Lack of effect of the alpha2C-adrenoceptor Del322-325 polymorphism on inhibition of cyclic AMP production in HEK293 cells.

BACKGROUND AND PURPOSE: The alpha(2C)-adrenoceptor has multiple functions, including inhibiting release of noradrenaline from presynaptic nerve terminals. A human alpha(2C) polymorphism, Del322-325, a potential risk factor for heart failure, has been reported to exhibit reduced signalling in CHO cells. To further understand the role of the Del322-325 polymorphism on receptor signalling, we attempted to replicate and further study the reduced signalling in HEK293 cells. EXPERIMENTAL APPROACH: Human alpha(2C) wild-type (WT) and Del322-325 adrenoceptors were stably transfected into HEK293 cells. Radioligand binding was performed to determine affinities for both receptors. In intact cells, inhibition of forskolin-stimulated cyclic AMP production by WT and Del322-325 clones with a range of receptor densities (200-2320 fmol.mg(-1) protein) was measured following agonist treatment. KEY RESULTS: Noradrenaline, brimonidine and clonidine exhibited similar binding affinities for WT and Del322-325. Brimonidine and clonidine also had similar efficacies and potencies for both receptors for the inhibition of cyclic AMP production at all receptor densities tested. A linear regression analysis comparing efficacy and potency with receptor expression levels showed no differences in slopes between WT and Del322-325. CONCLUSIONS AND IMPLICATIONS: The alpha(2C) WT and Del322-325 adrenoceptors exhibited similar binding properties. Additionally, inhibition of cyclic AMP production by Del322-325 was similar to that of WT over a range of receptor densities. Therefore, in intact HEK293 cells, the alpha(2C)-Del322-325 polymorphism does not exhibit reduced signalling to adenylyl cyclase and may not represent a clinically important phenotype.

A two-year randomized, placebo-controlled trial of dornase alfa in young patients with cystic fibrosis with mild lung function abnormalities.

OBJECTIVE: Our objective was to determine whether long-term treatment of young patients with cystic fibrosis (CF) with dornase alfa maintains lung function and reduces respiratory tract exacerbations. STUDY DESIGN: This was a 96-week, randomized, double-blind, placebo-controlled trial involving 49 CF centers. Inclusion criteria were age 6 to 10 years and forced vital capacity > or = 85% predicted. Patients were excluded for hospitalization for complications of CF within 2 months and use of dornase alfa within 6 months. Patients were treated with dornase alfa 2.5 mg or placebo once daily with a jet nebulizer and a compressor. RESULTS: Patients were randomized, 239 to dornase alfa and 235 to placebo. At baseline the mean age was 8.4 years, the mean forced expiratory volume in 1 second 95% predicted, the mean forced expiratory flow, midexpiratory phase 85% predicted, and the mean forced vital capacity 102% predicted. At 96 weeks the treatment benefit for dornase alfa compared with placebo in percent predicted (mean +/- SE) was 3.2 +/- 1.2 for forced expiratory volume in 1 second (P =.006), 7.9 +/- 2.3 for forced expiratory flow between 25% and 75% of vital capacity (P =.0008), and 0.7 +/- 1.0 for forced vital capacity (P =.51). The risk of respiratory tract exacerbation was reduced by 34% in patients who received dornase alfa (relative risk 0.66, P =.048). There was no statistically significant difference between the groups in changes in weight-for-age percentile. Adverse event profiles for the treatment groups were similar. CONCLUSIONS: Treatment of young patients with CF with dornase alfa maintains lung function and reduces the risk of exacerbations over a 96-week period.

Sleep-disordered breathing in Duchenne muscular dystrophy: a preliminary study of the role of portable monitoring.

Although Duchenne muscular dystrophy (DMD) is often associated with sleep disordered breathing (SDB), it is not standard clinical practice to routinely test this population for SDB, and the optimal timing and methodology for such testing has not been established. Our objectives were: 1) to examine the concordance between laboratory polysomnography (PSG) and two portable monitoring systems, and 2) to identify clinical factors associated with the onset of SDB. We performed a cross-sectional pilot study of patients with DMD who were 6 years of age or older, and who were registered at the Alberta Children's and Calgary General Hospitals. Patient symptom and functional rating scores were calculated, and pulmonary function tests, awake oxygen saturation, and capillary blood gases were obtained. PSG was performed according to standard methods, and results were compared with Snoresat(R) (Saga Tech Electronics, Inc.) and EdenTec(R) (Nellcor Puritan Bennett) portable home monitors. Eleven boys were studied. Ten of 11 subjects had normal awake oxygen saturation and capillary blood gases. Median forced vital capacity (FVC) was 70% of predicted values (15-104%). PSG identified 3 boys with severe hypoventilation occurring throughout REM sleep. Reported symptom severity did not predict the patients with significant SDB. All 3 boys with SDB had a severe functional disability and severely reduced FVCs. Portable monitoring in the home identified all patients with abnormal PSG. One additional patient was falsely identified by the EdenTec(R) monitor. We conclude that initial results using Snoresat(R) or EdenTec(R) monitoring equipment for the identification of SDB are promising, but further validation of portable home monitoring is required in this group of patients.

Histamine challenge in young children using computerized lung sounds analysis.

The use of the histamine challenge test (HCT) for the diagnosis of asthma has so far been limited to older children who can perform spirometry consistently. Recently, wheeze detection by tracheal auscultation with analog recording into a tape recorder has been utilized in young children in place of spirometry. Wheezing can also be identified using computerized lung sounds analysis (LSA) by a typical pattern on spectral analysis. Our aim was to develop a practical computerized system in which the response to histamine could be identified in an objective manner and documented on hard copy. Lung sounds were recorded with a Hewlett-Packard HP 21050A contact sensor placed over the right upper anterior chest. Sounds were amplified, band-filtered (50 to 2,000 Hz), and digitized at a sampling rate of 5.5 kHz into a Macintosh SE computer, and spectral LSA was performed. To validate our method, six older children (ages 9 to 16 years) with mild or moderate asthma underwent HCT. The identification of typical wheezing pattern (discrete, high-amplitude power peaks) on LSA was compared to 20 percent fall in FEV1 (PC20) and symptoms (cough, wheeze, chest tightness). In five children, the histamine concentration required to produce the characteristic wheezing pattern on LSA was half that required to produce a 20 percent fall in FEV1. In the sixth patient, wheezing on LSA and PC20 occurred at the same histamine concentration. To determine the technique's applicability to young children, we then studied six young asthmatic children (age 2 to 5 years). All children showed the wheezing pattern at a histamine concentration of 25 percent or 50 percent (one or two steps prior) to that producing symptoms (cough, wheeze, chest tightness) or wheezing on tracheal auscultation. Six age- and sex-matched nonasthmatic children (control subjects) did not show this pattern on LSA and had no symptoms or tracheal wheeze with HCT. We describe a sensitive method enabling application of HCT to young children who are unable to perform spirometry. This method is as sensitive as, and often more sensitive than, conventional PC20 with spirometry or tracheal auscultation.

Causes of death in hemolytic uremic syndrome.

One hundred and four children developed typical post-diarrhea hemolytic uremic syndrome (D+ HUS) over an 11-year period. Four of the children died in the acute stage. Two of the children died from a central nervous system complication which was attributable to the HUS process. One child died suddenly without any explanation. The 4th child died following a cardiovascular collapse, perhaps related to septic shock. The literature on the causes of death in children with HUS is reviewed.