedly upregulated on mature DCs obtained with different stimulations (lipopolysaccharides [LPS], Poly:IC, cytokines). In line with the functional role of CTLA-4 in T cells, treatment of mDCs with an agonistic anti-CTLA-4 mAb significantly enhanced secretion of regulatory interleukin (IL)-10 but reduced secretion of IL-8/IL-12 pro-inflammatory cytokines, as well as autologous CD4+ T-cell proliferation in response to stimulation with recall antigen purified protein derivative (PPD) loaded-DCs. Neutralization of IL-10 with an anti-IL-10 antibody during the mDCs-CD4+ T-cell co-culture partially restored the ability of anti-CTLA-4-treated mDCs to stimulate T-cell proliferation in response to PPD. Taken together, our data provide the first evidence that CTLA-4 receptor is expressed by human monocyte-derived mDCs upon their full activation and that it exerts immune modulatory effects.

Identification of key processes that control tumor necrosis factor availability in a tuberculosis granuloma.

Tuberculosis (TB) granulomas are organized collections of immune cells comprised of macrophages, lymphocytes and other cells that form in the lung as a result of immune response to Mycobacterium tuberculosis (Mtb) infection. Formation and maintenance of granulomas are essential for control of Mtb infection and are regulated in part by a pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF). To characterize mechanisms that control TNF availability within a TB granuloma, we developed a multi-scale two compartment partial differential equation model that describes a granuloma as a collection of immune cells forming concentric layers and includes TNF/TNF receptor binding and trafficking processes. We used the results of sensitivity analysis as a tool to identify experiments to measure critical model parameters in an artificial experimental model of a TB granuloma induced in the lungs of mice following injection of mycobacterial antigen-coated beads. Using our model, we then demonstrated that the organization of immune cells within a TB granuloma as well as TNF/TNF receptor binding and intracellular trafficking are two important factors that control TNF availability and may spatially coordinate TNF-induced immunological functions within a granuloma. Further, we showed that the neutralization power of TNF-neutralizing drugs depends on their TNF binding characteristics, including TNF binding kinetics, ability to bind to membrane-bound TNF and TNF binding stoichiometry. To further elucidate the role of TNF in the process of granuloma development, our modeling and experimental findings on TNF-associated molecular scale aspects of the granuloma can be incorporated into larger scale models describing the immune response to TB infection. Ultimately, these modeling and experimental results can help identify new strategies for TB disease control/therapy.

Gene expression profiling and antigen mining of the tuberculin production strain Mycobacterium bovis AN5.

Control of bovine tuberculosis (bTB) relies on regular testing of cattle with a crude preparation of mycobacterial antigens termed purified protein derivative (PPD). Worldwide production of bovine PPD uses the Mycobacterium bovis AN5, a strain that was originally isolated circa 1948 in Great Britain (GB). Despite its worldwide use, the AN5 strain is poorly characterised. AN5 was adapted to grow on glycerol in a process similar to that used for the derivation of the BCG vaccine strains; during this process, it is known that BCG deleted the genes for some potent antigens. Our previous analysis of the genome of M. bovis AN5 showed that it had not suffered extensive gene deletion events during in vitro adaptation. However, glycerol adaptation of AN5 strain may have caused differences in its global gene expression profile that could affect antigen expression. To assess this, we determined the transcriptome profile of AN5 and compared it to expression data for two endemic GB strains of M. bovis that account for approximately 61% of all GB bTB cases. Genes expressed at lower levels in AN5 compared to M. bovis field isolates were then screened for antigenicity in naturally infected animals. Using this approach a number of genes were found to be expressed at lower levels in AN5, including those for known antigens. Our results show that field strains of M. bovis show some significant differences in gene expression to AN5, and that this differential gene expression may impact on the antigen profiles expressed by AN5 during in vitro culture.

Immunogenicity of neonatal BCG vaccination in children entering primary school.

This study has been designed to evaluate the immunogenicity of neonatal BCG-vaccination in children at the age of 7 to 8 years, by skin test using Purified Protein Derivative (PPD), as BCG vaccination at birth is a part of routine program of immunization in our country, Iran; we decided to study its efficacy and also tried to determine if there is any correlation between PPD-test results and BCG scar size. This is a comparative study on 150 children (94 males and 56 females) at the age of 7 to 8 years, who possess neonatal-BCG scar. They were chosen from several primary schools in Tabriz-Iran, by simple random sampling and tested with 0.1 mL of 5-unit-PPD solution (a product of Iran Institute of Razi); then observations recorded. The average diameter of BCG scars were 7.03 mm in girls, 5.45 mm in boys and 6.05 for all. The diameter of induration area resulted from PPD-test after 72 h was less than 5 mm in 95.33% and 5-9 mm in 4.66% of studied children; there was no case with induration area of 10 mm or more at all. Every child who developed an induration area of 5 mm or more by PPD test, had a BCG scar with the diameter of 5 mm or more. There was a statistically meaningful direct correlation between sizes of neonatal-BCG scar and diameter of induration area after PPD-test (r = 0.21 and p = 0.008). This study shows that reactivity to PPD test (and probably immunity against tuberculosis) decreases as age increases; therefore it seems to be necessary to repeat BCG-vaccination in children at the age of entering primary school.

Prueba de la tuberculina y riesgo de tuberculosis. No sólo el tamaño es importante / The tuberculin skin test and risk of tuberculosis: size is not the only issue

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