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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., SCOPUS: ar.j, info:eu-repo/semantics/published

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., SCOPUS: ar.j, info:eu-repo/semantics/published

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 theMycobacterium tuberculosisreservoir. 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 theirM. tuberculosisinfection status (LTBI, TB infection, undeterminedM. tuberculosisinfection 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 ofMycobacterium bovisBCG vaccination during infancy, and a relative discrimination between LTBI and TB infection. Whereas the commercialized IGRAs show a greater sensitivity for recent than for remoteM. tuberculosisinfections, 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.

The clonal CD3(-)CD4(+) T-cell population characterizing lymphocytic variant hypereosinophilic syndrome (L-HES) persists for years, with a subgroup of patients ultimately progressing to T lymphoma. The molecular changes associated with the premalignant clone and the emergence of malignant subclones are unknown, precluding the development of targeted therapy for this HES variant. In this study, we used whole genome arrays to examine gene expression in the CD3(-)CD4(+) T cells and found that 850 genes were differentially regulated during chronic disease compared with CD3(+)CD4(+) T cells from healthy donors. Changes in the expression of 349 genes were altered in association with the clinical progression from chronic L-HES to T lymphoma in 1 patient, with 87 of 349 genes representing further changes in genes whose expression was altered in all chronic disease patients (87 of 850). Array analysis after CD2/CD28-mediated activation revealed that the major gene expression changes observed in the CD3(-)CD4(+) T cells do not reflect activation induced alterations but rather pathways involved in T-cell homeostasis, including transforming growth factor-beta signaling, apoptosis, and T-cell maturation, signaling, and migration. Examination of microRNA expression in the CD3(-)CD4(+) T cells from patients with chronic disease identified 23 microRNAs that changed significantly, among which miR-125a further decreased in association with one patient's evolution to T lymphoma.

The TET enzymes convert methylcytosine to the newly discovered base hydroxymethylcytosine. While recent reports suggest that TETs may play a role in response to oxidative stress, this role remains uncertain, and results lack in vivo models. Here we show a global decrease of hydroxymethylcytosine in cells treated with buthionine sulfoximine, and in mice depleted for the major antioxidant enzymes GPx1 and 2. Furthermore, genome-wide profiling revealed differentially hydroxymethylated regions in coding genes, and intriguingly in microRNA genes, both involved in response to oxidative stress. These results thus suggest a profound effect of in vivo oxidative stress on the global hydroxymethylome., info:eu-repo/semantics/published

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., SCOPUS: ar.j, info:eu-repo/semantics/published

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45(+) cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45(+) cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

Assessment of T-cell activation is pivotal for evaluation of cancerimmunotherapy. We initiated a clinical trial in patients with MAGE-A1and/or -A3 tumors using autologous DC pulsed with MAGE peptides aimedat analyzing T-cell-derived, IFN-γ secretion by cytokine flowcytometry and ELISPOT. We also tested whether further KLH additioncould influence this response favorably. Monocyte-derived DC weregenerated from leukapheresis products. They were pulsed with therelevant MAGE peptide(s) alone in group A (n=10 pts) andadditionally with KLH in group B (n=16 pts). A specific buttransient increase in the number of peripheral blood T lymphocytessecreting IFN-γ in response to the vaccine peptide(s) was observed in6/8 patients of group A and in 6/16 patients of group B. We concludethat anti-tumor vaccination using DC pulsed with MAGE peptides inducesa potent but transient anti-MAGE, IFN-γ secretion that is notinfluenced by the additional delivery of a nonspecific, T-cellhelp.

Antigen-specific and polyclonally induced T cell responses were analyzed in 10 HIV-infected individuals and in 14 controls by enumerating the numbers of tetanus toxoid (TT)-specific and phytohemagglutinin (PHA)-induced IFN-gamma-secreting cells (SC) and IL-4-SC using an enzyme-linked immunospot assay. Whereas the numbers of IFN-gamma-SC in HIV-infected patients were not different from those of the controls in response to an in vitro stimulation with PHA, they were significantly decreased in response to an in vitro stimulation with TT, both before and after a TT booster. Cell depletion experiments indicated that this difference was related to an impairment of CD4(+) T-cell-mediated TT-specific IFN-gamma secretion. Concerning IL-4, the numbers of both polyclonally induced IL-4-SC and TT-specific IL-4-SC were significantly lower in HIV-infected patients than in the controls. It is concluded that secretion of antigen-specific cytokines of both Th1 and Th2 types is depressed in HIV-infected patients.

Background—Cytokines secreted by intestinal T lymphocytes probably play a critical role in regulation of the gut associated immune responses.Aims—To quantify interferon γ (IFN-γ) and interleukin 4 (IL-4) secreting cells (SC) among human intraepithelial (IEL) and lamina propria (LPL) lymphocytes from the duodenum and right colon in non-pathological situations and in the absence of in vitro stimulation.Patients—Duodenal and right colonic biopsy specimens were obtained from patients with no inflammation of the intestinal mucosa.Methods—Intraepithelial and lamina propria cell suspensions were assayed for numbers of cells spontaneously secreting IFN-γ and IL-4 by a two site reverse enzyme linked immunospot technique (ELISPOT).Results—The relatively high proportion of duodenal lymphocytes spontaneously secreting IFN-γ (IEL 3.6%; LPL 1.9%) and IL-4 (IEL 1.3%; LPL 0.7%) contrasted with the very low numbers of spontaneously IFN-γ SC and the absence of spontaneously IL-4 SC among peripheral blood mononuclear cells. In the basal state, both IFN-γ and IL-4 were mainly produced by CD4+ cells. Within the colon, only 0.2% of IEL and LPL secreted IFN-γ in the basal state, and 0.1% secreted IL-4.Conclusions—Compared with peripheral lymphocytes substantial proportions of intestinal epithelial and lamina propria lymphocytes spontaneously secrete IFN-γ and/or IL-4. These cytokines are probably involved in the normal homoeostasis of the human intestinal mucosa. Disturbances in their secretion could play a role in the pathogenesis of gastrointestinal diseases.

CD4+ helper T (Th) cells are critical regulators of immune responses but their role in breast cancer is currently unknown. The goal of this project was to characterize CD4+ T lymphocytes infiltrating primary human breast tumors (TIL), analyze T cell signaling pathways influenced by the tumor microenvironment, correlate differential gene expression with the presence of a specific CD4+ T cell subset(s) and identify a profile predictive of positive patient outcome. Affymetrix U133 Plus 2.0 arrays were used to analyze CD4+ T cells freshly isolated from the primary tumor, axillary lymph node (LN) and blood (PB) of patients with ER+ and ER- invasive breast carcinoma in the discovery set (10 patients). Microarrays, flow cytometry and/or qRT-PCR were used to extend and confirm the data in CD4+ TIL isolated from >60 primary breast tumors as well as purified CD4+ T cells from healthy donors PB treated with supernatant (SN) derived from fresh breast tumor tissue. Assessment of the relative distribution of infiltrating mononuclear cells revealed that the CD4+ T cell subset was consistently the principal component. We detected gene expression changes in the CD4+ TIL compared to patient's LN and PB counterparts that reflected altered cellular signaling pathways, including significant suppression of the TCR/CD3 complex and downstream signaling molecules along with a highly restricted pattern of Th cytokine and chemokine expression. A comparison with publically available Th profiling microarray data revealed that the TIL have characteristics of an activated memory effector CD4+ T cell phenotype, with all the major subsets (Th1, Th2, Th17, Treg, Tfh) represented. Although very few differences were detected in a direct comparison between CD4+ TIL from ER+ and ER- tumors, important changes in TIL (including a Th subset skew and differential cytokine expression) from extensive versus minimally infiltrated tumors were observed, with those containing an extensive CD4+ T cell infiltrate associated with a better clinical outcome. Gene expression profiles of freshly isolated TIL were also compared with TIL that had been rested overnight. These experiments revealed that the rested TIL reverse expression in a number of critical cytokine and signaling genes. PB CD4+ T cells from healthy donors treated with primary tumor SN also displayed significant changes in gene expression, gaining >75% similarity to freshly isolated CD4+ TIL. These experiments further found that activation of CD4+ T cells in conjunction with tumor SN treatment rapidly suppressed stimulation-induced changes in gene expression. Together our data suggest that although activated CD4+ T cells infiltrating breast tumors are quickly suppressed, tumors that recruit higher levels of CD4+ T cells, even in the face of rapid immunosuppression, produce a better memory T cell pool that is ultimately capable of maintaining effective anti-tumor immunity in patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3541. doi:1538-7445.AM2012-3541

The influence of Broncho-Vaxom (BV) on different immune parameters was investigated in vitro on human peripheral blood mononuclear cells (PBMC). It was found that BV enhances the natural killer (NK) activity of PBMC and increases their spontaneous and phytohemagglutin (PHA)-induced production of tumor-necrosis factor--alpha and interleukin-2 as well as the PHA-stimulated production of interferon-gamma. These immunostimulating actions of BV on NK activity and cytokine production can contribute to the understanding of the enhancement of the body's defense mechanisms against respiratory tract infections.

Broncho-Vaxon (OM-85 BV) is a bacterial extract of eight bacterias usually involved in the respiratory tract infections. Since Broncho-Vaxom is clinically active in decreasing the incidence of such infections, its immunological effect was investigated, in vitro, using peripheral blood mononuclear cells (PBMC). The experimental data indicate that Broncho-Vaxom can modulate various immune functions. It was shown, using a radioimmunoassay for these cytokines, that Broncho-Vaxom will spontaneously enhance TNF alpha and IL-2 production whereas it has no action on IF gamma production. However, when the PBMC are stimulated with PHA, an increased production for IF gamma, TNF alpha and IL-2 was observed suggesting that, under appropriate conditions, Broncho-Vaxom enhances the production of these cytokines. In other experiments, Broncho-Vaxom was shown to markedly increase the natural killer activity of PBMC. All these results demonstrate that Broncho-Vaxom is an immunomodulator affecting multiple immunological mechanisms including the activation of natural killer cells, of monocytes and of T cells through direct mechanisms or through the cytokine cascade.

OM-89 is a bacterial extract of E. coli which is clinically used in the treatment of urinary tract infections. Since immunological mechanisms may play a role in its action, its immunological effects were evaluated in vitro on peripheral blood mononuclear cells. The results showed that OM-89 enhances natural killer activity, as well as the spontaneous production of alpha-TNF and IL2. In the presence of various mitogens, it enhanced the production of IL1, gamma-IF, alpha-TNF and IL2. These data indicate that OM-89 acts on cell-mediated mechanisms by a direct and/or an indirect effect on monocytes, T cells and natural killer cells. These data suggest that the clinical activity by OM-89 may be related to its immunological properties.