Adaptive Immune Response to Mycobacterium abscessus Complex (MABSC) in Cystic Fibrosis and the Implications of Cross-Reactivity.

Background: We aimed to characterise the adaptive immune response to Mycobacterium abscessus complex (MABSC) and its cross-reactivity with Mycobacterium avium complex (MAC) and Mycobacterium bovis (Bacille Calmette-Guérin, BCG) in cystic fibrosis (CF) patients and non-CF controls in terms of lymphocyte proliferation and immunophenotyping, cytokine production and anti-MABSC IgG plasma levels. Methods: In this cross-sectional analysis, peripheral blood mononuclear cells (PBMC) from CF patients with MABSC (CF/MABSC, n=12), MAC infection history (CF/MAC, n=5), no NTM history (CF/NTM-, n=15), BCG-vaccinated (C/BCG+, n=9) and non-vaccinated controls (C/BCG-, n=8) were cultured for four days under stimulation with an in-house MABSC lysate and we used flow cytometry to assess lymphocyte proliferation (given by lymphoblast formation) and immunophenotypes. Cytokine production was assessed after overnight whole blood stimulation with the same lysate, and anti-MABSC IgG levels were measured in plasma from non-stimulated blood. Results: All CF/MABSC patients had increased CD3+ and CD19+ lymphoblast formation upon PBMC stimulation with MABSC lysate. There was a higher rate of CD3+ than CD19+ lymphoblasts, predominance of CD4+ over CD8+ lymphoblasts, IFN-γ, TNF-α and IL-2 production, low production of the Th17-associated IL-17, and discrete or no production of Th2/B cell-associated cytokines soluble CD40 ligand (CD40L), IL-4 and IL-5, indicating a Th1-dominated phenotype and infection restricted to the lungs. A similar pattern was seen in C/BCG+ controls, and CF/MAC patients, pointing to cross-reactivity. MABSC-IgG levels were higher in CF/MABSC patients than in both control groups, but not CF/NTM- patients, most of whom also had CD3+ and/or CD19+ lymphoblast formation upon PBMC stimulation, indicating previous exposure, subclinical or latent infection with MABSC or other NTM. Conclusion: The anti-MABSC immune response is Th1-skewed and underlines the cross-reactivity in the anti-mycobacterial immune response. The results, together with published clinical observations, indicate that BCG vaccination may cross-react against NTM in CF patients, and this should be investigated. Due to cross-reactivity, it would also be interesting to investigate whether a combination of MABSC-induced cytokine production by blood cells and anti-MABSC IgG measurement can be useful for identifying latent or subclinical infection both with MABSC and other NTM in CF patients.

Patients with treated autoimmune hepatitis and persistent suppression of plasmacytoid dendritic cells: A different point of view.

Objectives: Plasmacytoid dendritic cells (pDCs) have been shown to have a role in autoimmune diseases, but their role in Autoimmune Hepatitis (AIH) is not completely clear. In the present study, we assessed the frequency of pDCs in peripheral blood of AIH patients under long-term standard immunosuppressive therapy. Methods: This cross-sectional analysis enrolled 27 AIH patients and 27 healthy controls. We analyzed and compared their proportion of pDCs, CD4+, CD8+, γδ T cells, CD25+ regulatory T (Treg) cells, FoxP3+, Foxp3+CD39+ Treg cells, total B (CD19+) cells, and plasma cells (CD38+) in peripheral blood using flow cytometry immunophenotyping. Results: AIH patients had a lower percentage of pDCs (median frequencies of 0.2% vs. 0.4%; p = .002) and higher expression of CD8 T cells (32.5% vs 28.6%; p = 0.008) in peripheral blood, when compared to healthy controls. We did not find statistically significant differences between the groups regarding the other cell subtypes.Conclusion: Our data suggest a persistent suppression of pDCs in AIH patients, along with increased CD8 T cell activity, years after AIH diagnosis and despite of good clinical response to treatment, thus pointing to a role of pDCs in the AIH pathogenesis.

Effective combined antiretroviral therapy provides partial immune recovery to mycobacterial antigens in vertically infected, BCG-vaccinated youth living with HIV.

BACKGROUND: We assessed the cytokine response by PBMC of youth living with HIV (YLHIV) under combined antiretroviral therapy (cART) to Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis (BCG) antigens. METHODS: PBMC from 20 Brazilian YLHIV under cART with long-term (≥1 year) virological control, and 20 healthy controls were cultured for 24-96 h under stimulation with BCG, Mtb lysates, ESAT-6 and SEB. We measured TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-10 and IL-17 in culture supernatants using a cytometric bead array. RESULTS: Controls had higher IFN-γ production at 24, 48, 72 and 96 h upon stimulation with BCG lysate, plateauing at 48 h (Median = 1991 vs. 733 pg/mL; p = 0.01), and after 48-72 h of stimulation with Mtb lysate, plateauing at 48 h (3838 vs. 2069 pg/mL; p = 0.049). YLHIV had higher TNF-α production at all time points upon stimulation with ESAT-6, with highest concentration at 36 h (388 vs. 145 pg/mL; p = 0.02). Within the YLHIV group, total CD4 T cell count and CD4/CD8 ratio were associated with IFN-γ response to Mtb lysate and ESAT-6, respectively. CONCLUSIONS: Even under long-term cART, YLHIV seem to have a suboptimal T-helper-1 response to mycobacterial antigens. This can be explained by early immunodeficiency in vertical infection, with lasting damage.

Preservation of cytotoxic granule production in response to mycobacterial antigens by T-lymphocytes from vertically HIV-infected Brazilian youth on effective combined antiretroviral therapy.

BACKGROUND: HIV infection harms adaptive cellular immunity mechanisms. Long-term virological control by combined antiretroviral therapy (cART) reduces the risk of mycobacterial infections. Thus, we aimed to study cellular responses to mycobacterial antigens in 20 HIV-infected adolescents with at least one year of virological control (HIV-RNA <40 copies/mL) and 20 healthy adolescents. METHODS: We evaluated CD8 and γδ T-cell degranulation by measurement of CD107a membrane expression after stimulation with lysates from BCG (10 µg/mL) and H37RA Mycobacterium tuberculosis (Mtb, 10 µg/mL). Immune activation and antigen-presenting ability were also assessed by determination of HLA-DR, CD80, and CD86 markers. RESULTS: TCR γδ T-cell CD107a expression was similar between groups in response to mycobacterial antigens, and lower in the HIV-infected group in response to mitogen. Higher baseline HLA-DR expression and lower mycobacterial-stimulated expression was found within the HIV-infected group. CONCLUSIONS: Similar degranulation in stimulated CD8+ and TCR γδ T-cells from HIV-infected adolescents, when compared to healthy controls suggests long-term immunological preservation with immune reconstitution under successful cART. However, differences in HLA-DR expression may represent ongoing inflammation and lower specific responses in HIV-infected youth. These features may be relevant in the context of the precocity and severity of vertically acquired HIV infection.

Secretory IgA-mediated immune response in saliva and early detection of Pseudomonas aeruginosa in the lower airways of pediatric cystic fibrosis patients.

Pseudomonas aeruginosa (Pa) detection in the paranasal sinuses may help to prevent or postpone bacterial aspiration to the lower airways (LAW) and chronic lung infection in cystic fibrosis (CF). We assessed the ability of an ELISA test for measurement of specific Pa secretory IgA (sIgA) in saliva (a potential marker of sinus colonization) to early detect changes in the Pa LAW status (indicated by microbiological sputum or cough swab culture and specific serum IgG levels) of 65 patients for three years, in different investigation scenarios. Increased sIgA levels were detected in saliva up to 22 months before changes in culture/serology. Patients who remained Pa-positive had significantly increased sIgA levels than patients who remained Pa-negative, both at the baseline (39.6 U/mL vs. 19.2 U/mL; p = 0.02) and at the end of the follow-up (119.4 U/mL vs. 25.2 U/mL; p < 0.001). No association was found between sIgA levels in saliva and emergence or recurrence of Pa in the LAW. A positive median sIgA result in the first year of follow-up implied up to 12.5-fold increased risk of subsequent Pa exposure in the LAW. Our test detected early changes in the P. aeruginosa LAW status and risk of exposure to P. aeruginosa in the LAW with two years in advance. Comparison with sinus culture is needed to assess the test's ability to identify CF patients in need of a sinus approach for Pa investigation, which could provide opportunities of Pa eradication before its aspiration to the lungs.

Assessment of IgG antibodies to Pseudomonas aeruginosa in patients with cystic fibrosis by an enzyme-linked immunosorbent assay (ELISA).

BACKGROUND: The usefulness of serological tests for detection of P. aeruginosa pulmonary infection in cystic fibrosis (CF) is controversial. Here, we assessed the value of detecting anti-P. aeruginosa IgG by a quantitative enzyme-linked immunosorbent assay (ELISA) for identification of P. aeruginosa infection in patients with cystic fibrosis. METHODS: Serum concentrations of anti-P. aeruginosa IgG were assessed in 117 CF patients classified according to their P. aeruginosa colonization/infection status (never colonized; free of infection; intermittently colonized and chronically infected) and in 53 healthy subjects by the ELISA test standardized with the St-Ag:1-17 antigen. RESULTS: The rate of IgG seropositivity and the median of IgG concentrations of this antibody in patients chronically infected were significantly higher than those found in the other CF groups and in the healthy control group. CONCLUSION: Detection of anti-P. aeruginosa IgG can be an useful tool for identification of P. aeruginosa chronic infection in patients with CF. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_158.

Immunogenicity of a Whole-Cell Pertussis Vaccine with Low Lipopolysaccharide Content in Infants

The lack of a clear correlation between the levels of antibody to pertussis antigens and protection against disease lends credence to the possibility that cell-mediated immunity provides primary protection against disease...