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A variant of severe combined immunodeficiency syndrome (SCID) with a selective inability to produce CD8 single positive T cells and a signal transduction defect in peripheral CD4+ cells has recently been shown to be the result of mutations in the ZAP-70 gene. T cell receptor (TCR) signaling requires the association of the ZAP-70 protein tyrosine kinase with the TCR complex. Human T cell leukemia virus type I-transformed CD4+ T cell lines were established from ZAP-70-deficient patients and normal controls. ZAP-70 was expressed and appropriately phosphorylated in normal T cell lines after TCR engagement, but was not detected in T cell lines from ZAP-70-deficient patients. To determine whether signaling could be reconstituted, wild-type ZAP-70 was introduced into deficient cells with a ZAP-70 retroviral vector. High titer producer clones expressing ZAP-70 were generated in the Gibbon ape leukemia virus packaging line PG13. After transduction, ZAP-70 was detected at levels equivalent to those observed in normal cells, and was appropriately phosphorylated on tyrosine after receptor engagement. The kinase activity of ZAP-70 in the reconstituted cells was also appropriately upregulated by receptor aggregation. Moreover, normal and transduced cells, but not ZAP-70-deficient cells, were able to mobilize calcium after receptor ligation, indicating that proximal TCR signaling was reconstituted. These results indicate that this form of SCID may be corrected by gene therapy.

Recently, a severe combined immunodeficiency syndrome with a deficiency of CD8+ peripheral T cells and a TCR signal transduction defect in peripheral CD4+ T cells was associated with mutations in ZAP-70. Since TCR signaling is required in developmental decisions resulting in mature CD4 (and CD8) T cells, the presence of peripheral CD4+ T cells expressing TCRs incapable of signaling in these patients is paradoxical. Here, we show that the TCRs on thymocytes, but not peripheral T cells, from a ZAP-70-deficient patient are capable of signaling. Moreover, the TCR on a thymocyte line derived from this patient can signal, and the homologous kinase Syk is present at high levels and is tyrosine phosphorylated after TCR stimulation. Thus, Syk may compensate for the loss of ZAP-70 and account for the thymic selection of at least a subset of T cells (CD4+) in ZAP-70-deficient patients.

Rationale: Severe respiratory syncytial virus (RSV) bronchiolitis has been associated with deficient IFN-gamma production in humans, but the role of this cytokine in determining the outcome of reinfection is unknown.Objectives: To define the role of IFN-gamma in the development of RSV-mediated airway hyperresponsiveness (AHR) and lung histopathology in mice.Methods: Wild-type (WT) and IFN-gamma knockout mice were infected with RSV in the newborn or weaning stages and reinfected 5 weeks later. Airway responses were assessed on Day 6 after the primary or secondary infection.Measurements and Main Results: Both WT and IFN-gamma knockout mice developed similar levels of AHR and airway inflammation after primary infection. After reinfection, IFN-gamma knockout mice, but not WT mice, developed AHR, airway eosinophilia, and mucus hyperproduction. Intranasal administration of IFN-gamma during primary infection but not during reinfection prevented the development of these altered airway responses on reinfection in IFN-gamma knockout mice. Adoptive transfer of WT T cells into IFN-gamma knockout mice before primary infection restored IFN-gamma production in the lungs and prevented the development of altered airway responses on reinfection. Treatment of mice with IFN-gamma during primary neonatal infection prevented the enhancement of AHR and the development of airway eosinophilia and mucus hyperproduction on reinfection.Conclusions: IFN-gamma production during primary RSV infection is critical to the development of protection against AHR and lung histopathology on reinfection. Provision of IFN-gamma during primary infection in infancy may be a potential therapeutic approach to alter the course of RSV-mediated long-term sequelae. [ABSTRACT FROM AUTHOR]

There are >22 million Americans with asthma. Chronic asthma is a worldwide problem with an increasing socioeconomic burden on individuals and on society. Recent advances have been made in diagnostic lung imaging, defining control of asthma, as well as in the education of patients with asthma. Accurate diagnosis of the cause of chronic cough in adults and of asthma in elderly individuals will help affected individuals receive appropriate treatment. Inhaled corticosteroids are the recommended first-line therapy for persistent asthma and can help prevent exacerbations in patients with asthma that are not well controlled. Early intervention and improved management can significantly reduce the socioeconomic burden of asthma. Patient education is an essential part of asthma management. [ABSTRACT FROM AUTHOR]

Rationale: Airway hyperresponsiveness is a critical feature of asthma. Substantial epidemiologic evidence supports a role for female sex hormones in modulating lung function and airway hyperresponsiveness in humans.Objectives: To examine the role of estrogen receptors in modulating lung function and airway responsiveness using estrogen receptor-deficient mice.Methods: Lung function was assessed by a combination of whole-body barometric plethysmography, invasive measurement of airway resistance, and isometric force measurements in isolated bronchial rings. M2 muscarinic receptor expression was assessed by Western blotting, and function was assessed by electrical field stimulation of tracheas in the presence/absence of gallamine. Allergic airway disease was examined after ovalbumin sensitization and exposure.Measurements and Main Results: Estrogen receptor-alpha knockout mice exhibit a variety of lung function abnormalities and have enhanced airway responsiveness to inhaled methacholine and serotonin under basal conditions. This is associated with reduced M2 muscarinic receptor expression and function in the lungs. Absence of estrogen receptor-alpha also leads to increased airway responsiveness without increased inflammation after allergen sensitization and challenge.Conclusions: These data suggest that estrogen receptor-alpha is a critical regulator of airway hyperresponsiveness in mice. [ABSTRACT FROM AUTHOR]

Rationale: Ambient particulate concentrations have been associated with variable physiologic effects in children with persistent asthma taking controller medications. Objective: To determine whether exposure to particulate matter has immediate effects on asthma control in children with persistent disease. Methods and Measurements: In a school-based cohort, 73 children, primarily with moderate and severe asthma, were followed daily over one or two winters (2001-2002, 2002-2003) in Denver, Colorado. The association among ambient fine particulate, bronchodilator use, and urinary leukotriene E(4) levels was assessed. Results: Daily concentrations of fine particulate peaked in the morning hours when children were commuting to school. In a multivariable analysis that controlled for meteorology, time trends, and upper respiratory infections, an increase of one interquartile range in morning maximum fine particulate levels was related to an average increase of 3.8% in bronchodilator usage at school (95% confidence interval [CI], 0.2-7.4; p = 0.04). Children with severe asthma demonstrated significantly stronger associations (8.1% increase; 95% CI, 2.9-13.4; p = 0.003) than those with mild/moderate disease (1.6% increase; 95% CI, -2.2-5.4; p = 0.41; p = 0.03 for difference between groups). Morning maximum fine particulate levels were also associated with urinary leukotriene E(4) measured during school hours (average increase of 6.2% per interquartile range increase; 95% CI, 1.9-10.5; p = 0.006). These associations were not discernable when 24-h averaged concentrations were used. Conclusions: Peak concentrations of ambient fine particulate are associated with early increases in bronchodilator use and urinary leukotriene E(4) levels among children with persistent asthma, despite the use of controller medications. [ABSTRACT FROM AUTHOR]

Rationale: Spleen tyrosine kinase (Syk) is important for Fc and B-cell receptor-mediated signaling. Objective: To determine the activity of a specific Syk inhibitor (R406) on mast cell activation in vitro and on the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation in vivo. Methods: AHR and inflammation were induced after 10 d of allergen (ovalbumin [OVA]) exposure exclusively via the airways and in the absence of adjuvant. This approach was previously established to be IgE, FcepsilonRI, and mast cell dependent. Alternatively, mice were passively sensitized with OVA-specific IgE, followed by limited airway challenge. In vitro, the inhibitor was added to cultures of IgE-sensitized bone marrow-derived mast cells (BMMCs) before cross-linking with allergen. Results: The inhibitor prevented OVA-induced degranulation of passively IgE-sensitized murine BMMCs and inhibited the production of interleukin (IL)-13, tumor necrosis factor alpha, IL-2, and IL-6 in these sensitized BMMCs. When administered in vivo, R406 inhibited AHR, which developed in BALB/c mice exposed to aerosolized 1% OVA for 10 consecutive d (20 min/d), as well as pulmonary eosinophilia and goblet cell metaplasia. A similar inhibition of AHR was demonstrated in mice passively sensitized with OVA-specific IgE and exposed to limited airway challenge. Conclusion: This study delineates a functional role for Syk in the development of mast cell- and IgE-mediated AHR and airway inflammation, and these results indicate that inhibition of Syk may be a target in the treatment of allergic asthma. [ABSTRACT FROM AUTHOR]

Rationale: There is conflicting information about the development and resolution of airway inflammation and airway hyperresponsiveness (AHR) after repeated airway exposure to allergen in sensitized mice.Methods: Sensitized BALB/c and C57BL/6 mice were exposed to repeated allergen challenge on 3, 7, or 11 occasions. Airway function in response to inhaled methacholine was monitored; bronchoalveolar lavage fluid inflammatory cells were counted; and goblet cell metaplasia, peribronchial fibrosis, and smooth muscle hypertrophy were quantitated on tissue sections. Bone marrow-derived dendritic cells were generated after differentiation of bone marrow cells in the presence of growth factors.Results: Sensitization to ovalbumin (OVA) in alum, followed by three airway exposures to OVA, induced lung eosinophilia, goblet cell metaplasia, mild peribronchial fibrosis, and peribronchial smooth muscle hypertrophy; increased levels of interleukin (IL)-4, IL-5, IL-13, granulocyte-macrophage colony-stimulating factor, transforming growth factor-beta(1), eotaxin-1, RANTES (regulated on activation, normal T-cell expressed and secreted), and OVA-specific IgG1 and IgE; and resulted in AHR. After seven airway challenges, development of AHR was markedly decreased as was the production of IL-4, IL-5, and IL-13. Levels of IL-10 in both strains and the level of IL-12 in BALB/c mice increased. After 11 challenges, airway eosinophilia and peribronchial fibrosis further declined and the cytokine and chemokine profiles continued to change. At this time point, the number of myeloid dendritic cells and expression of CD80 and CD86 in lungs were decreased compared with three challenges. After 11 challenges, intratracheal instillation of bone marrow-derived dendritic cells restored AHR and airway eosinophilia.Conclusions: These data suggest that repeated allergen exposure leads to progressive decreases in AHR and allergic inflammation, through decreases in myeloid dendritic cell numbers. [ABSTRACT FROM AUTHOR]

Background: To date, little has been published describing the pathology of severe childhood asthma. The currently accepted model of asthma holds that persistent airway inflammation leads to various symptoms of asthma, airway hyperresponsiveness, and airway remodeling that ultimately results in permanent loss of lung function.Methods: Evaluation of six children referred to the National Jewish Medical and Research Center with difficult-to-control asthma, despite aggressive anti-inflammatory therapy, who underwent bronchoscopy with endobronchial biopsy to better characterize their disease.Results: In every case, endobronchial biopsies revealed changes consistent with airway remodeling characterized by thickening of the basement membrane, smooth-muscle hypertrophy, with varying degrees of goblet-cell and submucous gland hyperplasia. The degree of subbasement membrane thickening did not appear to correlate with baseline FEV(1), ultimate FEV(1) following aggressive therapy, or lability in lung function. In five of six cases, there was minimal to no histologic evidence for airway inflammation with mild and patchy submucosal lymphocytic infiltration noted; eosinophils and neutrophils were not present. Further, the majority of the patients achieved normal FEV(1) values despite significant subbasement membrane thickening, counter to the current beliefs regarding airway remodeling and irreversible loss of lung function.Conclusions: This case report highlights some of the shortcomings of the current inflammatory paradigm for severe asthma. Despite little evidence of ongoing airway inflammation, many of the subjects displayed significant lung function lability. The lack of inflammation argues against steroid resistance at a cellular level, although it could be argued that inflammation may have been distal to the site sampled. Additionally, normal to nearly normal lung function was achieved despite the presence of significant remodeling. These findings suggest the need to look beyond inflammation to fully treat severe asthma and ultimately alter its progression. [ABSTRACT FROM AUTHOR]