Functional variants in the thromboxane A2 receptor gene are associated with lung function in childhood-onset asthma.

BACKGROUND: The thromboxane A2 receptor (TBXA2R) gene is associated with asthma, but no functional genetic variations are known to associate with the disease or its related phenotypes. OBJECTIVE: To investigate the association of TBXA2R polymorphisms with asthma susceptibility and related phenotypes and to identify functionally relevant polymorphisms. METHODS: We performed comprehensive sequencing of the TBXA2R gene in 48 Japanese control subjects and found a set of variants (SNP1 G>T rs2238634, SNP2 T>G rs2238633, SNP3 C>T rs2238632 and SNP4 G>A rs2238631) in intron 1 in linkage disequilibrium with c.795 T>C rs1131882, which was previously reported to be associated with asthma and related phenotypes. To investigate the effect of four common haplotypes (H1, H2, H3 and H4) on transcriptional activity, we performed a luciferase assay in primary bronchial smooth muscle cells (BSMCs) and human airway epithelial cells (BEAS-2B). We also studied the haplotype association with lung function, TBXA2R mRNA levels, and eosinophil fraction/count in peripheral blood in childhood-onset asthma patients and/or controls. RESULTS: H2 and H4, containing minor alleles of SNP2 and SNP3, had significantly higher transcriptional activities than H1 consisting of major alleles (P < 0.001 in BSMCs and BEAS-2B). Homozygotes for redefined haplotype h2 corresponding to minor alleles of SNP2 and SNP3 were associated with lower lung function in childhood-onset asthma patients compared to other zygotes (baseline Forced expiratory volume in one second (FEV1)/ Forced vital capacity (FVC) and Forced expiratory flow between 25% and 75% of the FVC (%FEF(25-75%)): P = 0.00201 and 0.0128, respectively, and post-bronchodilator FEV1/FVC and %FEF(25-75%): P = 0.00224 and 0.0393 respectively). Haplotype h2 was also associated with higher mRNA levels in control peripheral blood cells and higher blood eosinophil fractions and counts in female controls. CONCLUSIONS AND CLINICAL RELEVANCE: Genetic variants were identified in the TBXA2R gene that influenced transcriptional activity and were associated with asthma-related phenotypes. Thromboxane pathways may therefore play important roles in airway inflammation and remodelling in asthma patients.

Respiratory syncytial virus infection suppresses IFN-gamma production of gammadelta T cells.

The immunological mechanisms by which respiratory syncytial virus (RSV) contributes to the development of asthma are poorly understood. gammadelta T cells are important in mucosal defence, and may contribute to the establishment of primary immune responses by producing cytokines early during respiratory infections. Thus, we used flow cytometry and intracellular cytokine staining to investigate the expression of interferon (IFN)-gamma and interleukin (IL)-4 by mitogen-stimulated gammadelta T cells from the peripheral blood of 15 hospitalized infants with RSV bronchiolitis, seven rotavirus-infected infants and eight normal controls. gammadelta T cells from RSV-infected infants had a lower proportion of IFN-gamma-producing cells (median, 4.00%; range, 0.58-6.60%) and a slightly but significantly higher proportion of IL-4-producing cells (median, 0.40%; range, 0.13-2.76%) than rotavirus-infected infants (median, 32.10%; range, 14.43-61.21%; P < 0.01, median, 0.00%; range, 0.00-0.00%; P < 0.05) in the acute phase. By contrast, differences in cytokine production by total CD3+ T cells did not differ significantly between patient groups. Thus, reduced IFN-gamma-production by gammadelta T cells in the peripheral blood of RSV-infected infants is accompanied by increased Th2 cytokine production during the acute phase of disease. At follow-up, eight children had recurrent episodes of wheezing. The frequencies of IFN-gamma-producing gammadelta T cells were significantly lower in patients who developed recurrent wheezing (median, 0.65%; range, 0.02-1.75%) than in patients without recurrent wheezing (median, 6.90%; range, 5.25-10.98%; P < 0.005). Cytokine production by gammadelta T cells may therefore be important in the pathogenesis of acute RSV disease, and play a part in the development of recurrent childhood wheezing after bronchilolitis.

Circadian variation in nasal reactivity in children with allergic rhinitis: correlation with the activity of eosinophils and basophilic cells.

BACKGROUND: In allergic rhinitis, the major symptoms of runny nose, sneezing, and stuffy nose tend to become worse upon waking up in the morning, and yet the mechanisms underlying this phenomenon are poorly understood. We investigated whether the worsening of allergic rhinitis in the morning is associated with changes in the activity of inflammatory cells. METHODS: Nasal reactivity to methacholine was assessed twice in 8 children with allergic rhinitis and 8 healthy control subjects at 6.00 a.m. and 3.00 p.m. The amounts of eosinophil cationic protein (ECP), histamine and tryptase in induced nasal secretions and peripheral blood were also measured. RESULTS: Nasal reactivity to methacholine was higher at 6.00 a.m. not only in patients but also in healthy controls. Serum ECP and plasma histamine levels showed no circadian patterns. On the other hand, significantly higher levels of inflammatory activation products were found in nasal secretions at 6.00 a.m., thus showing a direct association with nasal reactivity. CONCLUSION: These results suggest that the circadian variation in nasal reactivity is associated with changes in the activity of eosinophils and basophilic cells in the nasal mucosa.

[Flow cytometric analysis for the activation of peripheral immunoregulatory cells from patients with bronchial asthma].

By use of flow cytometry, we have investigated intracellular activated eosinophil cationic protein (ECP) in eosinophils and mitogen-induced cytokine production of T cells in peripheral blood from children with acute severe asthma. In addition, we measured ECP releasability (serum ECP/lysate ECP) as a maker of activated eosinophils. The monoclonal antibody EG2 (anti-activated ECP/EPX antibody) was used for measuring the amount of intracellular activated ECP. ECP releasability and mean fluorescence intensity (MFI) values of EG2-positive eosinophils increased at the time of asthmatic attack and reduced after treatment with improvement in peak expiratory flow. Furthermore, the frequency of T cells which produced IL-4, IL-5 and IFN-gamma stimulated with phorbol myristate acetate and ionomycin increased and reduced in parallel with MFI of EG2-positive cells. These observations suggest that flow cytometric analysis for intracellular ECP and mitogen-induced cytokine production reflects the activation of T cells in bronchial mucosa, and is useful for monitoring airway inflammation in bronchial asthma.

[Antigenic determinants on ovalbumin and ovomucoid: comparison of the specificity of IgG and IgE antibodies].

To delineate the antigenic determinants on ovalbumin (OA) and ovomucoid (OVM) recognized by specific IgG and IgE antibodies in sera from patients with allergy to hen's egg, we studied the binding activities of IgG and IgE antibodies to native OA or OVM and seven different OA or OVM preparations, i.e. heated OA or OVM (100 degrees C for 3 min and 80 degrees C for 30 min) or 0.3 M NaOH, dithiothreitol (DTT), sodium dodecyl sulfate (SDS), 6 M urea or HCl treated OA or OVM. Eight patients with IgE anti-OA antibodies and 12 patients with IgE anti-OVM antibodies were used in these studies. The binding activities of IgG and IgE antibodies to physically or chemically denatured OA were partially decreased compared with those to native OA, whereas IgG and IgE antibodies in sera from patients bound well to denatured OVM with similar binding activities to native OVM. These results strongly suggest that antibodies to OA recognize partially conformational antigenic determinants on OA, whereas antibodies to OVM mainly recognize sequential antigenic determinants on OVM, and that antigenic determinants of OVM recognized by antibodies in sera from patients are more stable than those of OA under these denaturation conditions. In addition, the binding activities of IgE antibodies to denatured OA or OVM were significantly different from those of IgG antibodies to these OA or OVM, suggesting that the specificities of IgE antibodies to OA or OVM may be different from those of IgG antibodies to OA or OVM.

Special therapy in an institutional hospital.

I would like to introduce institutional therapy, a special therapeutic method used in our country for severe bronchial asthmatic children. Institutional therapy is mainly being used in national and municipal medical facilities. Approximately 2,300 patients receive this treatment yearly. Of the 158 patients who received this treatment in our institution in the past 4 years, 56.3% of them are leading normal lives after hospital discharge. Comparing those with a good prognosis to those with a bad prognosis, the time from onset of the serious stage to hospitalization is shorter and EIA levels are increased in the former group. Adequate amounts of physical training are effective in improving the patients' functional abilities and promoting confidence; therefore this therapy is highly effective.