Identification and seasonality of rhinovirus and respiratory syncytial virus in asthmatic children in tropical climate.

INTRODUCTION: Asthma is a disease that has been associated with the presence of different genetic and socio-environmental factors. OBJECTIVE: To identify and evaluate the seasonality of respiratory syncytial virus (RSV) and human rhinovirus (RV) in asthmatic children and adolescents in tropical climate, as well as to assess the socioeconomic and environmental factors involved. METHODS: The study was conducted in a referral hospital, where a total of 151 children were recruited with a respiratory infection. The International Study of Asthma and Allergies in Childhood (ISAAC) protocol and a questionnaire were applied, and a skin prick test was performed. The nasal swab was collected to detect RV and RSV through molecular assay. National Meteorological Institute (INMET) database was the source of climatic information. RESULTS: The socio-environmental characterization of asthmatic children showed the family history of allergy, disturbed sleep at night, dry cough, allergic rhinitis, individuals sensitized to at least one mite. We identified RV in 75% of children with asthma and 66.7% of RSV in children with asthma. There was an association between the presence of RV and the dry season whereas the presence of the RSV was associated with the rainy season. Contributing to these results, a negative correlation was observed between the RSV and the wind speed and the maximum temperature (T. Max) and a positive correlation with precipitation. CONCLUSIONS: The results suggest a high prevalence of RV and RSV in asthmatic children and the seasonality of these viruses were present in different climatic periods. This has significant implications for understanding short- and long-term clinical complications in asthmatic patients.

BAY 41-2272 inhibits human neutrophil functions.

BAY 41-2272 is a guanylyl cyclase (GC) stimulator derived from YC-1 (3-[(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole]). Previous studies by our group showed that BAY 41-2272 activates human monocytes via soluble guanylyl cyclase (sGC) and cGMP. In this study, we investigated the effect of BAY 41-2272 on human neutrophil function and found that 30 µM BAY 41-2272 inhibits neutrophil migration (1.82-fold lower than FMLP, P < 0.05 by one-way ANOVA followed by Tukey's test), oxidative burst (1.70-fold lower than PMA, P < 0.05 by one-way ANOVA followed by Tukey's test), and IL-8 cytokine production (1.80-fold lower than PMA, P < 0.05 by one-way ANOVA followed by Tukey's test). Our results suggest that these effects are independent of the sGC pathway but dependent instead on cGMP production, as the response induced by 30 µM BAY 41-2272 was 6.40-fold greater than that observed in our negative control (P < 0.05 by parametric t-test). 1H-[1, 2, 4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), which is an irreversible inhibitor of sGC, was unable to reverse the effects of BAY 41-2272 on human neutrophils, indicating that this drug acts independently of sGC. Our results confirm the immunomodulatory effect of BAY 41-2272 on human neutrophils.

Soluble CD40L is associated with increased oxidative burst and neutrophil extracellular trap release in Behçet's disease.

BACKGROUND: Studies have suggested that soluble factors in plasma from patients with active (aBD) and inactive (iBD) Behçet's disease (BD) stimulate neutrophil function. Soluble CD40 ligand (sCD40L) is an important mediator of inflammation in BD. Its expression and effect on neutrophil oxidative burst and neutrophil extracellular trap (NET) release have not been characterized. In this study, we sought to investigate the role of plasma and the CD40L pathway on NET release and the oxidative burst profile in patients with aBD and iBD. METHODS: Neutrophils and peripheral blood mononuclear cells (PBMCs) were obtained from patients with aBD (n = 30), patients with iBD (n = 31), and healthy control subjects (HCs; n = 30). sCD40L plasma concentration was determined in individual samples. A pool of plasma for each group was created. In some experiments, plasma pools were treated with recombinant CD40 (rhCD40-muIg) for sCD40L blockade. NET release and H2O2/O2- production were determined after stimulation with phorbol 12-myristate 13-acetate, sCD40L, or plasma pool. Flow cytometric analysis was performed to evaluate the expression of (1) CD40, Mac-1, and phosphorylated NF-κB p65 on neutrophils and monocytes and (2) CD40L on activated T cells and platelets. CD40L gene expression in PBMCs was determined by qRT-PCR. RESULTS: sCD40L plasma levels were significantly higher in patients with iBD (median 17,234, range 2346-19,279 pg/ml) and patients with aBD (median 18,289, range 413-19,883 pg/ml) than in HCs (median 47.5, range 33.7-26.7 pg/ml; p < 0.001). NET release was constitutively increased in BD compared with HC. NET release and H2O2/O2- were higher after stimulation with sCD40L or BD plasma and decreased after sCD40L blockade. Mac-1 expression was constitutively increased in neutrophils of patients with aBD (88.7 ± 13.2% of cells) and patients with iBD (89.2 ± 20.1% of cells) compared with HC (27.1 ± 18.8% of cells; p < 0.01). CD40 expression on phagocytes and CD40L expression on platelets were similar in the three groups. PBMCs as well as nonactivated and activated CD4+ T cells from patients with BD showed higher CD40L expression. CONCLUSIONS: Plasma from patients with aBD exerts a stimulus on NET release and oxidative burst, probably induced by sCD40L.

Behçet's disease heterogeneity: cytokine production and oxidative burst of phagocytes are altered in patients with severe manifestations.

OBJECTIVES: To test the hypothesis that classical phagocytic functions are constitutively stimulated in patients with Behçet's disease (BD). METHODS: Four study groups were analysed: active BD (aBD; n=30), inactive BD (iBD; n=31); septic patients (SP; n=25); healthy controls (HC; n=30). Microbicide activity against Streptococcus pneumoniae, Streptococcus sanguinis and Candida albicans was determined by means of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and absorbance read by ELISA. Flow cytometry analysis evaluated phagocytosis (zymosan particles and microrganisms) and oxidative burst by dihidrorhodamine oxidation before and after stimulation with phorbol myristate acetate (PMA). The supernatant of PBMC cultures under TLR or microbial stimuli and of neutrophil cultures under PMA, LPS or microbial stimuli were used for determination of cytokine production by ELISA. RESULTS: We found no significant differences between the BD patient groups and control groups with regard to oxidative burst, phagocytic activity, microbicide activity or cytokine production. However, the cells from patients with severe BD (based on clinical manifestation) exhibit significantly higher oxidative burst activity, both before and after PMA stimulation, compared to cells from patients with mild BD. Furthermore, we found significant correlations between the BD patients' scores on the simplified Behçet's Disease Current Activity Form adapted for Portuguese (BR-BDCAFs) and Streptococcus sanguinis-stimulated production of IL23 by PBMC and IL8 by neutrophils, and between BR-BDCAFs score and constitutive production of TNF-α, IFNγ, IL6 and IL23 by PBMC. CONCLUSIONS: Patients with severe active BD do exhibit phagocytic dysfunction and some evidence of constitutive activation regarding oxidative burst and cytokine production.

BAY 41-2272 activates host defence against local and disseminated Candida albicans infections.

In our previous study, we have found that 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]-pyrimidin-4-ylamine (BAY 41-2272), a guanylate cyclase agonist, activates human monocytes and the THP-1 cell line to produce the superoxide anion, increasing in vitro microbicidal activity, suggesting that this drug can be used to modulate immune functioning in primary immunodeficiency patients. In the present work, we investigated the potential of the in vivo administration of BAY 41-2272 for the treatment of Candida albicans and Staphylococcus aureus infections introduced via intraperitoneal and subcutaneous inoculation. We found that intraperitoneal treatment with BAY 41-2272 markedly increased macrophage-dependent cell influx to the peritoneum in addition to macrophage functions, such as spreading, zymosan particle phagocytosis and nitric oxide and phorbol myristate acetate-stimulated hydrogen peroxide production. Treatment with BAY 41-2272 was highly effective in reducing the death rate due to intraperitoneal inoculation of C. albicans, but not S. aureus. However, we found that in vitro stimulation of peritoneal macrophages with BAY 41-2272 markedly increased microbicidal activities against both pathogens. Our results show that the prevention of death by the treatment of C. albicans-infected mice with BAY 41-2272 might occur primarily by the modulation of the host immune response through macrophage activation.

BAY 41-2272 activates host defence against local and disseminated Candida albicans infections

In our previous study, we have found that 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]-pyrimidin-4-ylamine (BAY 41-2272), a guanylate cyclase agonist, activates human monocytes and the THP-1 cell line to produce the superoxide anion, increasing in vitro microbicidal activity, suggesting that this drug can be used to modulate immune functioning in primary immunodeficiency patients. In the present work, we investigated the potential of the in vivo administration of BAY 41-2272 for the treatment of Candida albicans and Staphylococcus aureus infections introduced via intraperitoneal and subcutaneous inoculation. We found that intraperitoneal treatment with BAY 41-2272 markedly increased macrophage-dependent cell influx to the peritoneum in addition to macrophage functions, such as spreading, zymosan particle phagocytosis and nitric oxide and phorbol myristate acetate-stimulated hydrogen peroxide production. Treatment with BAY 41-2272 was highly effective in reducing the death rate due to intraperitoneal inoculation of C. albicans, but not S. aureus. However, we found that in vitro stimulation of peritoneal macrophages with BAY 41-2272 markedly increased microbicidal activities against both pathogens. Our results show that the prevention of death by the treatment of C. albicans-infected mice with BAY 41-2272 might occur primarily by the modulation of the host immune response through macrophage activation. .

Dendritic cells from X-linked hyper-IgM patients present impaired responses to Candida albicans and Paracoccidioides brasiliensis.

BACKGROUND: Patients with X-linked hyper-IgM syndrome (X-HIGM) due to CD40 ligand (CD40L) mutations are susceptible to fungal pathogens; however, the underlying susceptibility mechanisms remain poorly understood. OBJECTIVE: To determine whether monocyte-derived dendritic cells (DCs) from patients with X-HIGM exhibit normal responses to fungal pathogens. METHODS: DCs from patients and controls were evaluated for the expression of costimulatory (CD80 and CD86) and MHC class II molecules and for their ability to produce IL-12 and IL-10 in response to Candida albicans and Paracoccidioides brasiliensis. We also evaluated the ability of C albicans- and P brasiliensis-pulsed mature DCs to induce autologous T-cell proliferation, generation of T helper (T(H)) 17 cells, and production of IFN-γ, TGF-ß, IL-4, IL-5, and IL-17. RESULTS: Immature DCs from patients with X-HIGM showed reduced expression of CD80, CD86, and HLA-DR, which could be reversed by exogenous trimeric soluble CD40L. Most important, mature DCs from patients with X-HIGM differentiated by coculturing DCs with fungi secreted minimal amounts of IL-12 but substantial amounts of IL-10 compared with mature DCs from normal individuals. Coculture of mature DCs from X-HIGM patients with autologous T cells led to low IFN-γ production, whereas IL-4 and IL-5 production was increased. T-cell proliferation and IL-17 secretion were normal. Finally, in vitro incubation with soluble CD40L reversed the decreased IL-12 production and the skewed T(H)2 pattern response. CONCLUSION: Absence of CD40L during monocyte/DC differentiation leads to functional DC abnormalities, which may contribute to the susceptibility to fungal infections in patients with X-HIGM.