FDA-approved drug screening in patient-derived organoids demonstrates potential of drug repurposing for rare cystic fibrosis genotypes.

BACKGROUND: Preclinical cell-based assays that recapitulate human disease play an important role in drug repurposing. We previously developed a functional forskolin induced swelling (FIS) assay using patient-derived intestinal organoids (PDIOs), allowing functional characterization of CFTR, the gene mutated in people with cystic fibrosis (pwCF). CFTR function-increasing pharmacotherapies have revolutionized treatment for approximately 85% of people with CF who carry the most prevalent F508del-CFTR mutation, but a large unmet need remains to identify new treatments for all pwCF. METHODS: We used 76 PDIOs not homozygous for F508del-CFTR to test the efficacy of 1400 FDA-approved drugs on improving CFTR function, as measured in FIS assays. The most promising hits were verified in a secondary FIS screen. Based on the results of this secondary screen, we further investigated CFTR elevating function of PDE4 inhibitors and currently existing CFTR modulators. RESULTS: In the primary screen, 30 hits were characterized that elevated CFTR function. In the secondary validation screen, 19 hits were confirmed and categorized in three main drug families: CFTR modulators, PDE4 inhibitors and tyrosine kinase inhibitors. We show that PDE4 inhibitors are potent CFTR function inducers in PDIOs where residual CFTR function is either present, or created by additional compound exposure. Additionally, upon CFTR modulator treatment we show rescue of CF genotypes that are currently not eligible for this therapy. CONCLUSION: This study exemplifies the feasibility of high-throughput compound screening using PDIOs. We show the potential of repurposing drugs for pwCF carrying non-F508del genotypes that are currently not eligible for therapies. ONE-SENTENCE SUMMARY: We screened 1400 FDA-approved drugs in CF patient-derived intestinal organoids using the previously established functional FIS assay, and show the potential of repurposing PDE4 inhibitors and CFTR modulators for rare CF genotypes.

Tobramycin and vestibulotoxicity: retrospective analysis of four cases.

Over a course of 7 months, four patients developed vestibulotoxicity after treatment with intravenous tobramycin. Since vestibulotoxicity is a serious adverse effect which can be irreversible, an investigation was undertaken to determine if there was a cause for the toxicity and whether the quality of care had been inadequate. In this period, 26 patients with cystic fibrosis were treated with tobramycin according to valid guidelines, of which four experienced acute dizziness which disrupted their daily activities. Two patients experienced irreversible bilateral vestibular hypofunction and two unilateral loss of the right labyrinth, with decreasing dizziness over time. No apparent cause for the vestibulotoxicity was found in these four patients and the simultaneous occurrence was not due to a lack in quality of care. Symptoms of dizziness and balance disorders should be recognised by patients and caretakers at an early stage so additional diagnostics can be done to prevent further deterioration.

Functional Restoration of CFTR Nonsense Mutations in Intestinal Organoids.

BACKGROUND: Pharmacotherapies for people with cystic fibrosis (pwCF) who have premature termination codons (PTCs) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are under development. Thus far, clinical studies focused on compounds that induce translational readthrough (RT) at the mRNA PTC location. Recent studies using primary airway cells showed that PTC functional restoration can be achieved through combining compounds with multiple mode-of-actions. Here, we assessed induction of CFTR function in PTC-containing intestinal organoids using compounds targeting RT, nonsense mRNA mediated decay (NMD) and CFTR protein modulation. METHODS: Rescue of PTC CFTR protein was assessed by forskolin-induced swelling of 12 intestinal organoid cultures carrying distinct PTC mutations. Effects of compounds on mRNA CFTR level was assessed by RT-qPCRs. RESULTS: Whilst response varied between donors, significant rescue of CFTR function was achieved for most donors with the quintuple combination of a commercially available pharmacological equivalent of the RT compound (ELX-02-disulfate or ELX-02ds), NMD inhibitor SMG1i, correctors VX-445 and VX-661 and potentiator VX-770. The quintuple combination of pharmacotherapies reached swelling quantities higher than the mean swelling of three VX-809/VX-770-rescued F508del/F508del organoid cultures, indicating level of rescue is of clinical relevance as VX-770/VX-809-mediated F508del/F508del rescue in organoids correlate with substantial improvement of clinical outcome. CONCLUSIONS: Whilst variation in efficacy was observed between genotypes as well as within genotypes, the data suggests that strong pharmacological rescue of PTC requires a combination of drugs that target RT, NMD and protein function.

Comparison of microbial composition of cough swabs and sputum for pathogen detection in patients with cystic fibrosis.

BACKGROUND: With the continued advancement of CFTR modulator therapies there is likely to be a burgeoning population of adult cystic fibrosis (CF) patients unable to expectorate sputum. Consequently, the detection and surveillance of pulmonary colonisation, previously reliant on sputum culture, needs re-examining. We hypothesised that cough swabs analysed with culture-independent analysis of the 16S gene could serve as a surrogate for colonisation of the lower airways. METHODS: Cough swabs and sputum samples were prospectively collected from consecutive adults and children with CF across two sites at regular outpatient appointments. Conventional culture analysis and next generation sequencing were used to compare paired same day samples. RESULTS: Twenty-two adults and 8 paediatric patients provided 75 paired cough swabs and sputum samples. Alpha diversity measures showed increased bacterial richness in sputum, while evenness and Simpson's diveristy index were higher in cough swabs. Within each sampling technique, microbial composition showed greater similarity when considering intra-patient variation. Poor concordance was observed between culture independent cough swabs and culture dependent/independent sputum analysis for specific pathogens, with cough swabs unable to accurately identify commonly associated CF pathogens (AUROCC range: 0.51 to 0.64). CONCLUSION: Culture independent analysis of cough swabs provides an inaccurate diagnosis of lower respiratory tract colonisation and should not be used as a diagnostic test in patients with CF.

Effectiveness of pulmonary rehabilitation at high-altitude compared to sea-level in adults with severe refractory asthma.

BACKGROUND: Beneficial effects of pulmonary rehabilitation at high-altitude (HAPR) in patients with severe refractory asthma have been reported earlier, but evidence for the effectiveness is limited. AIM: To investigate the effectiveness of high-altitude pulmonary rehabilitation to comparable treatment at sea-level (LAPR) on patient outcome parameters. METHODS: Adults with severe refractory asthma living in The Netherlands were included. Treatment consisted of a 12-week personalized multidisciplinary rehabilitation program either at high-altitude (Davos Switzerland) (n = 93) or in a tertiary lung center at sea-level in The Netherlands (n = 45). At baseline, after treatment, and during 12 months follow-up asthma related quality of life (AQLQ), asthma control (ACQ), pulmonary function and OCS-dose were assessed. Patients could not be randomized resulting in different asthma populations. Groups were compared using linear regression analysis (ANCOVA) adjusted for baseline values, in addition to age, atopy, smoking history, BMI and gender. RESULTS: After treatment, and at 12 months follow-up, improved AQLQ(0.92,p < 0.001 and 0.82,p = 0.001, respectively), ACQ(-0.87,p < 0.001 and -0.69,p = 0.008, respectively) and lower maintenance OCS dose (Unadjusted linear regression analysis-5.29 mg, p = 0.003 and Crude Odds Ratio-1.67, p = 0.003, respectively) were observed in the HAPR-group compared to the LAPR group. Patients receiving HAPR also had less asthma exacerbations (≥1 exacerbation: 20% vs 60%,p < 0.001) and showed improvement in lung function (%predFEV1 3.4%,p = 0.014) compared to the LAPR group, but at 12 months no differences between groups were observed. CONCLUSION: HAPR resulted in a larger improvement in patient outcome parameters compared to LAPR, on the long run the improvement in patient reported symptoms and lower maintenance OCS-dose persists. Underlying factors that explain this observed effect need to be investigated.

Predictors of benefit from high-altitude climate therapy in adults with severe asthma.

BACKGROUND: High-altitude climate therapy has been shown to benefit patients with severe asthma but it is not known which patients benefit most from this treatment. In the current study we aimed to identify clinical, functional and inflammatory predictors of favourable outcome of high-altitude climate therapy. METHODS: This is a secondary analysis of a prospective cohort including 136 adult patients with a diagnosis of severe refractory asthma, referred to the Dutch Asthma Centre in Davos (1600 metres above sea level), Switzerland. They had assessments of medication usage, asthma-related quality of life (Asthma-related Quality of Life Questionnaire, AQLQ), asthma control, body mass index (BMI), sino-nasal symptoms, fatigue, lung function (forced expiratory volume in one second, FEV1), exercise tolerance, allergy and inflammation (fraction of exhaled nitric oxide, blood eosinophils) at entry and after 12 weeks of treatment. Five clinically relevant outcomes were considered: AQLQ, oral corticosteroid dose, FEV1, body mass index and blood eosinophils. Independent predictors of beneficial outcome were identified by multiple linear regression analysis. RESULTS: Lower blood eosinophil counts (p < 0.01), younger age (p = 0.02) and poorer asthma control (p < 0.01) were independently associated with greater reduction in the dose of oral corticosteroids. Lower fatigue score at baseline (p = 0.01) was associated with greater weight loss (reduction in BMI). Higher levels of total IgE at baseline (p < 0.01), and higher doses of inhaled corticosteroids (p = 0.03) were associated with greater decreases in blood eosinophils. There were no predictors for improvement in AQLQ or FEV1. CONCLUSIONS: The beneficial effect of high-altitude climate therapy in adults with severe asthma can be predicted by patient characteristics, such as age, blood eosinophils and degree of asthma control before admission.

Emerging understanding of the mechanism of action of Bronchial Thermoplasty in asthma.

Bronchial Thermoplasty (BT) is an endoscopic treatment for moderate-to-severe asthma patients who are uncontrolled despite optimal medical therapy. Effectiveness of BT has been demonstrated in several randomized clinical trials. However, the asthma phenotype that benefits most of this treatment is unclear, partly because the mechanism of action is incompletely understood. BT was designed to reduce the amount of airway smooth muscle (ASM), but additional direct and indirect effects on airway pathophysiology are expected. This review will provide an overview of the different components of airway pathophysiology including remodeling, with the ASM as the key player. Current concepts in the understanding of BT clinical effectiveness with a focus on its impact on airway remodeling will be reviewed.

4-month omalizumab efficacy outcomes for severe allergic asthma: the Dutch National Omalizumab in Asthma Registry.

BACKGROUND: Omalizumab is licensed as add-on therapy for patients with severe allergic asthma. Response is in most studies scored by the physician's global evaluation of treatment effectiveness (GETE). A good clinical and validated parameter for treatment response is currently missing. Also, there are no established criteria for identifying patients who will respond to omalizumab based on pre-treatment characteristics. The Dutch National Omalizumab in Asthma Registry was developed in 2011 to better evaluate inclusion criteria and measure treatment response after 4 months. METHODS: This is a "real world" prospectively designed, observational data registry in which the outcomes of patients who received omalizumab between 2012 and 2015 were evaluated. Data were collected from all centers in the Netherlands comprising demographic features, criteria for starting treatment, GETE, FEV1, oral corticosteroid use and ACQ. RESULTS: 65.5% of the 403 patients had a good or excellent response to omalizumab after 16 weeks according to the treating physician GETE. 64.5% fulfilled all the criteria for prescribing omalizumab at baseline. The mean ACQ improved from 2.96 at baseline to 1.83 at 16 weeks (p < 0.001). 75.3% of the responders showed more than 0.5 points improvement in the ACQ. The mean FEV1 increased from 71.58 to 79.06 (p < 0.001). There was no relationship between patients with a FEV1 <80 and ≥80% at baseline and response (p = 0.981). Most of the responders had a considerable improvement of FEV1 either/or ACQ or OCS use (88.3%). While 86.7% of the responders had an improvement of either ACQ or FEV1. 75.4% of the responders had an improvement of ACQ, while 50.4% had an improvement of FEV1. Finally 11.7% of the patients with no improvement of FEV1, ACQ or OCS use were considered to have a good response. CONCLUSIONS: This registry of 403 inadequately controlled severe allergic asthma patients in the Netherlands showed a good or excellent response of 65.5% to omalizumab after 16 weeks, in accordance with previous studies. The assumption that careful registration would lead to higher response rates could not be supported by the data from this registry. Improvement of ACQ appears to be a useful additional assessment tool to measure response in omalizumab treated patients.

Detection of Airway Colonization by Aspergillus fumigatus by Use of Electronic Nose Technology in Patients with Cystic Fibrosis.

Currently, there is no noninvasive test that can reliably diagnose early invasive pulmonary aspergillosis (IA). An electronic nose (eNose) can discriminate various lung diseases through an analysis of exhaled volatile organic compounds. We recently published a proof-of-principle study showing that patients with prolonged chemotherapy-induced neutropenia and IA have a distinct exhaled breath profile (or breathprint) that can be discriminated with an eNose. An eNose is cheap and noninvasive, and it yields results within minutes. We determined whether Aspergillus fumigatus colonization may also be detected with an eNose in cystic fibrosis (CF) patients. Exhaled breath samples of 27 CF patients were analyzed with a Cyranose 320. Culture of sputum samples defined the A. fumigatus colonization status. eNose data were classified using canonical discriminant analysis after principal component reduction. Our primary outcome was cross-validated accuracy, defined as the percentage of correctly classified subjects using the leave-one-out method. The P value was calculated by the generation of 100,000 random alternative classifications. Nine of the 27 subjects were colonized by A. fumigatus. In total, 3 subjects were misclassified, resulting in a cross-validated accuracy of the Cyranose detecting IA of 89% (P = 0.004; sensitivity, 78%; specificity, 94%). Receiver operating characteristic (ROC) curve analysis showed an area under the curve (AUC) of 0.89. The results indicate that A. fumigatus colonization leads to a distinctive breathprint in CF patients. The present proof-of-concept data merit external validation and monitoring studies.

External validation of blood eosinophils, FE(NO) and serum periostin as surrogates for sputum eosinophils in asthma.

BACKGROUND: Monitoring sputum eosinophils in asthma predicts exacerbations and improves management of asthma. Thus far, blood eosinophils and FE(NO) show contradictory results in predicting eosinophilic airway inflammation. More recently, serum periostin was proposed as a novel biomarker for eosinophilic inflammation. OBJECTIVES: Quantifying the mutual relationships of blood eosinophils, FE(NO), and serum periostin with sputum eosinophils by external validation in two independent cohorts across various severities of asthma. METHODS: The first cohort consisted of 110 patients with mild to moderate asthma (external validation cohort). The replication cohort consisted of 37 patients with moderate to severe asthma. Both cohorts were evaluated cross-sectionally. Sputum was induced for the assessment of eosinophils. In parallel, blood eosinophil counts, serum periostin concentrations and FENO were assessed. The diagnostic accuracy of these markers to identify eosinophilic asthma (sputum eosinophils ≥3%) was calculated using receiver operating characteristics area under the curve (ROC AUC). RESULTS: In the external validation cohort, ROC AUC for blood eosinophils was 89% (p<0.001) and for FE(NO) level 78% (p<0.001) to detect sputum eosinophilia ≥3%. Serum periostin was not able to distinguish eosinophilic from non-eosinophilic airway inflammation (ROC AUC=55%, p=0.44). When combining these three variables, no improvement was seen. The diagnostic value of blood eosinophils was confirmed in the replication cohort (ROC AUC 85%, p<0.001). CONCLUSIONS: In patients with mild to moderate asthma, as well as patients with more severe asthma, blood eosinophils had the highest accuracy in the identification of sputum eosinophilia in asthma. The use of blood eosinophils can facilitate individualised treatment and management of asthma. TRIAL REGISTRATION: NTR1846 and NTR2364.

Three phenotypes of adult-onset asthma.

RATIONALE: Adult-onset asthma differs from childhood-onset asthma in many respects. It is more heterogeneous, often severe and frequently associated with loss of lung function. To identify underlying mechanisms of adult-onset asthma and to capture predictors of disease progression, detailed characterization and phenotyping is necessary. OBJECTIVES: To characterize adult-onset asthma and identify subphenotypes of adult-onset asthma. METHODS: A cohort of 200 patients with adult-onset (>18 year) asthma (age 54 (26-75) year) was recruited from one academic and three nonacademic pulmonary outpatient clinics in Amsterdam, the Netherlands. These patients were fully characterized with respect to clinical, functional and inflammatory markers. After data reduction, K-means nonhierarchical cluster analysis was performed to identify clusters of adult-onset asthma. MEASUREMENTS AND MAIN RESULTS: Patients with adult-onset asthma were predominately female (61%) and nonatopic (55%). Within this group of patients were identified three clusters of adult-onset asthma. Cluster 1 (n = 69) consisted of patients with severe eosinophilic inflammation-predominant asthma and persistent airflow limitation despite high-intensity anti-inflammatory treatment, with relatively low symptom scores. The second cluster was characterized by obese women with frequent symptoms, high healthcare utilization and low sputum eosinophils. The third cluster consisted of patients with mild-to-moderate, well-controlled asthma with normal lung function and low inflammatory markers. Repeatability accuracy was 98.2%. CONCLUSIONS: Amongst patients with adult-onset asthma, three subphenotypes can be identified with distinct clinical and inflammatory characteristics. These subphenotypes help to understand the underlying pathobiology and provide clinicians with directions for personalized management.

Non-atopic males with adult onset asthma are at risk of persistent airflow limitation.

BACKGROUND: Patients with asthma have on average a more rapid decline in FEV (1) as compared with the general population. Recent cluster analysis has revealed different asthma phenotypes that can be distinguished by age of onset and reversibility of airflow limitation. OBJECTIVE: This study aimed at detecting risk factors associated with persistent airflow limitation in patients with the adult onset asthma phenotype. METHODS: We recruited 88 patients with adult onset (≥ 18 years) asthma from an academic pulmonary outpatient clinic in the Netherlands. The associations of age, age of asthma onset, asthma duration, gender, race, atopy, smoking pack-years, BMI, use of oral corticosteroids with post-bronchodilator FEV (1) /FVC were investigated. RESULTS: Multiple linear regression analysis showed an association of absence of atopy (r = -0.27, B = -0.26, P = 0.01) and male gender (r = 0.31, B = 0.30, P = 0.004) with post-bronchodilator FEV (1) /FVC. Multiple logistic regression analysis showed that male patients were 10.8 (CI: 2.6-45.2) times the odds than women to have an FEV (1) /FVC < 0.7, and non-atopic patients were 5.2 (CI: 1.3-20.3) times the odds to have an FEV (1) /FVC < 0.7 than atopic patients. CONCLUSIONS AND CLINICAL RELEVANCE: We conclude that in patients with adult onset asthma, male gender and absence of atopy are associated with persistent airflow limitation. This might suggest that amongst patients with adult onset asthma, non-atopic male patients are at increased risk of accelerated decline in lung function.

Synbiotics reduce allergen-induced T-helper 2 response and improve peak expiratory flow in allergic asthmatics.

BACKGROUND: Previous studies suggest that pre/probiotics can be used in the prevention and treatment of early allergic disease in newborns and young children. OBJECTIVE: To determine the effect of treatment with synbiotics (90% short-chain galacto-oligosaccharides, 10% long-chain fructo-oligosaccharides: Immunofortis(®) and Bifidobacterium breve M-16V) on allergic responses in adults with established allergic asthma. Primary outcome was allergen-induced bronchial inflammation as represented by eosinophil counts. METHODS: Twenty-nine patients with asthma and house dust mite (HDM) allergy were randomized in a double-blind, parallel design to receive placebo or synbiotics for 4 weeks. At study entry and after treatment, a bronchial allergen challenge with HDM was performed, followed by lung function tests, collection of blood (in/ex vivo IL-5) and induced sputum (inflammatory parameters). During treatment, a diary was kept with peak expiratory flow (PEF) and asthma scores. RESULTS: Treatment did not affect the allergen-induced increase in sputum eosinophils at 6 and 24 h after challenge. Likewise, other parameters for bronchial inflammation and early and late changes in lung function did not differ upon treatment. Both the morning and evening PEF, however, significantly increased during synbiotics treatment (morning P = 0.003, evening P = 0.011). Also, the increase in serum IL-5 after allergen challenge was significantly inhibited by synbiotics (P = 0.034), as was ex vivo allergen-induced Th2-cytokine (IL-5 and IL-4+ IL-13) production by PBMCs (P = 0.046). In vivo (24 h) and ex vivo IL-5 production were associated. CONCLUSION: Four-week treatment with synbiotics had no effect on bronchial inflammation and LAR, but did significantly reduce systemic production of Th2-cytokines after allergen challenge and improved PEF.

Health-related quality of life and functional status in end-stage COPD: a longitudinal study.

Since there is still a dearth of information about the end stage of chronic obstructive pulmonary disease (COPD), the main aim of this study was to examine the development of health-related quality of life (HRQoL) and functional status over time in COPD patients in Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage IV. 82 Dutch COPD patients completed the St George's Respiratory Questionnaire (SGRQ) for HRQoL and the Groningen Activities for Daily Living Restriction Scale (GARS) for functional status every 3 months during the year following enrolment. Survival was followed up to 5 yrs after enrolment. Data were analysed by stratifying the study population into severity subgroups according to the lowest, intermediate and highest tertile of SGRQ and GARS at baseline. Outcome measures were change in SGRQ and GARS scores over time and survival time. In the majority of patients, scores on the SGRQ and GARS declined gradually over time. In the subgroup of 32 patients that died within 2 yrs of enrolment, these scores also declined gradually, without steep deteriorations. In patients with end-stage COPD, HRQoL and functional status deteriorated gradually over time, indicating that clinicians did not gain much additional support for differentiating the end stage of COPD by considering HRQoL and functional status using the SGRQ and GARS.

A case-control study of the relation between plasma selenium and asthma in European populations: a GAL2EN project.

BACKGROUND: There is evidence that selenium levels are relatively low in Europe and may be falling. Low levels of selenium or low activity of some of the enzymes dependent on selenium have been associated with asthma. METHODS: The GA(2)LEN network has organized a multicentre case-control study in Europe to assess the relation of plasma selenium to asthma. The network compared 569 cases in 14 European centres with a diagnosis of asthma and reporting asthma symptoms in the last 12 months with 576 controls from the same centres with no diagnosis of asthma and no asthmatic symptoms in the last 12 months. RESULTS: All cases and controls were selected from the same population defined by age and place of residence. Mean plasma selenium concentrations among the controls ranged from 116.3 microg/l in Palermo to 67.7 microg/l in Vienna and 56.1 microg/l among the children in Oslo. Random effects meta-analysis of the results from the centres showed no overall association between asthma and plasma selenium [odds ratio (OR)/10 microg/l increase in plasma selenium: 1.04; 95% confidence interval (CI): 0.89-1.21] though there was a significantly protective effect in Lodz (OR: 0.48; 95% CI: 0.29-0.78) and a marginally significant adverse effect in Amsterdam (OR: 1.68; 95% CI: 0.98-2.90) and Ghent (OR: 1.35; 95% CI: 1.03-1.77). CONCLUSION: This study does not support a role for selenium in protection against asthma, but effect modification and confounding cannot be ruled out.

The relation between paracetamol use and asthma: a GA2LEN European case-control study.

Studies from the UK and USA suggest that frequent use of paracetamol (acetaminophen) may increase the risk of asthma, but data across Europe are lacking. As part of a multicentric case-control study organised by the Global Allergy and Asthma European Network (GA(2)LEN), it was examined whether or not frequent paracetamol use is associated with adult asthma across Europe. The network compared 521 cases with a diagnosis of asthma and reporting of asthma symptoms within the last 12 months with 507 controls with no diagnosis of asthma and no asthmatic symptoms within the last 12 months across 12 European centres. All cases and controls were selected from the same population, defined by age (20-45 yrs) and place of residence. In a random effects meta-analysis, weekly use of paracetamol, compared with less frequent use, was strongly positively associated with asthma after controlling for confounders. There was no evidence for heterogeneity across centres. No association was seen between use of other analgesics and asthma. These data add to the increasing and consistent epidemiological evidence implicating frequent paracetamol use in asthma in diverse populations.

Seasonal variations in cyclic AMP production by peripheral blood mononuclear cells in allergic asthmatics.

BACKGROUND: Dysfunction of the beta-adrenoceptor (betaAR)/adenylyl cyclase (AC) system can impair the response of different cell types, including lymphocytes. In asthma, impairment of this system as well as changes in cytokine production by lymphocytes have been described. Because the severity of asthma can change over the year, a circannual pattern of the betaAR/AC system activity may also exist. OBJECTIVES: We set out to examine the activity of this betaAR/AC signal transduction system in peripheral blood mononuclear cells (PBMCs) of allergic asthmatics to asses whether differences existed between seasons. We investigated whether changes were associated with asthma severity and circannual changes in serum cortisol levels. METHODS: During 19 months, 41 allergic asthmatics (mean age 28 years) with nocturnal airway obstruction were enrolled in the study. AC activity was measured by cyclic AMP production. Resting, stimulated and potentiated AC activities and their relationships with clinical parameters, seasonal influences and serum cortisol levels were assessed. RESULTS: The AC activity in resting, stimulated and potentiated cells varied during the year. AC activity was relatively low in the periods June-August and September-November, and higher in December-February and March-May. Receptor-mediated and potentiated responses expressed as percentage of the resting response were equivalent throughout the year. Serum cortisol levels were positively related to AC activity. No relationships were found between clinical parameters and AC activity or serum cortisol levels. CONCLUSIONS: These results indicate that AC activity in PBMCs of allergic asthmatics shows a seasonal variation. However, seasonal differences in AC activity seems to be unrelated with clinical parameters. Other factors such as serum cortisol levels may have a modulating influence on AC activity. Future studies of AC systems in blood cells of asthmatic patients need to take into account these seasonal influences.

T-Cell repertoire in the blood and lungs of atopic asthmatics before and after ragweed challenge.

T cells play a pivotal role in initiating and orchestrating allergic responses in asthma. The goal of this work was to learn whether ragweed challenge in the lungs alters the T-cell repertoire expressed in the blood and lungs of atopic asthmatics. Analyses of cell numbers, differentials, and T-cell subsets in bronchoalveolar lavage (BAL) fluids showed that ragweed challenge was associated with preferential recruitment of CD4+ T cells into the lungs. A reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify T-cell receptor (TCR) gene transcripts from unfractionated, CD4+, and CD8+ T cells in blood and BAL fluids. As judged by RT-PCR, the usage of TCR Valpha and Vbeta gene families in BAL fluids was similar to that in blood. Ragweed challenge did not change the levels of expression of these V gene families. The clonality of T cells was estimated by analyzing the diversity of TCR V-(D)-J junctional region nucleotide lengths associated with each Valpha and Vbeta gene family, using sequencing gel electrophoresis. Most V gene families in blood and BAL fluids were associated with multiple junctional region lengths before and after ragweed challenge, indicating polyclonal expression. Some V gene families were expressed in an oligoclonal manner in unfractionated, CD4+, and CD8+ T cells in BAL fluids before ragweed challenge, as indicated by a few predominant junctional region lengths. The majority of these V gene families became polyclonal after challenge, compatible with polyclonal T-cell influx during inflammation immediately after ragweed challenge. However, some V gene families became oligoclonal or developed a new oligoclonal pattern of junctional region lengths in BAL T cells after ragweed challenge. Surprisingly, this occurred in both CD4+ and CD8+ T cells. In one of these instances, DNA sequencing of Vbeta21 junctional regions in CD8+ T cells confirmed a change from polyclonal to oligoclonal expression after ragweed challenge. These findings show that ragweed challenge is associated with polyclonal influx and oligoclonal activation of both CD4+ and CD8+ T cells in the lungs.