J. Ammar

NKT Cells in the Induced Sputum of Severe Asthmatics

To determine whether there was a specific inflammatory process in severe asthmatics, the phenotypic characteristics of induced sputum immune cells were analysed among patients with severe asthma. Twenty-two induced sputa (10 severe asthmatics) were studied. Flow cytometric analysis was performed using immune cells of the sputum and monoclonal antibodies to CD3, CD4, CD8, CD56, CD25, and TCRγδ. The number of NKT (CD3+CD56+) cells was significantly higher in the sputum of severe asthmatics compared with mild asthmatic and healthy control groups (P < .05). CD8+CD56+ cells were the predominant subtype of the increased NKT cells in severe asthmatics. CD3+CD56+Vα24+, TCRγδ+ CD56+, and CD4+CD25+ T cells were significantly increased in severe asthmatic patients. These results suggest that the immunopathogenesis of severe asthmatics vary between severe and mild asthmatics, and that CD8+CD56+ NKT cells may play an important role in the immunopathogenesis of severe asthma.

Association of vitamin D receptor gene polymorphisms with susceptibility to asthma in Tunisian children: A case control study.

BACKGROUND Vitamin D and its nuclear receptor (VDR) are linked to asthma in a genetic and immunologic basis. Polymorphisms in the VDR gene may alter the actions of vitamin D and then influence the development and the severity of asthma. AIMS We aimed at elucidating the genetic association of VDR gene polymorphisms with susceptibility to asthma in Tunisian children and with serum vitamin D levels. METHODS The study included 155 patients recruited from Abderrahmen MAMI hospital in Tunisia and two hundred twenty five healthy individuals matched with patients in age and sex for comparison. VDR genotypes were determined by PCR-RFLP method using endonuclease FokI, BsmI, TaqI and ApaI and vitamin D was assessed with a radioimmunoassay kit. RESULTS The distribution of genotype frequencies differed significantly between asthmatics and controls (FokI: P=0.04; BsmI: P=0.006; TaqI: P=0.006). Haplotype analyses revealed a significant association between bAt and bat haplotypes and asthma (P=0.00076, P=0.016). When patients were stratified according to atopic status and stage of severity, no significant association was detected with VDR variants. No association was found between VDR SNPs and serum 25-hydroxyvitamin D levels. CONCLUSION Our study shows a relation between VDR gene polymorphisms and susceptibility to asthma in children.

The impact of vitamin D deficiency on immune T cells in asthmatic children: a case-control study

Background Vitamin D exerts profound effects on both adaptive and innate immune functions involved in the development and course of autoimmune and inflammatory diseases. As the incidence of vitamin D insufficiency is surprisingly high in the general population, experimental studies have started to investigate whether vitamin D levels (measured as serum 25 hydroxy vitamin D-25[OH]D) are correlated with immune cells and clinical parameters. Purpose The aim of the present research was to investigate serum vitamin D status in a case-control study in children with asthma and to study associations between vitamin D levels and certain immunological parameters. Materials and methods A case control study of thirty-nine children with clinically controlled asthma was enrolled to assess the relationship between serum vitamin D concentrations and disease activity. Vitamin D was assayed with a radioimmunoassay kit. We evaluated the relationship between vitamin D concentrations and forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), and the FEV1/FVC ratio. Correlations between inflammatory mediators, Th1, Th2, Th17, and regulatory T cells (Treg) and vitamin D were investigated. Results Only 15.38% of our asthmatic children had a sufficient serum 25(OH)D (≥30 ng/mL) whereas 80% of healthy children expressed sufficient levels. Deficient values of vitamin D (<20 ng/mL) were observed in 17 (43.59%) asthmatic patients (14.40 ± 3.30 ng/mL; P = 0.0001). Deficiency was not observed in controls. Th1/Th2 ratio was significantly correlated to 25(OH) D level (r = 0.698; P = 0.0001). A significant negative correlation was observed between serum interleukin-17 and vitamin D levels in young asthmatics (r = −0.617; P = 0.001). A significant correlation was observed between CD25+Foxp3+ Treg cells and vitamin D values in asthmatics (r = 0.368; P = 0.021). Conclusion Even in a southern Mediterranean country, hypovitaminosis D is frequent in children with asthma. Our findings suggest that vitamin D is an important promoter of T cell regulation in vivo in young asthmatics.

Anti-inflammatory activity of IL-37 in asthmatic children: Correlation with inflammatory cytokines TNF-α, IL-β, IL-6 and IL-17A.

BACKGROUND The aim of this study was to assess interleukin (IL)-37 production in asthmatic children in serum and induced sputum and to look to the impact of IL-37 on pro-inflammatory cytokines production (TNF-α, IL-6, IL-1β and IL-17). METHODS Forty children with well-controlled asthma (20 moderate and 20 mild asthmatics) were studied. IL-37 was measured by ELISA in serum and induced sputum (IS) samples, and compared with 22 age- and sex-matched healthy controls. Real-time quantitative PCR was used to determine IL-37 mRNA expression in induced sputum cells. Induced sputum mononuclear cells from 10 moderate asthmatics and 10 healthy controls were stimulated either with lipopolysaccharides (LPS) or LPS plus recombinant IL-37 (rIL-37) comparing pro-inflammatory cytokines production. TNF-α, IL-1β, IL-6 and IL-17 were measured by RT-PCR and ELISA. FINDINGS The expression of IL-37 mRNA in asthmatic patients was significantly lower than that observed in healthy controls (P=0.0001). IL37 mRNA expression depended on asthma severity. Serum and IS IL-37 levels were significantly lower in asthma patients compared to healthy controls. LPS-stimulated sputum cells from asthma patients produced higher levels of IL-1β, IL-6, and TNF-α than those from HC. Adding rIL-37 suppressed TNF-α, IL-1β and IL-6 production in IS cells. In the same way, stimulating IS CD4(+) T cells in the presence of rIL-37 inhibited IL-17 production both in asthma patients and HC. IL-37 effect on IL-17 was more pronounced in patients than controls. INTERPRETATION The decrease in IL-37 level observed in IS was found to correlate with disease severity. The increased pro-inflammatory cytokines production from asthma IS cells was abrogated by the addition of rIL-37. IL-37 could be an important cytokine in the control of asthma by suppressing the production of inflammatory cytokines.

Transcriptional characteristics of CD4 T cells in young asthmatic children: RORC and FOXP3 axis.

Background Asthma is a chronic inflammatory disorder, hypothetically caused by autoreactive Th2 cells, whereas Th1 and regulatory T cells may confer protection. The development of Th subpopulations is dependent on the expression of lineage-specific transcription factors. Purpose This study aimed to assess the balance of CD4+ T cell populations in asthmatic children. Methods Peripheral blood mononuclear cells (PBMC) mRNA expression was assessed in 30 asthmatic children (18 patients with mild asthma and 12 with moderate asthma). Real-time polymerase chain reaction (RT-PCR) quantified TBX21, GATA-3, RORC, FOXP3, and EBI3 mRNA expression. Intracellular cytokine expression of IL-2, IL-4, IL-10, and IFN-γ in CD4+ T cells in asthmatic children was measured by flow cytometry. IL-6 and IL-17 cytokines were assessed in serum by enzyme-linked immunosorbent assay (ELISA). Results A significant increase was found in the percentage of CD4+ and CD8+ T cell-producing IL-4, IL-6, and IL-17. A decreased percentage of CD4+ producing IFN-γ in asthmatic children was found. Expression of GATA-3 (Th2), retinoid-related orphan receptor C (RORC) (Th17), and EBI3 were increased in asthmatic patients compared to healthy controls. Expression of FOXP3 (Treg) and TBX21 (Th1) were decreased (P < 0.0001 and P < 0.0001) in asthmatic children. Analysis of transcription factor ratios revealed an increase in the RORC/FOXP3 (P = 0.0001), and a significant decrease of TBX21/GATA-3 (P = 0.0001) ratios in patients with asthma. Conclusion Young asthmatics were characterized by increased IL-4 production and low IFN-γ synthesis. The increased serum IL-17 and IL-6 levels sustained an inflammatory environment in young asthmatics. The results indicate that FOXP3 and RORC mRNA expression could be associated with the sustained inflammatory process, transduced by low immune tolerance by Treg cells. The TBX21/GATA-3 and RORC/FOXP3 ratios dysregulation in asthmatics is consistent with the plasticity existing between Th1, Th17, and Treg cells during inflammation.

Inflammatory Process of CD8 + CD28 − T Cells in Induced Sputum From Asthmatic Patients

Previously unreported CD8(+) CD28(-) and CD8(+) CD28(+) T-cell subsets occur in healthy individuals and expand in patients suffering from autoimmune disease. Here we studied, for the first time, the expression of CD8(+) CD28(+) , CD8(+) CD28(-) , and CD8(+) CD56(+) subpopulations in induced sputum from asthmatics. Using sputum samples, purified CD8(+) T cells were stained for surface antigen CD28, CD56, FITC-conjugated anti-perforin, and anti-IFN-gamma. Cytotoxic activity was evaluated in a chromium releasing test. Induced sputum CD8(+) CD28(-) T cells were found to be more expanded and expressed low levels of IFN-gamma in severe asthmatics than mild asthma and age-matched healthy controls. The predominance of CD8(+) CD28(-) T cells can be in part explained by the expansion of CD8(+) CD56(+). CD8(+) CD28(-) T cells from severe asthmatics produced high intracytoplasmic perforin and exerted a potent cytotoxic activity. Considering their phenotyping and functional properties, the CD8(+) CD28(-) T-cell subset may constitute an intermediate phenotype in the process of CD8(+) T-cell differentiation of effector-type cells in severe asthmatics. Functional studies showed that CD8(+) CD28(-) T cells had cytotoxic function.

IL-17A and IL-17F genes variants and susceptibility to childhood asthma in Tunisia

Abstract Objectives: IL-17A and IL-17F are new pro-inflammatory cytokines implicated in neutrophilic inflammation and thus, involved in the pathogenesis of asthma. We investigated the possible association among asthma and IL-17A -197G/A (rs2275913), IL-17F 7488A/G (rs763780) and IL-17F 7383A/G (rs2397084). Methods: The study was performed in 171 patients with asthma (mean age 9.5 years, 105 boys, and 66 girls) and 171 healthy individuals matched with patients in age and sex. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to detect genes’ polymorphisms. Results: IL-17A -197G/A and IL-17F 7383A/G were associated with asthma in children (p = 0.008, p = 0.001, respectively). No association was found with IL-17F 7488A/G polymorphism. Haplotype analysis revealed a significant association between GA and AG haplotypes and asthma (p = 0.004, p = 0.02). When patients were stratified according to the atopic status, no significant association was detected with any of the three studied variants. Conclusion: Our results suggested that SNPs in IL-17A and IL-17F confer susceptibility to childhood asthma in Tunisia.

Conservative management of postoperative bronchopleural fistulas.

OBJECTIVE A bronchopleural fistula (BPF) is a serious complication after pulmonary resection and carries a high mortality rate. It remains a therapeutic challenge. The lack of a consensus suggests that no optimal therapy is available; however, endoscopic closure of a fistula may avoid extensive and potentially risky surgery. METHODS Seventeen patients (15 men and 2 women) with a BPF after a pneumonectomy (n = 2) or a lobectomy (n = 15), seen between 1995 and 2010, were reviewed. Their median age was 50 years (range, 14-75 years). Underlying diseases were malignant (n = 4) and nonmalignant (n = 13). RESULTS The mean interval between surgery and fistula development was 20 days (range, 5-270 days). Clinical symptoms leading to a diagnosis of BPF were a persistent air leak (n = 2), a persistent air leak associated with pleural empyema (n = 3), pleural empyema alone (n = 11), and dyspnea (n = 1). Mean fistula size was 3.3 mm (range, 2-9 mm). Treatment consisted of oriented pleural drainage, adequate antibiotic therapy, and endoscopic closure of the fistula with local application of silver nitrate through a flexible bronchoscope (3-15 sessions, 3 times per week). Fistula closure was successful in 16 patients, but failed in 1 patient, who died from acute respiratory distress. CONCLUSIONS BPF is a severe complication in thoracic surgery. The combination of pleural drainage, adequate antibiotic treatment, and mucosal application of silver nitrate, through a flexible bronchoscope, is an efficient alternative and avoids extensive surgical intervention.

Regulatory T cells in induced sputum of asthmatic children: association with inflammatory cytokines.

Background and objectiveCD4+CD25+ regulatory T (Treg) cells play an essential role in maintaining immune homeostasis. In this study, we investigated whether the induced sputum (IS) pool and the function of CD4+CD25+ Treg cells are altered in asthma pediatric patients.MethodsTreg activity was studied in the IS of 40 asthmatic children. CD3+ cells were analyzed for the expression of FoxP3 mRNA by real time reverse transcription-polymerase chain reaction (RT-PCR). IS cells from asthmatics and controls were stained for Treg markers and analyzed by flow cytometry. We also studied the ability of Treg cells to differentiate monocytes toward alternatively activated macrophages (AAM), and to suppress proinflammatory cytokines.Results(i) Mild and moderate asthmatics had significantly decreased expression of FoxP3/β-actin mRNA and decreased proportions of CD4+CD25highFoxP3+ cells compared to healthy children; (ii) patients with moderate asthma had even lower proportions of FoxP3 expression compared to mild asthmatic patients; (iii) monocytes cultured with Treg cells displayed typical features of AAM, including up-regulated expression of CD206 (macrophage mannose receptor) and CD163 (hemoglobin scavenger receptor), and an increased production of chemokine ligand 18 (CCL18). In addition, Treg cells from asthmatics have a reduced capacity to suppress LPS-proinflammatory cytokine production from monocytes/macrophages (IL-1, IL-6 and TNF-α).ConclusionAsthma pediatric patients display a decreased bronchial Treg population. The impaired bronchial Treg activity is associated with disease severity.RiasssuntoFinalitàLe cellule T regolatorie (Treg) CD4+CD25+ svolgono un ruolo fondamentale nel mantenere l’omeostasi dell’immunità. In questo studio si è ricercato se la quantità e la funzione dellecellule Treg CD4+CD25+ risultino alterate nello sputo indotto (IS) di pazienti asmatici in età pediatricaMetodiL’attività dei Treg è stata studiata prendendo in esame l’IS di 40 bambini asmatici. Le cellule CD3+ sono state analizzate per l’espressione di FoxP3 mRNA attraverso la retro trascrizionedella reazione a catena della polimerasi (RT–PCR) in tempo reale. Le cellule dell’IS di asmatici e soggetti di controllo sono state colorate specificamente per i Treg marker e analizzatecon la citometria a flusso. Si è studiata altresì la capacità delle cellule Treg di differenziare i monociti in macrofagi alternativamente attivati (AAM), e di sopprimere citochine proinfiammatorieRisultatiRisultati principali ottenuti: (i) Pazienti affetti da asma lieve e moderato mostravano una considerevole riduzione dell’espressione di FoxP3/β-actin mRNA e una ridotta proporzionedi cellule CD4+CD25highFoxP3+ in confronto a bambini sani; (ii) Pazienti affetti da asma moderato presentavano proporzioni ancora più basse di espressione di FoxP3 rispetto ai pazienti con asma lieve; (iii) Monociti coltivati con le cellule Treg hanno mostrato i tratti caratteristici di AAM, comprese leespressioni up-regolate di CD206 (recettore macrofagico per ilmannosio) e CD163 (recettore “scavenger” per l’emoglobina), e un aumento della produzione di ligando 18 delle chemochine(CCL18)Inoltre i Treg degli asmatici evidenziano una ridotta capacità di sopprimere la produzione delle citochine LPS-proinfiammatorie da monociti/macrofagi (IL-1, IL-6 e TNF-α)ConclusioniI pazienti pediatrici con asma presentano undecremento della popolazione bronchiale di Treg. La compromissione dell’attività bronchiale dei Treg è proporzionale al livello di gravità della malattia

Expression of Fas antigen and Fas ligand in bronchoalveolar lavage from silicosis patients.

OBJECTIVE: To understand the role of apoptosis through Fas/Fas ligand (FasL) interaction in the pathogenesis of silicosis, we examined the expression of Fas antigen, FasL and apoptosis in bronchoalveolar lavage fluid lymphocytes obtained from patients with silicosis. MATERIALS AND METHODS: Ten patients with silicosis, and 10 healthy controls were studied. Non-adherent cells were separated and analysed by cytometry for the expression of Fas antigen, FasL, and the co-expression of Fas/FasL. By double staining, we studied the FasL expression on CD4, CD8, CD56 and CD45RO-positive cells. DNA fragmentation was investigated by the terminal deoxy(d) UTP nick end labelling (TUNEL) method. RESULTS: We have found Fas and FasL expression in silicosis patients to be significantly higher than those in healthy controls. Interestingly, 6-18% of lymphocytes from silicosis patients co-expressed Fas and FasL. In silicosis patients, FasL was highly expressed on CD4+, CD56+ and CD45RO+ bronchoalveolar lavage cells. Fas antigen expressing cells showed DNA fragmentation characteristic for apoptosis. CONCLUSION: FasL was significantly expressed on cytotoxic effector and memory cells. The Fas/FasL system is implicated in the inflammatory process observed in silicosis patients.