Allergen Valency, Dose, and FcεRI Occupancy Set Thresholds for Secretory Responses to Pen a 1 and Motivate Design of Hypoallergens.

Ag-mediated crosslinking of IgE-FcεRI complexes activates mast cells and basophils, initiating the allergic response. Of 34 donors recruited having self-reported shrimp allergy, only 35% had significant levels of shrimp-specific IgE in serum and measurable basophil secretory responses to rPen a 1 (shrimp tropomyosin). We report that degranulation is linked to the number of FcεRI occupied with allergen-specific IgE, as well as the dose and valency of Pen a 1. Using clustered regularly interspaced palindromic repeat-based gene editing, human RBLrαKO cells were created that exclusively express the human FcεRIα subunit. Pen a 1-specific IgE was affinity purified from shrimp-positive plasma. Cells primed with a range of Pen a 1-specific IgE and challenged with Pen a 1 showed a bell-shaped dose response for secretion, with optimal Pen a 1 doses of 0.1-10 ng/ml. Mathematical modeling provided estimates of receptor aggregation kinetics based on FcεRI occupancy with IgE and allergen dose. Maximal degranulation was elicited when ∼2700 IgE-FcεRI complexes were occupied with specific IgE and challenged with Pen a 1 (IgE epitope valency of ≥8), although measurable responses were achieved when only a few hundred FcεRI were occupied. Prolonged periods of pepsin-mediated Pen a 1 proteolysis, which simulates gastric digestion, were required to diminish secretory responses. Recombinant fragments (60-79 aa), which together span the entire length of tropomyosin, were weak secretagogues. These fragments have reduced dimerization capacity, compete with intact Pen a 1 for binding to IgE-FcεRI complexes, and represent a starting point for the design of promising hypoallergens for immunotherapy.

Chitinase 3-like-1 regulates both visceral fat accumulation and asthma-like Th2 inflammation.

RATIONALE: Obesity, especially truncal obesity, is a risk factor for asthma incidence, prevalence, and severity. Chitinase 3-like-1 (Chi3l1) is an evolutionarily conserved moiety that plays a critical role in antipathogen and Th2 responses. However, the mechanisms that underlie the association between asthma and obesity and the role(s) of Chi3l1 in fat accumulation have not been defined. OBJECTIVES: To determine whether Chi3l1 is regulated by a high-fat diet (HFD) and simultaneously plays an important role(s) in the pathogenesis of asthma and obesity. METHODS: We evaluated the regulation of Chi3l1 by an HFD and Th2 inflammation. We also used genetically modified mice to define the roles of Chi3l1 in white adipose tissue (WAT) accumulation and Th2 inflammation and blockers of sirtuin 1 (Sirt1) to define its roles in these responses. Finally, the human relevance of these findings was assessed with a case-control study involving obese and lean control subjects and those with asthma. MEASUREMENTS AND MAIN RESULTS: These studies demonstrate that an HFD and aeroallergen challenge augment the expression of WAT and pulmonary Chi3l1. Chi3l1 also played a critical role in WAT accumulation and lung Th2 inflammation. In addition, Chi3l1 inhibited Sirt1 expression, and the deficient visceral fat and Th2 responses in Chi3l1 null mice were reversed by Sirt1 inhibition. Finally, serum and sputum Chi3l1 were positively associated with truncal adiposity, and serum Chi3l1 was associated with persistent asthma and low lung function in obese subjects with asthma. CONCLUSIONS: Chi3l1 is induced by an HFD and Th2 inflammation, and simultaneously contributes to the genesis of obesity and asthma.

High sputum total adiponectin is associated with low odds for asthma.

OBJECTIVE: Adipose tissue produces adiponectin, an anti-inflammatory protein. High systemic total adiponectin is associated with a low risk for incident asthma but the association with lung adiponectin is not known. Our objective was to evaluate the association between sputum total adiponectin and asthma. METHODS: This case-control study included 44 cases with objectively-confirmed asthma and an equal number of body mass index (BMI) and sex-matched controls. Serum and sputum adiponectin were estimated by ELISA and Western Blot technique, respectively. While Fisher's exact test, t-test and Spearman's correlations were used for univariate analyses, Spearman and regression analyses were performed for multivariable analyses. RESULTS: While high-molecular-weight adiponectin was the dominant isoform in serum, medium-molecular-weight isoform was dominant in sputum. Sputum total adiponectin was not correlated with serum adiponectin or BMI. Sputum total adiponectin was lower among asthmatics than controls (p = 0.03), although individual sputum isoforms were not similarly associated. High sputum total adiponectin was associated with lower odds for asthma (OR 0.33, 95% C.I. 0.12, 0.91), even after adjustment for systemic adiposity measures including serum adiponectin. CONCLUSIONS: High sputum total adiponectin predicted lower odds for asthma, even after adjustment for serum adiponectin. Although not studied, it is possible that pharmacological modulation of sputum adiponectin may suggest new ways to prevent and/or treat asthma.

Body mass index is a stronger predictor than the metabolic syndrome for future asthma in women. The longitudinal CARDIA study.

RATIONALE: It is hypothesized that the metabolic syndrome explains the association between body mass index (BMI) and asthma in adults. OBJECTIVES: Our objective was to longitudinally compare the relative strengths of the associations of the metabolic syndrome and BMI with incident asthma in adults. METHODS: We included 4,619 eligible participants in the Coronary Artery Risk Development in Young Adults (CARDIA) cohort followed over 25 years. Incident asthma was defined by a new self-reported provider asthma diagnosis plus either the presence of asthma symptoms and/or use of asthma medications. Cox proportional hazard analyses were performed. MEASUREMENTS AND MAIN RESULTS: Six hundred two subjects (417 women and 185 men) developed incident asthma over 25 years of follow-up. Metabolic syndrome predicted incident asthma among women but not men (unadjusted hazard ratios, 1.50 and 0.98; P = 0.01 and 0.93, respectively). BMI had a similar predictive association among women but not men (unadjusted hazard ratios, 1.19 and 1.04 per 5 units of BMI; P < 0.001 and 0.60, respectively). The association of metabolic syndrome with incident asthma in women was no longer statistically significant after adjustment for BMI (P = 0.44). In contrast, the association of BMI with incident asthma in women remained statistically significant after adjusting for the metabolic syndrome (P = 0.01). In a stepwise model, BMI was a stronger predictor than the metabolic syndrome (P = 0.001). CONCLUSIONS: BMI is a stronger predictor of incident asthma among women than the metabolic syndrome. Other obesity-associated factors that are not a part of the metabolic syndrome may play a role in the BMI-asthma association in women.

Effect of allergen inhalation on airway oxidant stress, using exhaled breath condensate 8-isoprostane, in mild asthma.

OBJECTIVE: Exhaled breath condensate (EBC) 8-isoprostane concentrations are increased in asthma, but it is not known if they acutely change following bronchoprovocation. The objective of this study was to evaluate EBC 8-isoprostane concentrations following allergen-induced bronchoprovocation in asthma. METHODS: This comparison study included eight mild atopic asthmatics and six controls. Asthmatics were challenged with inhaled specific allergen, methacholine, and irrelevant allergen in random order. Controls were challenged with irrelevant allergen. EBCs collected at 0, 3, 6, 9, and 23 hours by the R-tube method were measured for 8-isoprostanes by ELISA technique. Repeated measures ANOVA technique was used for analysis. RESULTS: EBC 8-isoprostane concentrations did not change following any inhalational challenge, as compared to baseline, in either asthmatics or controls. CONCLUSIONS: EBC 8-isoprostane concentrations do not acutely change following bronchoprovocation in subjects with mild asthma.

Low serum adiponectin predicts future risk for asthma in women.

RATIONALE: Our previous cross-sectional study showed that serum adiponectin is inversely associated with asthma among women. However, it is not known if serum adiponectin predicts future development of asthma or if asthma affects subsequent serum adiponectin concentrations among women. OBJECTIVES: To determine longitudinal association between serum adiponectin and incident asthma among women. METHODS: We used data from examinations at Years 10, 15, and 20 of the Coronary Artery Risk Development in Young Adults (CARDIA) cohort. In our primary analysis, the association of CARDIA Year 15 serum adiponectin concentration with Year 20 incident asthma was evaluated. In our secondary analysis, the converse direction, that is, the association of CARDIA Year 10 prevalent asthma with Year 15 serum adiponectin, was evaluated, using logistic regression techniques. MEASUREMENTS AND MAIN RESULTS: Our primary analysis included 1,450 women, mostly premenopausal. Multivariable analyses demonstrated that the lowest tertile of Year 15 serum adiponectin concentration (<7 mg/L) predicted significantly higher risk for incident asthma at Year 20 among women (odds ratio, 2.07; 95% confidence interval, 1.05, 4.10), and particularly among current smokers (interaction P = 0.051). Further, low serum adiponectin was more important than body mass index in predicting the risk for incident asthma among women. We also showed that the converse relationship was not true; that is, Year 10 prevalent asthma did not predict Year 15 serum adiponectin concentrations in women. CONCLUSIONS: Serum adiponectin affects future risk for asthma in women and not vice versa. Measures that raise systemic adiponectin concentrations may lead to newer ways to prevent asthma among women, particularly among those who smoke.

Cigarette smoke suppresses Bik to cause epithelial cell hyperplasia and mucous cell metaplasia.

RATIONALE: Aberrant regulation of airway epithelial cell numbers in airways leads to increased mucous secretions in chronic lung diseases such as chronic bronchitis. Because the Bcl-2 family of proteins is crucial for airway epithelial homeostasis, identifying the players that reduce cigarette smoke (CS)-induced mucous cell metaplasia can help to develop effective therapies. OBJECTIVES: To identify the Bcl-2 family of proteins that play a role in reducing CS-induced mucous cell metaplasia. METHODS: We screened for dysregulated expression of the Bcl-2 family members. MEASUREMENTS AND MAIN RESULTS: We identified Bik to be significantly reduced in bronchial brushings of patients with chronic epithelial cell hyperplasia compared with nondiseased control subjects. Reduced Bik but increased MUC5AC mRNA levels were also detected when normal human airway epithelial cells (HAECs) were exposed to CS or when autopsy tissues from former smokers with and without chronic bronchitis were compared. Similarly, exposure of C57Bl/6 mice to CS resulted in increased numbers of epithelial and mucous cells per millimeter of basal lamina, along with reduced Bik but increased Muc5ac expression, and this change was sustained even when mice were allowed to recover in filtered air for 8 weeks. Restoring Bik expression significantly suppressed CS-induced mucous cell metaplasia in differentiated primary HAEC cultures and in airways of mice in vivo. Bik blocked nuclear translocation of phospho-ERK1/2 to induce apoptosis of HAECs. The conserved Leu61 within Bik and ERK1/2 activation were essential to induce cell death in hyperplastic mucous cells. CONCLUSIONS: These studies show that CS suppresses Bik expression to block airway epithelia cell death and thereby increases epithelial cell hyperplasia in chronic bronchitis.

Reduced FcepsilonRI-mediated release of asthma-promoting cytokines and chemokines from human basophils during omalizumab therapy.

BACKGROUND: Treating asthmatics with the humanized IgE-scavenging antibody, omalizumab (rhuMAb-E25, Xolair, reduces airways inflammation and asthma symptoms. Previously, omalizumab was shown to cause a dramatic and reversible loss of cell surface high-affinity IgE receptors, FcepsilonRI, from the peripheral blood basophils of asthmatics. The consequences of receptor loss for the FcepsilonRI-mediated synthesis and release of cytokines implicated in allergic asthma have not been examined. METHODS: Fifteen asthmatic volunteers each received omalizumab for 12 weeks. Peripheral blood basophils were isolated before, during, 2 weeks after and 6 months after omalizumab. Basophils were assayed for the basal and anti-IgE-stimulated release of cytokines, chemokines and histamine. Pooled data were analyzed by repeated measures ANOVA and by paired t tests. RESULTS: Anti-IgE-stimulated human basophils synthesize and release Th2 cytokines (IL-4, IL-13) and chemokines (IL-8, RANTES). The anti-IgE-stimulated release of IL-4, IL-13 and IL-8 was reduced during omalizumab treatment and returned to pretreatment levels after omalizumab withdrawal. Omalizumab did not alter basophil histamine levels or basal and anti-IgE-stimulated histamine release. CONCLUSIONS: Omalizumab may reduce asthma symptoms in part by suppressing the FcepsilonRI-mediated production by basophils of Th2 cytokines and selected chemokines. Anti-IgE-stimulated basophil cytokine synthesis appears more sensitive than histamine release to the loss of FcepsilonRI caused by omalizumab treatment.

Do electronic health records affect the patient-psychiatrist relationship? A before & after study of psychiatric outpatients.

BACKGROUND: A growing body of literature shows that patients accept the use of computers in clinical care. Nonetheless, studies have shown that computers unequivocally change both verbal and non-verbal communication style and increase patients' concerns about the privacy of their records. We found no studies which evaluated the use of Electronic Health Records (EHRs) specifically on psychiatric patient satisfaction, nor any that took place exclusively in a psychiatric treatment setting. Due to the special reliance on communication for psychiatric diagnosis and evaluation, and the emphasis on confidentiality of psychiatric records, the results of previous studies may not apply equally to psychiatric patients. METHOD: We examined the association between EHR use and changes to the patient-psychiatrist relationship. A patient satisfaction survey was administered to psychiatric patient volunteers prior to and following implementation of an EHR. All subjects were adult outpatients with chronic mental illness. RESULTS: Survey responses were grouped into categories of "Overall," "Technical," "Interpersonal," "Communication & Education,," "Time," "Confidentiality," "Anxiety," and "Computer Use." Multiple, unpaired, two-tailed t-tests comparing pre- and post-implementation groups showed no significant differences (at the 0.05 level) to any questionnaire category for all subjects combined or when subjects were stratified by primary diagnosis category. CONCLUSIONS: While many barriers to the adoption of electronic health records do exist, concerns about disruption to the patient-psychiatrist relationship need not be a prominent focus. Attention to communication style, interpersonal manner, and computer proficiency may help maintain the quality of the patient-psychiatrist relationship following EHR implementation.

Classification of hypersensitivity pneumonitis: a hypothesis.

BACKGROUND: Regardless of the causative antigen, hypersensitivity pneumonitis (HP) is usually classified as 'acute', 'subacute' or 'chronic'. Considerable confusion still surrounds this classification because there are no widely accepted criteria to distinguish the various stages. The objective of this study was to determine whether the current classification of HP truly reflects categories of patients with distinct clinical features. METHODS: Data obtained from a large prospective multicenter cohort study (the HP Study) were used to divide a cohort of patients with HP into a limited number of categories (clusters) with maximally differing clinical patterns, without prejudgment. The results of this cluster analysis were compared with the current classification of HP (acute, subacute or chronic). RESULTS: 168 patients were included in the analysis. A 2-cluster solution best fitted the data. Patients in cluster 1 (41 patients) had more recurrent systemic symptoms (chills and body aches) and normal chest radiographs than those in cluster 2 (127 patients) who showed significantly more clubbing, hypoxemia, restrictive patterns on pulmonary function tests and fibrosis on high-resolution computed tomography (HRCT). All p values were <0.0001, using Fisher's exact test. Nodular opacities were seen on HRCT as often in cluster 1 as in cluster 2. There was considerable disagreement between the current classification of HP and the results of our analysis. CONCLUSION: The current classification of acute, subacute and chronic HP is not supported by our analysis. Subacute HP is particularly difficult to define.

A comparison of inflammatory mediators released by basophils of asthmatic and control subjects in response to high-affinity IgE receptor aggregation.

BACKGROUND: In human blood basophils, cross-linking the high-affinity IgE receptor Fc epsilonRI with multivalent antigen activates a signaling pathway leading to secretion of inflammatory mediators and cytokine production. Basophils are known to play an important role in the pathogenesis of asthma but there has been no comprehensive examination of the effectors these cells produce. Here a study of the transcription and release of a selection of chemokines and cytokines from basophils was undertaken. METHODS: A Cartesian antibody array provided an effective method of assaying for multiple cytokines and chemokines simultaneously. Results were verified by RT-PCR and ELISA assays. This allowed the comparison of freshly prepared peripheral blood basophil responses to cross-linking of the high-affinity IgE receptor, with and without preincubation with IL-3. RESULTS: Evidence that human blood basophils produce the chemokines MIP-5, eotaxin and GM-CSF was provided by antibody array and RT-PCR analyses. Preincubation with IL-3 enhanced the expression and release of IL-13, IL-8 and mRNA transcripts encoding MIP-5 and GATA2 in basophils from both asthmatic and control subjects. Leptin mRNA transcription, storage and release in basophils are described for the first time. CONCLUSIONS: Surveying cytokine and chemokines stored and released by peripheral blood basophils shows that asthmatic and control subjects share similar profiles even when their degranulation responses are distinct. Evidence is provided for the production of leptin, GM-CSF, eotaxin and MIP-5 by peripheral blood basophils. IL-3 preincubation enhances the production and release of IL-8 upon IgE receptor cross-linking.

Immune response to hepatitis B vaccine in asthmatic children.

Asthma is a disease that demonstrates chronic Th2 lymphocyte-mediated pulmonary inflammation. We hypothesized that cytokines produced by asthmatic lung inflammation bias the immune response to antigens administered systemically toward a Th2 response, as assessed by serum IgE antibody and lymphocyte-secreted IL-4 and IL-5. We also hypothesized that treatment of asthmatic children with local anti-inflammatory agents reduces this cytokine-mediated Th2 influence. We systemically immunized groups of asthmatic children (n=29) who were participating in a long-term, randomized, placebo-controlled clinical trial of inhaled anti-inflammatory therapy (Childhood Asthma Management Program) and nonasthmatic children (n=12) with hepatitis B (Hep B) antigen, and examined their antigen-specific antibody and lymphocyte cytokine secretion profiles. The asthmatic population demonstrated an increased amount of Th2-mediated serum IgE anti-Hep B antibody, as compared to nonasthmatic children; but comparable amounts of IgG1, IgG2, IgG3, IgA, and IgM anti-Hep B antibody and lymphocyte IFNgamma, IL4, and IL5. There was no significant difference of antibody isotype or cytokine production between asthmatic subjects receiving treatment with budesonide or nedocromil, as compared to placebo. In conclusion, there is a subtle bias in responses to systemic immunization in children with asthma, but anti-inflammatory therapy does not affect this bias. The findings support the concept that the Th2 bias may be largely genetic. Importantly, we confirmed that children with asthma, including even those on inhaled corticosteroids, responded to Hep B immunization as well as did nonasthmatic children with the major isotypes of anti-Hep B antibody, suggesting that vaccine protection against hepatitis B is not influenced by inhaled steroid therapy.

The APO E4 allele and cognition in New Mexico Hispanic elderly.

OBJECTIVE: To determine if the apolipoprotein E E4 (APO E4) allele is associated with cognitive performance in New Mexico Hispanic elderly. METHOD: We performed a cross-sectional survey of 105 community volunteers, aged 60 years and older, born in New Mexico, with both parents of Hispanic descent. Subjects were excluded for medical conditions that could influence cognitive performance. We also performed a longitudinal analysis on 18 participants who were re-tested over a 3-year interval. The main outcome measures for both the cross-sectional and longitudinal analysis were scores on 5 cognitive tests comparing subjects with the E4 allele to those without the E4 allele. RESULTS: The mean age was 69 years, with a range of 60 to 91. For the cross-sectional analysis, there were no significant differences between the 2 groups on the cognitive tests, although subjects with an E4 allele did not perform as well on color trails A (P=.09). In the longitudinal analysis we found that the variability of cognitive test scores tended to be higher in the E4 group on most cognitive measures. The time needed to complete color trails A (indicating slower performance) was significantly greater (P<.05) in the E4 group. For the total recall portion of the Fuld Object Memory test, the E4 group did not perform as well on follow-up (P=.08). CONCLUSION: We found no significant cross-sectional association between the APO E4 allele and cognitive performance. In our longitudinal analysis, the time needed to complete color trails A was significantly greater in the E4 group, and the E4 group did not perform as well on total recall.

Adult-onset asthma becomes the dominant phenotype among women by age 40 years. the longitudinal CARDIA study.

RATIONALE: Although asthma is usually considered to originate in childhood, adult-onset disease is being increasingly reported. OBJECTIVES: To contrast the proportion and natural history of adult-onset versus pediatric-onset asthma in a community-based cohort. We hypothesized that asthma in women is predominantly of adult onset rather than of pediatric onset. METHODS: This study used data from the Coronary Artery Risk Development in Young Adults (CARDIA) cohort in the United States over a 25-year period. Adult- and pediatric-onset asthma phenotypes were studied, as defined by age at onset of 18 years or older. Subjects with asthma were categorized by sex, obesity, atopy, smoking, and race by mean age/examination year, using a three-way analysis of covariance model. Natural history of disease was examined using probabilities derived from a Markov chain model. MEASUREMENTS AND MAIN RESULTS: Asthma of adult onset became the dominant (i.e., exceeded 50%) phenotype in women by age 40 years. The age by which adult-onset asthma became the dominant phenotype was further lowered for obese, nonatopic, ever-smoking, or white women. The prevalence trend with increasing time for adult-onset disease was greater among subjects with nonatopic than atopic asthma among both sexes. Furthermore, adult-onset asthma had remarkable sex-related differences in risk factors. In both sexes, the quiescent state for adult-onset asthma was less frequent and also "less stable" over time than for pediatric-onset asthma. CONCLUSIONS: Using a large national cohort, this study challenges the dictum that most asthma in adults originates in childhood. Studies of the differences between pediatric- and adult-onset asthma may provide greater insight into the phenotypic heterogeneity of asthma.

Roles for the High Affinity IgE Receptor, FcεRI, of Human Basophils in the Pathogenesis and Therapy of Allergic Asthma: Disease Promotion, Protection or Both?

The role of basophils, the rarest of blood granulocytes, in the pathophysiology of allergic asthma is still incompletely understood. Indirect evidence generated over many decades is consistent with a role for basophils in disease promotion. Recent improvements in procedures to purify and analyze very small numbers of human cells have generally supported this view, but have also revealed new complexities. This chapter focuses on our analyses of Fcε R1 function in basophils in the context of understanding and treating human allergic asthma. In long-term studies, we demonstrated that asthmatic subjects have higher circulating numbers of basophils than non-atopic non-asthmatic subjects and that their basophils show higher rates of both basal and anti-IgE or antigen-stimulated histamine release. These results hint at a direct role for basophils in promoting asthma. Supporting this interpretation, the non-releaser phenotype that we linked to the excessive proteolysis of Syk via the ubiquitin/proteasomal pathway is less common in basophils from asthmatic than non-asthmatic donors. The discovery of a basophil-specific pathway regulating Syk levels presents a clear opportunity for therapy. Another route to therapy was revealed by evidence that basophil FcεRI signaling can be downregulated by co-crosslinking the ITAM-containing IgE receptor, FcγRI, to the ITIM-containing IgG receptor, FcγRIIB. Based on this discovery, hybrid co-crosslinking fusion proteins are being engineered as potential therapies targeting basophils. A third distinguishing property of human basophils is their high dependence on IgE binding to stabilize membrane FcεRI. The circulating IgE scavenging mAb, Omalizumab, reduces FcεRI expression in basophils from asthmatics by over 95% and produces a substantial impairment of IL-4, IL-8 and IL-13 production in response to the crosslinking of residual cell surface IgE-FcεRI. A search for small molecule inhibitors that similarly impair high affinity IgE binding to basophils may yield reagents that mimic Omalizumab's therapeutic benefits without the potential for immune side effects. Although studies on allergen and FcεRI-mediated basophil activation all point to a role in promoting disease, a case can also be made for an alternative or additional role of basophil FcεRI in protection against allergic asthma. Human basophils have high affinities for IgE, they upregulate receptor levels over a >100-fold range as circulating IgE levels increase and they have short half-lives in the circulation. Thus, when allergen is absent, basophil FcεRI could serve as scavengers of serum IgE and therefore protectors against mast cell IgE-mediated inflammatory responses. Further studies are clearly needed to determine if FcεR-expressing basophils play pathogenic or protective roles - or both - in human allergic asthma and other IgE-mediated inflammatory disorders.

Effect of specific allergen inhalation on serum adiponectin in human asthma.

BACKGROUND: Adiponectin is associated with asthma. The direction of this association is not known in humans. In mice, this association is bidirectional: allergen inhalation affects serum adiponectin, and exogenous adiponectin administration affects asthma. We sought to evaluate whether allergen inhalation affects serum adiponectin in human asthma. METHODS: This study included eight sensitized subjects with mild asthma and six healthy control subjects. Asthmatic subjects were challenged with inhaled specific allergen (positive allergen skin test), methacholine, and irrelevant allergen (negative allergen skin test). Control subjects were challenged with irrelevant allergen. Sequential serum samples were obtained before and nine times after each challenge. Serum adiponectin- (primary outcome), leptin-, adiponectin-to-leptin ratio-, eotaxin-, and tumor necrosis factor-alpha-response curves, area under the curves, and baseline and peak concentrations were evaluated. Statistical analysis used repeated-measures analysis of variance and paired t tests. RESULTS: There were no significant differences in outcome measures among the challenges in asthmatic subjects or when compared to control subjects. Type II error is an unlikely explanation for these findings because the study was adequately powered to detect changes in serum adiponectin, as reported in the literature. Further, pooled data showed that serum adiponectin diurnal variation curves were lower in asthmatic subjects than in control subjects. CONCLUSIONS: Serum adiponectin concentrations are lower in asthmatic subjects than in control subjects. Specific allergen inhalation in asthmatic subjects does not acutely affect serum adiponectin concentrations. The reverse association (ie, effect of adiponectin on asthma) needs further study. If future studies prove adiponectin to be a protective factor for asthma, modulating adiponectin may open a new approach toward managing asthma.

Expression of the proapoptotic protein Bax is reduced in bronchial mucous cells of asthmatic subjects.

The present studies were designed to determine whether our findings in mice showing that the Bcl-2-associated protein X (Bax), which plays a role in the resolution of allergen-induced mucous cell metaplasia, can be applied to asthma in humans. Immunostaining of autopsy tissues from mild and severe asthmatic subjects showed a significant reduction in the percentage of Bax-positive mucous cells compared with those from nonasthmatic controls. To exclude the possibility that postmortem changes may have affected Bax expression, Bax mRNA levels in airway epithelial cells obtained from nonsmoking asthmatic subjects were compared with those from nonasthmatic controls. Because the number of cells obtained by bronchial brushings is limited, we developed a robust preamplification procedure of cDNA before quantitative real-time PCR to allow detection of 100 gene targets from limited sample size, even when it was prepared from partially degraded RNA. cDNA was prepared by reverse transcription from RNA isolated from bronchial epithelial cells obtained by bronchial brushings from well-characterized subjects without lung disease and from subjects with mild asthma. Quantitative analysis showed that Bax mRNA levels were significantly reduced in samples obtained from asthma patients compared with nonasthma controls. Furthermore, Bax mRNA levels were reduced when primary airway epithelial cells from 10 individuals were treated in culture with the T helper 2 cytokine IL-13. These studies show that Bax expression is reduced in airway epithelial cells of even mild asthmatic subjects and suggest that restoring Bax expression may provide a clinical approach for restoring the normal numbers of epithelial cells and reduced mucous hypersecretion in asthma.