Test-retest reliability, construct validity and determinants of 6-minute walk test performance in adult patients with asthma.

INTRODUCTION AND OBJECTIVES: Current knowledge regarding the measurement properties of the 6-minute walk test (6MWT) in patients with asthma is limited. Therefore, the aim of this study was to assess the test-retest reliability, measurement error and construct validity of the 6MWT and identify determinants of 6-minute walk distance (6MWD) in patients with asthma. PATIENTS AND METHODS: 201 asthma patients referred for pre-pulmonary rehabilitation assessment, were retrospectively analyzed (age 61±12 years, 42% male, FEV1 78±27% predicted). Patients performed two 6MWTs on subsequent days using a 30 m straight walking course. Other measurements included resting dyspnea, maximal exercise capacity, body composition, pulmonary function, pulmonary and quadriceps muscle strength and symptoms of anxiety and depression. Measurement error (absolute reliability) was tested using standard error of measurement (SEM), minimal detectable change at 95% confidence interval (MDC95%) and Bland and Altman 95% limits of agreement, whereas test-retest reliability (relative reliability) and construct validity were assessed using the intra-class correlation coefficient (ICC2,1) and correlations, respectively. RESULTS: The 6MWD showed excellent test-retest reliability (ICC2,1: 0.91). The mean change in 6MWD after the second 6MWT was 18m (95%CI 11-24m), with 73% of the patients walking further in the second test. The SEM and MDC95% for the 6MWT were 35 m and 98 m, respectively. The best 6MWD correlated strongly with peak oxygen uptake during CPET and resting dyspnea (r = 0.61-0.64) and had no-to-moderate correlations with body composition, pulmonary function, respiratory and quadriceps muscle strength and symptoms of anxiety and depression (r = 0.02-0.45). Multiple linear regression was able to identify maximal workload, BMI, rollator use, maximal expiratory pressure, FEV1 and DLCO as independent determinants of the best 6MWD (R2 = 0.58). CONCLUSIONS: The 6MWT was considered to be reliable and valid in patients with asthma, which strengthens its clinical utility. However, the majority of patients demonstrated a considerable learning effect in the second 6MWT, providing a strong rationale for performing two 6MWTs.

Impact of acute exacerbations of COPD on patients' health status beyond pulmonary function: A scoping review.

This scoping review summarized the evidence regarding the impact of acute exacerbations of COPD (AECOPD) on patients' health status beyond pulmonary function. PubMed, Embase, and Web of Science were searched. Prospective cohort studies assessing the health status of patients with COPD in a stable phase of the disease and after a follow-up period (where at least one AECOPD occurred) were included. An integrated assessment framework of health status (i.e., physiological functioning, complaints, functional impairment, quality of life) was used. Twenty-two studies were included. AECOPD acutely affected exercise tolerance, quadriceps muscle strength, physical activity levels, symptoms of dyspnoea and fatigue, and impact of the disease. Long-term effects on quadriceps muscle strength, symptoms of dyspnoea and depression, and quality of life were found. Repeated exacerbations negatively impacted the fat-free mass, levels of dyspnoea, impact of the disease and quality of life. Conflicting evidence was found regarding the impact of repeated exacerbations on exercise tolerance and physical activity levels. AECOPD have well-established acute and long-term adverse effects on health status beyond pulmonary function; nevertheless, the recovery trajectory and the impact of repeated exacerbations are still poorly studied. Further prospective research is recommended to draw firm conclusions on these aspects.

Carnosine, oxidative and carbonyl stress, antioxidants, and muscle fiber characteristics of quadriceps muscle of patients with COPD.

Oxidative/carbonyl stress is elevated in lower-limb muscles of patients with chronic obstructive pulmonary disease (COPD). Carnosine is a skeletal muscle antioxidant particularly present in fast-twitch fibers. The aims of the present study were to compare muscle carnosine, oxidative/carbonyl stress, antioxidants, and fiber characteristics between patients with COPD and healthy controls (HCs) and between patients after stratification for airflow limitation (mild/moderate vs. severe/very severe), as well as to investigate correlates of carnosine in patients with COPD. A vastus lateralis muscle biopsy was obtained from 40 patients with stable COPD and 20 age- and sex-matched HCs. Carnosine, oxidative/carbonyl stress, antioxidants, fiber characteristics, quadriceps strength and endurance (QE), V̇o2peak (incremental cycle test), and physical activity (PA) were determined. Patients with COPD had a similar carnosine concentration [4.16 mmol/kg wet weight (WW; SD = 1.93)] to HCs [4.64 mmol/kg WW (SD = 1.71)] and significantly higher percentage of fast-twitch fibers and lower QE, V̇o2peak, and PA versus HCs. Patients with severe/very severe COPD had a 31% lower carnosine concentration [3.24 mmol/kg WW (SD = 1.79); n = 15] versus patients with mild/moderate COPD [4.71 mmol/kg WW (SD = 1.83); n = 25; P = 0.02] and significantly lower V̇o2peak and PA versus patients with mild/moderate COPD. Carnosine correlated significantly with QE (rs = 0.427), V̇o2peak (rs = 0.334), PA (rs = 0.379), and lung function parameters in patients with COPD. In conclusion, despite having the highest proportion of fast-twitch fibers, patients with severe/very severe COPD displayed a 31% lower muscle carnosine concentration compared with patients with mild/moderate COPD. As no other markers of oxidative/carbonyl stress or antioxidants were affected, the observed carnosine deficiency is thought to be a possible first sign of muscle redox balance abnormalities.NEW & NOTEWORTHY Carnosine, particularly present in fast-twitch fibers, was investigated in the quadriceps of patients with chronic obstructive pulmonary disease (COPD). Carnosine concentration was similar between patients with COPD and healthy controls but was 31% lower in patients with severe/very severe COPD, despite their high proportion of fast-twitch fibers, versus patients with mild/moderate COPD. As no other markers of oxidative/carbonyl stress or antioxidants were affected, the observed carnosine deficiency is thought to be a possible first sign of muscle redox balance abnormalities.

The correlation between quadriceps muscle strength and endurance and exercise performance in patients with COPD.

To determine the association between quadriceps muscle strength (QMS) and endurance (QME) and exercise capacity in patients with COPD after stratification for sex and resting lung function (LF). Data were collected from 3,246 patients with COPD (60% men, 64 ± 9 yr), including measures of exercise capacity [peak aerobic capacity (peakV̇o2), 6-min walk distance (6MWD)] and isokinetic QMS and QME. Patients were stratified for sex, forced expiratory volume in 1 s (>50/≤50% predicted), single breath carbon monoxide diffusing capacity (>50/≤50% predicted), and residual volume (>140/≤140% predicted). After stratification for resting LF, QMS and QME were significantly associated with peakV̇o2 (r range: 0.47-0.61 and 0.49-0.65 for men and 0.53-0.66 and 0.48-0.67 for women, respectively) and 6MWD (r range: 0.29-0.42 and 0.44-0.55 for men and 0.25-0.54 and 0.34-0.55 for women, respectively) (P < 0.001). Regression models demonstrated that QMS and QME were significant determinants of peakV̇o2 (explained variance R2 range: 35.6%-48.8% for men and 36.8%-49.0% for women) and 6MWD (R2 range: 24.3%-43.3% for men and 28.4%-40.3% for women), independent of age and fat-free mass. Quadriceps muscle function was significantly associated with peakV̇o2 and 6MWD in male and female patients with COPD after stratification for resting LF, in which QME appear to be a more important determinant than QMS. This underlines the importance of systematically evaluating both quadriceps muscle strength and endurance in in all patients with COPD.NEW & NOTEWORTHY Our findings identified quadriceps muscle function as an important determinant of exercise capacity across a wide spectrum of lung function. Quadriceps muscle endurance appears to be a more important determinant than quadriceps muscle strength, underlining the importance of including both the measurement of quadriceps muscle strength and endurance in routine assessment for all patient with COPD.

Effects of a community-based pulmonary rehabilitation programme during acute exacerbations of chronic obstructive pulmonary disease - A quasi-experimental pilot study.

BACKGROUND: Pulmonary rehabilitation (PR) is a cornerstone intervention for the management of patients with stable chronic obstructive pulmonary disease (COPD). However, its role during acute exacerbations (AECOPD) is controversial since most studies have been conducted in hospitalised patients, when more than 80% of AECOPD are managed on an outpatient basis. This quasi-experimental pilot study assessed the effects of a community-based PR programme during mild-to-moderate AECOPD. METHODS: Outpatients were recruited from hospitals and allocated to experimental (EG) or control (CG) groups. EG received standard medication plus 3-weeks of PR. The CG received standard medication. Dyspnoea (mMRC), quadriceps muscle strength (QMS), functionality (5-repetition sit-to-stand test) and impact of the disease (COPD assessment test (CAT)) were assessed within 48h of the AECOPD onset and after PR. Symptoms of dyspnoea and fatigue (mBorg), heart and respiratory (RR) rates and peripheral oxygen saturation (SpO2) were assessed at rest and monitored in all PR sessions. Need for hospitalisation was monitored during the 3-weeks. RESULTS: Twelve patients (69±7 years, FEV1 52±27 pp) in the EG and eleven in the CG (66±9 years, FEV1 55±22 pp) were enrolled. The EG presented significant improvements on QMS (Pre 21.0 vs. Post 25.0, p=0.012), CAT (Pre 23.0 vs. Post 14.5, p=0.008), symptoms of dyspnoea at rest (Pre 3.0 vs. Post 1.0, p=0.008), SpO2 (Pre 94.0 vs. Post 96.0, p=0.031) and RR (Pre 24.0 vs. Post 20.5, p=0.004). No significant improvements were found in the CG. CONCLUSION: Adding PR to the management of mild-to-moderate AECOPD seems to result in improvements on parameters usually associated with an increased risk of re-exacerbation and poor prognosis. Randomised studies with larger samples are needed to confirm these results.

Higher food intake and appreciation with a new food delivery system in a Belgian hospital. Meals on Wheels, a bedside meal approach: a prospective cohort trial.

AIM: A new system of meal distribution called Meals on Wheels, allowing food ordering at mealtime and providing guidance by trained nutritional assistants, might show benefit in offering nutritional support. This study investigates whether Meals on Wheels improves total food intake per day and yielded improved appreciation of food quality and increased access to food and mealtimes. METHODS: In a prospective cohort trial where control and intervention groups were taken from all patients hospitalized at the respiratory disease department, age, sex, BMI, admission weight, height, reason for admission and discharge weight were noted, as was food intake, supplements, waste per meal and daily total. For food appreciation the questionnaire developed by Naithani et al. was used. The study included 83 patients in the control group and 106 patients in the Meals on Wheels group. RESULTS: Mean total daily food intake was 236 g higher in patients in the Meals on Wheels than in controls. There was higher intake of oral nutritional supplements in the Meals on Wheels group compared to controls, resulting in significantly less oral nutritional supplements wasted. There was also significantly less waste in the Meals on Wheels group. For food access and appreciation, patients appreciated Meals on Wheels more than the old system in terms of choice, hunger, food quality and organization. CONCLUSIONS: Meals on Wheels resulted in higher food intake during each meal, less waste and better use of oral nutritional supplements. Patients appreciated Meals on Wheels more than the old system in terms of choice, hunger, food quality and organization.

Exercise training after lung transplantation improves participation in daily activity: a randomized controlled trial.

The effects of exercise training after lung transplantation have not been studied in a randomized controlled trial so far. We investigated whether 3 months of supervised training, initiated immediately after hospital discharge, improve functional recovery and cardiovascular morbidity of patients up to 1 year after lung transplantation. Patients older than 40 years, who experienced an uncomplicated postoperative period, were eligible for this single blind, parallel group study. Sealed envelopes were used to randomly allocate patients to 3 months of exercise training (n = 21) or a control intervention (n = 19). Minutes of daily walking time (primary outcome), physical fitness, quality of life and cardiovascular morbidity were compared between groups adjusting for baseline assessments in a mixed models analysis. After 1 year daily walking time in the treated patients (n = 18) was 85 ± 27 min and in the control group (n = 16) 54 ± 30 min (adjusted difference 26 min [95%CI 8-45 min, p = 0.006]). Quadriceps force (p = 0.001), 6-minute walking distance (p = 0.002) and self-reported physical functioning (p = 0.039) were significantly higher in the intervention group. Average 24 h ambulatory blood pressures were significantly lower in the treated patients (p ≤ 0.01). Based on these results patients should be strongly encouraged to participate in an exercise training intervention after lung transplantation.

Determinants of physical activity in daily life in candidates for lung transplantation.

BACKGROUND: Participation in daily physical activity (PA) has never been objectively assessed in candidates for lung transplantation (LTx). The main research questions were: 1) How active are LTx-candidates in daily life? 2) What are determinants of activity behavior before LTX? METHODS: Ninety-six candidates for LTx (diagnosis of COPD or interstitial lung disease; mean age 55 ± 7 years) underwent measurements of PA, pulmonary function, 6-min walking distance (6MWD), muscle force and health-status (SF-36 scale). RESULTS: Patients were markedly inactive (5% of waking hours walking, 26% standing and 69% sedentary). Backward multiple regression identified 6MWD (expressed as % of predicted value; ß = 73.0 steps, partial r(2) = 0.36, p = 0.00), a higher score on the energy/fatigue scale of the SF-36 (ß = 28.6 steps, partial r(2) = 0.09, p = 0.00) and a higher expiratory muscle force (expressed as % of predicted value; ß = 11.8 steps, partial r(2) = 0.05, p = 0.02) as determinants of daily steps. Minutes of mild to moderate (≥2 METs) activity were determined by 6MWD (expressed as % of predicted value; ß = 2.14 min, partial r(2) = 0.30, p = 0.00), inspiratory muscle force (expressed as % of predicted value; ß = 0.33 min, partial r(2) = 0.04, p = 0.05) and seasonal influences (spring/summer vs. autumn/winter: ß = 18.95 min, partial r(2) = 0.04, p = 0.05). The overall fit of the models was r(2) = 0.50 and r(2) = 0.38, respectively. CONCLUSIONS: The 6MWD was the main determinant of an inactive lifestyle in these patients. Respiratory muscle force, energy and fatigue and seasonal variations explained some additional variability in activity behavior. Patients should be encouraged to participate in interventions aimed at improving physical fitness and participation in daily physical activity before LTx.

Rehabilitation and acute exacerbations.

Recent evidence indicates that acute exacerbations of chronic obstructive pulmonary disease aggravate the extrapulmonary consequences of the disease. Skeletal muscle dysfunction, a sustained decrease in exercise tolerance, enhanced symptoms of depression and fatigue are reported. Avoidance of physical activities is likely to be a key underlying mechanism and increases the risk of new exacerbations. Pulmonary rehabilitation is an intervention targeting these systemic consequences. Exercise strategies need to be adapted to the increased feelings of dyspnoea and fatigue. This review aims to describe the systemic consequences of acute exacerbations and compiles evidence for the feasibility and effectiveness of different rehabilitation strategies to counteract these consequences during and/or immediately after the acute phase of the exacerbation. Resistance training and neuromuscular electrical stimulation have been applied safely in frail, hospitalised patients and have the potential to prevent muscle atrophy. Comprehensive pulmonary rehabilitation, including general exercise training, can be implemented immediately after the exacerbation, leading to a reduction in hospital admissions and an increase in exercise tolerance and quality of life. Self-management strategies play a crucial role in changing disease-related health behaviour and preventing hospital admissions.

Guia para prática clínica: fisioterapia em pacientes com Doença Pulmonar Obstrutiva Crônica (DPOC): [review] / Clinical practice guideline for physical therapy in patients with Chronic Obstructive Pulmonary Disease (COPD): portuguese version: [review]

INTRODUÇÃO: No contexto da colaboração internacional para desenvolvimento de guias práticos (ou guidelines), a Sociedade Real Holandesa de Fisioterapia (Koninklijk Nederlands Genootschap voor Fysiotherapie, KNGF) se propôs a desenvolver um guia para esclarecimento sobre a prática clínica de Fisioterapia em pacientes com Doença Pulmonar Obstrutiva Crônica (DPOC), assim como também optou por estimular a sua tradução para outras línguas, a fim de torná-lo acessível para públicos internacionais. OBJETIVOS: O presente guia é a versão em língua portuguesa do Guia para Prática Clínica de Fisioterapia em pacientes com DPOC desenvolvido pela KNGF, que teve como objetivo descrever a Fisioterapia baseada em evidências para pacientes com DPOC que apresentam limitação da função pulmonar, da função muscular respiratória e periférica, da capacidade de exercício, da depuração mucociliar e da qualidade de vida, além de limitações em relação à atividade física na vida diária pela dispneia e/ou intolerância ao exercício. CONCLUSÃO: O guia propõe-se principalmente a prover recomendações terapêuticas práticas que auxiliem o fisioterapeuta a oferecer o melhor tratamento possível para pacientes com DPOC, consideradas as evidências científicas disponíveis na atualidade.

INTRODUCTION: In the context of international collaboration for the development of practice guidelines, the Royal Dutch Society for Physical Therapy (Koninklijk Nederlands Genootschap voor Fysiotherapie, KNGF) has developed guidelines for the clinical practice of physical therapy in patients with Chronic Obstructive Pulmonary Disease (COPD). It has also stimulated its translation into other languages to make it accessible to international audiences. OBJECTIVES: The present document brings the Portuguese version of the KNGF Clinical Practice Guidelines for physical therapy in COPD patients. Its purpose was to describe evidence-based physical therapy for COPD patients with impairments in pulmonary function, peripheral and respiratory muscle function, exercise capacity, mucus clearance and quality of life, in addition to limitations in physical activity in daily life due to dyspnea and/or exercise intolerance. CONCLUSION: The guideline provides practical and therapeutic recommendations based on currently available scientific evidence to help the physical therapist provide the best possible treatment to COPD patients.

A clinical practice guideline for physiotherapists treating patients with chronic obstructive pulmonary disease based on a systematic review of available evidence.

PURPOSE: Update of a clinical practice guideline for the physiotherapy management of patients with chronic obstructive pulmonary disease supporting the clinical decision-making process. METHODS: A systematic computerized literature search was performed on different modalities for improving physical exercise capacity, reducing exertional dyspnoea, improving airway clearance and encouraging changes in physical activity behaviour. Methodological quality was scored with the PEDro Scale. Scientific conclusions were graded according to the criteria of the; Dutch Evidence Based Guideline Development Platform'. These, together with practical considerations, were used to formulate recommendations for clinical practice. RESULTS: A total of 103 studies were included in the systematic review, consisting of five meta-analyses of randomized controlled trials, 84 randomized controlled trials and 14 uncontrolled studies. Twenty scientific conclusions supported six recommendations on physical exercise training. Nineteen scientific conclusions supported eight recommendations on interventions for reducing dyspnoea. Five scientific conclusions supported seven recommendations concerning treatment modalities to improve mucus clearance, and two scientific conclusions supported two recommendations on strategies for encouraging permanent changes in physical activity behaviour. CONCLUSIONS: Strong recommendations support the use of physical exercise training to improve health-related quality of life and functional exercise capacity. Future research should investigate whether additional interventions for reducing exertional dyspnoea have a place as adjuncts to physical exercise training in selected patients. In addition, treatment of impaired mucus clearance, especially during acute exacerbations, requires further research. With the advance of new technologies for objective measurements of physical activities in daily life more research is needed concerning interventions to initiate and maintain physical activity behaviour change during and after supervised physical exercise training programmes.

Voltage-dependent ion channels on human basophils: do they exist?

The presence of voltage-dependent ion channels (particularly Ca2+ channels) on the surface of 'non excitable' cells such as human basophils is a matter of debate. Indeed, in basophils, Ca2+ entry or mobilization is not sufficient by itself to trigger secretion, although enhanced cytosolic Ca2+ concentration increases it. In order to address this question, we used a two-signal model and we report here experiments which suggest the presence of voltage-dependent structures directly or indirectly linked to membrane Ca2+ pathways. Indeed, it is known that, in the presence of PMA at threshold concentration (1st signal), elevation of cytosolic Ca2+ (2nd signal) induces histamine release. We observed that a depolarizing external solution (high K+) induced a Ca(2+)-dependent release of histamine from PMA-treated human basophils. High K+ alone did not induce histamine release. Although the voltage-sensitive component and the physiological relevance of this mechanism remain to be defined, these results suggest that this voltage-dependent Ca2+ influx in the human basophil could contribute to the up-regulation of histamine release.

Regulation of human basophil activation; the role of Na+ and Ca2+ in IL-3-induced potentiation of IgE-mediated histamine release from human basophils.

The release of mediators from human basophils is strongly enhanced by IL-3. However, the signalling pathways of IL-3 are poorly defined in these cells. Since external Ca2+ and Na+ play important regulating roles in histamine release, the possibility that these cations could be involved in the potentiation by IL-3 of the anti-IgE-induced histamine release from human basophils was considered, and it was observed that: (i) IL-3 dramatically decreased the external Ca2+ requirement for IgE-mediated histamine release. However, histamine release from IL-3-treated basophils became only partially independent of external Ca2+, since addition of EGTA in the external medium abolished the effect of IL-3; (ii) decreasing Na+ influx by lowering external Na+ concentration in isosmotic medium inhibited the potentiating effect of IL-3 on IgE-mediated release; (iii) amiloride, an inhibitor of Na+/Ca2+ and Na+/H+ exchanges, and its derivative, benzamil, more specific for Na+/Ca2+ exchanges, inhibited the release potentiated by IL-3. In contrast, the amiloride derivative 5-(N,N-dimethyl)-amiloride, more specific for Na+/H+ exchanges, slightly increased the IL-3-enhanced release. Thus, the decreased requirement for external Ca2+ and the dependence on external Na+, taken with the effect of the Na+/Ca2+ exchange inhibitors, suggest that Na+/Ca2+ exchanges are involved in the IL-3-induced enhancement of IgE-mediated human basophil histamine release.

Regulation of human basophil activation. III. Impairment of the inhibitory effect of Na+ on IgE-mediated histamine release in patients with allergic rhinitis.

We recently observed that external Na+ inhibited the IgE-dependent human basophil histamine release (HR) in normal subjects. In this article we report differences in the Na+ effect on basophil HR between normal subjects (n = 16) and age matched patients with allergic rhinitis (AR) (n = 18). As expected, in vitro anti-IgE-stimulated basophils from the group with AR released greater amounts of histamine than basophils from the normal group. However, removal of external Na+ (and replacement by N-methyl-D-glucamine) abolished this difference between the two groups. HR in the normal group increased to the same high level as that of the group with AR. By contrast, the release of histamine in the group with AR was not further increased by Na+ removal. Although high releasers were more frequent in the group with AR, the absence of effect after Na+ removal was not due to the high basal release level (in the presence of Na+) because no effect after Na+ removal was also observed with medium releasers. These results strongly suggest that increased basophil HR in populations with AR, and possibly in other allergic populations, is linked to a defect in the inhibitory effect of Na+.

Modifications in tissue histamine levels in mice treated with chronic epicutaneous application of phorbol-12-myristate-13-acetate.

Chronic epicutaneous application of phorbol-12-myristate-13-acetate (PMA) on the right flank of C 57 BL/6 mice induces an inflammatory reaction and a selective increase in skin histamine level at the site of treatment. Tissue histamine levels were not increased at other sites such as contralateral skin, stomach rumen and skeletal muscle, and were decreased in stomach fundus and dorsal skin. These results are in agreement with the hypothesis that mast cells play a significant role in cutaneous inflammation induced by PMA.

Regulation of human basophil activation. II. Histamine release is potentiated by K+ efflux and inhibited by Na+ influx.

Na+ and K+ are the major extra- and intracellular cations, respectively. We have thus studied the role of these ions on human basophil histamine release by modifying their transmembrane gradients or by increasing membrane ion fluxes using ionophores. 1) When external Na+ (reduced to 4 mM) was replaced by the nonpermeating Na+ substitute N-methyl-D-glucamine, the release of histamine was enhanced in 2 mM Ca2+ (from 37.5 +/- 8.0% in 140 mM Na+ to 68.5 +/- 9.1% in low Na+) and became possible in the presence of low Ca2+ (at 1 microM Ca2+: from 0.6 +/- 0.7% in 140 mM Na+ to 36.2 +/- 8.0% in low Na+); moreover, in low Na+, the release of histamine became partly independent on Ca2+ influx. 2) Increasing the Na+ influx with the cation channel-forming gramicidin D inhibited the release of histamine by 33.2 +/- 13.6% (n = 6) in an external Na(+)-dependent manner. 3) Decreasing K+ efflux using K+ channel blockers (4-aminopyridine, quinine, sparteine) inhibited histamine release in a dose-response manner. 4) The K+ ionophore valinomycin, which increases K+ efflux, slightly enhanced IgE-mediated histamine release when used alone, whereas it potentiated the release of histamine from leukocytes previously treated with 4-aminopyridine by 57.0 +/- 18.6% (n = 7). 5) Decreasing K+ efflux by increasing external K+ inhibited IgE-mediated release in a similar manner as Na+ did. The inhibitory effects of Na+ and high K+ were not additive, thus suggesting that both cations inhibited the release by a common mechanism. In conclusion 1) our data evidence that histamine release from human basophils is inhibited by Na+ influx and potentiated by K+ efflux; 2) they suggest that K+ channels are present on the basophil membrane and that Na+ and K+ fluxes act on histamine release most probably via modulation of membrane potential.

Regulation of human basophil activation. IV. Dissociation between cationic dye binding and histamine release: role of Ca2+ ions.

In previous studies we observed that in vitro histamine release from human basophils could be dissociated from the loss of affinity of basophil granules for a cationic dye, toluidine blue. In the present study we further explored the intracellular signals leading to the decrease in toluidine blue positive basophil (TB+) numbers, with or without histamine release. Since Ca2+ mobilization is a crucial event in secretion and particularly in histamine release, we studied the role of Ca2+ in histamine release as compared to TB+ decrease. In the presence of external Ca2+ (2 mM): i) Ca2+ channel antagonists verapamil and nifedipine up to 10 microM were without effect on IgE-mediated histamine release and TB+ decrease; ii) loading of the leucocytes with Quin2 or preincubation with TMP-8, an internal Ca2+ antagonist, significantly inhibited the release of histamine and the decrease of TB+ basophils. In the absence of added external Ca2+:i) histamine release was abolished whereas the decrease of TB+ was not modified, even in the presence of EGTA;ii) the decrease of TB+ could be inhibited by prolonged EGTA preincubation, by Quin2 loading and incubation with TMB-8. We conclude that histamine release requires both external Ca2+ influx and mobilization of internal Ca2+. In contrast, no influx of external Ca2+ is required for TB+ decrease in which, however, internal Ca2+ mobilization appears to play an important role.

Regulation of human basophil activation. I. Dissociation of cationic dye binding from histamine release in activated human basophils.

Human basophil activation was demonstrated by histamine release (HR) and by the decrease of the toluidine blue-positive basophils (TB+). In four experimental systems, TB+ number decreased in the absence of HR (1) in basophils from atopic subjects stimulated by allergen concentrations below the threshold for HR, (2) in basophils sensitized by anti-2,4-dinitrophenyl IgE stimulated by noncovalently linked 2,4-dinitrobenzene sulfonic acid-human serum albumin (also, the threshold for decrease of TB+ required lower concentrations of sensitizing anti-2,4-dinitrophenyl IgE than for HR), (3) in low Ca++ medium, and (4) in the presence of the Na+/H+ exchanger, monensin. These results suggest that (1) there is a lower threshold for TB+ decrease than for HR in allergen concentration, number of membrane IgE molecules, and number of IgE cross-linkings; moreover, external Ca++ requirement is lower for decrease of TB+ than for HR and (2) TB+ decrease reflects either granule exocytosis or, in the absence of HR, biochemical changes (most probably cation exchanges) altering the interaction of the basic dye with the granules. Thus, monitoring decrease in TB+ allows detection of basophil activation in the absence of HR.