Eosinophil cationic protein and interleukin-8 levels in bronchial lavage fluid from children with asthma and infantile wheeze.

It has been shown previously that airway eosinophils characterize childhood asthma and neutrophils contribute to the pathophysiology of both infantile wheezing and asthma. Therefore, eosinophil cationic protein (ECP) and interleukin-8 (IL-8) levels in bronchoalveolar lavage fluid (BALF) from asthmatics (n = 16) and infantile wheezers (n = 30) were analyzed as markers of eosinophil- and neutrophil-mediated inflammation. To aid the interpretation, a control group of children (n = 10) with no lower airway pathology were included. Disease severity was assessed by using a symptom score. Surprisingly, no significant difference was found in IL-8 or ECP levels among asthma, infantile wheeze, and control groups. Asthma was characterized by: a correlation between ECP levels and eosinophil counts (r = 0.618, p = 0.014); a correlation between neutrophil number and IL-8 levels (r = 0.747, p = 0.002); and increasing IL-8 levels with symptom score (p = 0.03). In infantile wheezers, IL-8 levels were poorly related to neutrophil number but were significantly increased when neutrophils were > 10%. Although detectable levels were found in all but one symptomatic infant, IL-8 concentrations did not reflect the symptom score in infantile wheeze. ECP was unexpectedly correlated to neutrophil percentages (Rho = 0.832, p = 0.001), and a threshold of ECP>20 ng/ml was associated with persistent symptoms in these infantile wheezers. Hence, in accordance with BALF cellularity, activation of eosinophils was suggested by raised levels of ECP in childhood asthma, but not in infantile wheeze. Neutrophil-mediated inflammation appeared to better reflect the severity of asthma than that of infantile wheeze. Although its meaning remains to be elucidated, ECP was suggested to be a helpful indicator of persistent infantile wheeze. However, its utility as a marker predicting ongoing asthma remains to be established.

Soluble intercellular adhesion molecule-1 (sICAM-1) and interferon-gamma in bronchoalveolar lavage fluid from children with airway diseases.

We have previously described that in bronchoalveolar lavage fluid (BALF), eosinophils characterize asthma and neutrophils are more prominent in infantile wheeze. In this study, we hypothesized that intercellular adhesion molecule 1 (ICAM-1) and interferon-gamma (IFN-gamma) would have a role in promoting migration of both cell types into the airway. To investigate this, we measured soluble (s) ICAM-1 in 68 BALFs from infants and young children with various respiratory problems. Children with asthma were characterized by significantly raised sICAM compared with those with chronic cough without wheeze (p = 0.05) or control subjects with no lower airway pathology (p = 0.045). The levels correlated with disease severity (evaluated with a symptom score) and with lymphocyte numbers. IFN-gamma levels were also raised in children with asthma compared with those with chronic cough (p = 0.05), but there was no correlation with disease activity. Infantile wheeze was characterized by a linear correlation between sICAM-1 and IFN-gamma (r = 0.55; p = 0.002). sICAM-1 levels in infantile wheeze correlated with the severity of the disease and lymphocyte numbers. IFN-gamma levels were elevated in the wheezers treated with inhaled steroids compared with untreated infants (p = 0.03). Although sICAM-1 levels were increased in those with severe cough, no characteristic inflammatory profile was found in the group with chronic cough. Our study suggests that ICAM-1 and IFN-gamma play a role in the activity of the inflammatory process in asthma in childhood and possibly in some infant wheezers, in whom IFN-gamma may be one of the factors increasing the expression of ICAM-1. The role of IFN-gamma, a T helper-1 cytokine, in children with asthma remains to be fully understood.

Bronchoalveolar cell profiles in children with asthma, infantile wheeze, chronic cough, or cystic fibrosis.

Differential cell counts of bronchoalveolar lavage (BAL) have been reported in normal children but few data on cellular profiles in bronchial diseases in childhood are available. We determined the BAL cell profiles of 72 children divided into 5 groups: asthma (n = 14), chronic cough (n = 12), infantile wheeze (n = 26), cystic fibrosis (n = 10), and control (n = 10). The highest total cell, eosinophil, and neutrophil counts were found in children with cystic fibrosis. The cell profile of children with chronic cough was similar to that of control children. Asthma and infantile wheeze were characterized by a high median ratio of eosinophils (3%) and neutrophils (12%), respectively. In both diseases, epithelial shedding was suggested by an elevated epithelial cell count, 13.5 and 12%, respectively. Lymphocyte subset analysis showed a higher proportion of CD8 cells (58 versus 40%) and therefore a lower CD4/CD8 ratio (0.266 versus 0. 455) in children with asthma compared with infantile wheezers (p = 0. 02). Irrespective of the presence or absence of radiological abnormalities, a proportion of neutrophils > 10%, was found in one-third of the children with asthma and in half of the infantile wheezers, and was related to symptom severity. We suggest that neutrophil-mediated inflammation, with or without bacterial infection, may contribute to symptoms of asthma in childhood. Chronic cough, however, is not associated with the cell profiles suggestive of asthma and in isolation should not be treated with prophylactic antiasthma drugs.

Serological characteristics of peanut allergy.

BACKGROUND: Peanut is the most common cause of severe or fatal food-associated anaphylaxis. Studies indicate that peanut extracts contain many allergenic proteins. The identification of major and minor allergenic components is necessary for standardization of experimental and diagnostic extracts. OBJECTIVE: To identify further major and minor allergenic components of peanut extract using a large population of peanut allergics, and to relate serological findings to clinical parameters. METHODS: The crude peanut extract was fractionated by fast protein liquid chromatography and the IgE binding proteins identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis followed by western blotting. Serum from 89 peanut allergics with a positive history of peanut allergy and elevated specific IgE and control serum from four atopic and four non-atopic, non-peanut allergics were used. RESULTS: Nineteen peanut proteins were found to bind IgE from peanut allergic sera. Over 70% of subjects reacted to protein bands of 63 and 17 kDa (consistent with Ara h 1 and Ara h 2, respectively), confirming the importance of these two proteins as major allergens. A high proportion of patient sera also bound proteins at 15, 10, 30, 18 and 51 kDa in decreasing order. The percentage of cases with sensitivity to a 15 kDa protein was found to be higher in patient groups with severe reactions to peanut. CONCLUSION: This study highlights the diversity of peanut allergens. Diagnostic extracts containing a high proportion of the 15 kDa component may aid in diagnosis.

HLA class II DRB1, DQB1 and DPB1 genotypic associations with peanut allergy: evidence from a family-based and case-control study.

BACKGROUND: Peanut is one of the most common foods provoking allergic reactions and is the most frequent cause of fatal and near-fatal food-induced anaphylaxis. However, as yet, little is known of the genetic and immunological mechanisms which underly peanut allergy. OBJECTIVE: Based on findings in other allergic diseases, we have investigated whether particular human leucocyte antigen (HLA) class II genetic polymorphisms contribute to the development of peanut allergy. METHODS: All individuals from 37 families each containing one or more peanut allergic individuals, plus nine unrelated patients (161 individuals in total, defined as the study group) were typed for the HLA class II DRB1, DQB1 and DPB1 loci, by PCR-based techniques. Genotype frequencies were compared with those found in 293 unrelated controls. RESULTS: Four class II genotypes (DRB1*08 (13.7% vs 4.8%; Pc = 0.026), DRB1*08/12 tyr 16 (22.4% vs 8.2%; Pc = 0.021), DQB1*04 (12.2% vs 2.7%; Pc = 0.0026) and DPB1*0301 (49.1 vs 22.5%; Pc = 0.00062)) were present at a significantly higher frequency in the study group compared with controls. Three of these genotypes (DRB1*08 (18.0%; Pc = 0.027), DRB1*08/12 tyr16 (24.0%; Pc = 0.029) and DQB1*04 (16.7%; Pc = 0.0029)) were also significantly increased in peanut allergic individuals compared with controls. In addition, two genotypes (DPB1*0101 and 0201) were significantly decreased in frequency in the overall study group, but not specifically in peanut allergic individuals. CONCLUSION: While other genetic factors may be important, results from this study indicate that HLA class II genetic polymorphism may play a role in determining susceptibility to peanut allergy.

Peanut allergic subjects' peripheral blood mononuclear cell proliferative responses to crude peanut protein.

BACKGROUND: Peanut allergy is characterized by a high frequency of severe and occasionally fatal reactions. OBJECTIVE: To determine if there are features of the in vitro cellular response that may account for the observed severity of peanut allergy. METHODS: Skin-prick testing (SPT), RAST assay of serum peanut-specific and total IgE and mixed peripheral blood mononuclear cells (PBMC) proliferative responses to crude peanut protein were measured in 44 peanut allergics with varying severity of clinical reactions. PBMC responses of 13 non-peanut allergic controls (six atopic) were also studied. RESULTS: Subjects' PBMCs proliferated more than controls', even without stimulation. Subjects' PBMC proliferative responses did not correlate with clinical severity, SPT weal size or peanut-specific IgE levels. Controls' PBMCs did not respond to peanut. There was no correlation between PBMC response and time since last reaction to peanut. Subjects' PBMCs responded more than controls, to mitogen as well as allergen. Proliferation increased with increasing concentration of peanut protein (P < 0.0001). CONCLUSION: PBMCs of peanut allergics demonstrate a dose-dependent response to peanut which does not correlate with clinical severity, SPT reaction or levels of peanut specific IgE. The response is antigen-specific. Peanut protein is not mitogenic and is not acting as a superantigen. While there are non-specific differences in the PBMC responses of peanut allergic individuals compared with atopic and non-atopic controls, these differences do not explain the unique severity of peanut allergy.

Randomised, double blind, crossover challenge study of allergenicity of peanut oils in subjects allergic to peanuts.

OBJECTIVE: To determine the in vivo allergenicity of two grades of peanut oil for a large group of subjects with proved allergy to peanuts. DESIGN: Double blind, crossover food challenge with crude peanut oil and refined peanut oil. SETTING: Dedicated clinical investigation unit in a university hospital. SUBJECTS: 60 subjects allergic to peanuts; allergy was confirmed by challenge tests. OUTCOME MEASURES: Allergic reaction to the tested peanut oils. RESULTS: None of the 60 subjects reacted to the refined oil; six (10%) reacted to the crude oil. Supervised peanut challenge caused considerably less severe reactions than subjects had reported previously. CONCLUSIONS: Crude peanut oil caused allergic reactions in 10% of allergic subjects studied and should continue to be avoided. Refined peanut oil did not pose a risk to any of the subjects. It would be reasonable to recommend a change in labelling to distinguish refined from crude peanut oil.

Application of an electrophoretic methodology for the identification of low molecular weight proteins in foods.

Identification and characterisation of food proteins are core features of food allergy research. Current methods used to identify allergenic proteins in food have insufficient resolution and are unable to detect low molecular weight proteins. In this study we report the use of a simple SDS-PAGE method which allows resolution of small proteins. We have subsequently applied this method and reported presence of low molecular weight proteins in a range of hydrolysed milk formulae (Nutramigen, Pregistimil, Alfare, Pepti-Junior and Pregomin), and crude peanut protein extract. The molecular weight distribution for the peanut extract and the hydrolysates ranged between 5-200kDa and 2-17kDa respectively.

Peanut allergy in relation to heredity, maternal diet, and other atopic diseases: results of a questionnaire survey, skin prick testing, and food challenges.

OBJECTIVES: To determine rates of other atopic manifestations in people with peanut allergy and the prevalence of such allergy in their families. DESIGN: A survey of people with self reported peanut allergy and people referred by their general practitioner for suspected peanut allergy; survey and skin testing of 50 children with reported peanut allergy and their available first degree relatives. SUBJECTS: 622 adults and children with reported, suspected, or known peanut allergy. MAIN OUTCOME MEASURES: Prevalence of peanut allergy and other allergies in the families of people with peanut allergy. RESULTS: 622 valid completed questionnaires were returned out of the 833 questionnaires dispatched (74.7%). All forms of atopy were both more common in successive generations (P < 0.0001) and more common in maternal than paternal relatives (P < 0.0001). Peanut allergy was reported by 0.1% (3/2409) of grandparents, 0.6% (7/1213) of aunts and uncles, 1.6% (19/1218) of parents, and 6.9% (42/610) of siblings. Consumption of peanuts while pregnant or breast feeding was more common among mothers of probands aged < or = 5 years than mothers of probands aged > 5 years (P < 0.001). Age of onset correlated inversely with year of birth (r = -0.6, P < 0.001). Skin prick testing of 50 children with reported peanut allergy and their families: 7 probands (14%) had a negative result for peanut. Peanut allergy was refuted by food challenge in all those tested (5/7). No parent and 13% (5/39) of siblings had a positive result on skin prick testing for peanut. Two of these siblings had negative challenge with peanuts. The prevalence of peanut allergy in siblings is therefore 3/39 (7%). CONCLUSIONS: Peanut allergy is more common in siblings of people with peanut allergy than in the parents or the general population. Its apparently increasing prevalence may reflect a general increase of atopy, which is inherited more commonly from the mother. Peanut allergy is presenting earlier in life, possibly reflecting increased consumption of peanut by pregnant and nursing mothers.

Faecal interleukin-8 and tumour necrosis factor-alpha concentrations in cystic fibrosis.

Interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-alpha) concentrations were measured in faecal samples from nine patients with cystic fibrosis and nine healthy age matched controls. The patients were assessed with Shwachman score, apparent energy absorption, pancreatic enzyme dosage, simple spirometry, and presence of pseudomonal colonisation. Median (range) wet stool IL-8 and TNF-alpha concentrations in patients were 32,113 pg/g (21,656-178,128) and 3187 pg/g (368-17,611) respectively, compared with < 43.5 pg (IL-8)/g (< 22-4079) and 99 pg (TNF-alpha)/g (< 0.26-231) in controls. IL-8 concentration was negatively correlated with Shwachman score (r = -0.79) and pancreatic enzyme dosage (r = -0.77), but not with energy absorption. Seven patients were mature enough to cooperate with spirometry. Their IL-8 concentrations correlated with percentage predicted forced expiratory volume in one second (r = -0.78). IL-8 concentration was greater in four patients with, than five without, established pseudomonal colonisation: median difference 134,583 pg/g. TNF-alpha concentration was not correlated with measures of disease severity. Faecal IL-8 concentration might reflect the severity of pulmonary inflammation in cystic fibrosis and could provide an easily obtainable marker of disease activity.

Desensitisation of neutrophil responses by systemic interleukin 8 in cystic fibrosis.

BACKGROUND: Inflammation associated with neutrophil infiltration is a commonly observed feature of children with cystic fibrosis. Production of the major neutrophil chemotactic cytokine interleukin 8 (IL-8) is potentially of great importance in the pathology of cystic fibrosis. Concentrations of IL-8 in both sputum and bronchoalveolar lavage fluid have been found to be higher in children with cystic fibrosis than in controls. The IL-8 induced chemotactic response and numbers of IL-8 receptors on peripheral neutrophils obtained from children with cystic fibrosis have been compared with a control group of children. METHODS: Cells were isolated from 18 patients with cystic fibrosis (aged 4-20 years) and 13 controls (aged 5-12 years) by dextran centrifugation followed by separation on Lymphoprep. Chemotaxis was assayed using multiwell microchemotaxis chambers and 5 microns polycarbonate filters. Filters were fixed and stained with Haema-Gurr for counting. Results were expressed as numbers of neutrophils per high power field (HPF). RESULTS: At the optimum concentration (1 x 10(-8) mol/l) the number of cells migrating were similar for controls (150 (12)/HPF) and for the cystic fibrosis group (140 (14)/HPF)). At lower concentrations the numbers of neutrophils migrating were lower for the cystic fibrosis group. Scatchard analysis of 125I-labelled IL-8 binding revealed lower numbers of receptors on neutrophils from patients with cystic fibrosis (22,000 per cell) than from controls (75,000 per cell). CONCLUSIONS: Reduced responsiveness to IL-8 of neutrophils from patients with cystic fibrosis is associated with receptor desensitisation as a result of exposure to high systemic levels of IL-8.

Passive smoking amongst asthmatic children. Questionnaire or objective assessment?

An increasing number of studies have implicated passive smoking as a definite threat to non-smokers' health. Self-reports of smoking status may not always be reliable, particularly in situations where the smoker feels under pressure to give up smoking. In this study questionnaire and salivary cotinine, an objective measure of nicotine exposure, were studied in asthmatic and age-matched control children. We have consequently developed a sensitive assay for cotinine using high performance liquid chromatography (HPLC) to quantitate environmental tobacco smoke (ETS) exposure in a group of 5- to 7-year-old asthmatic and control children. We chose to use mixed unstimulated saliva collected by absorption into dental rolls in the mouth for 5 min. Our modified extraction procedure was highly reproducible with a > 90% retrieval rate of cotinine from spiked saliva. The parents were asked to fill in a questionnaire on atmospheric pollutants to obtain an estimate of declared ETS exposure in the home. Results showed that 31% of the asthmatic patients were exposed to ETS, according to the parents, but by HPLC 69% had been so exposed (n = 19). From the control group the figures were 40% and 50% of patients, respectively. Therefore, an objective assessment is essential as ETS is more ubiquitous than is apparent from the questionnaire alone. Finally, in this small number of individuals our objective assessment demonstrates that ETS exposure is more prevalent in asthmatic children (69%) than age-matched controls (51%).

Incorporation of alphaxalone into different types of liposomes.

The poor solubility of steroid anaesthetics in water has been a serious drawback in the development of clinically acceptable intravenous formulations. The use of Cremophor EL to solubilize steroids such as alphaxalone led to unacceptable hypersensitivity reactions and consequent withdrawal of this anaesthetic. In principle, liposomes can act as a safe solvent for the intravenous administration of alphaxalone. We report the incorporation of [14C]acetylated alphaxalone in both multilamellar vesicles and stable plurilamellar vesicles prepared from a range of amphiphiles including synthetic polyhydroxyl lipids. For both types of preparations, addition of cholesterol to phosphatidylcholine-based lipids caused an increase in encapsulation efficiency. Maximum encapsulation was achieved with the stable plurilamellar vesicle preparation of 1-stearyl-2-myristylglycerate-3, N-methylglucamine:cholesterol:egg phosphatidylcholine (78%). The rate of efflux of this anaesthetic from a range of liposomes was measured in serum. The highest rate (85% after 30 min) was observed with an equimolar egg phosphatidylcholine:cholesterol stable plurilamellar vesicle preparation. From these studies it can be concluded that liposomes offer a suitable alternative for intravenous delivery of steroidal anaesthetics.

Interleukin-8 concentrations are elevated in bronchoalveolar lavage, sputum, and sera of children with cystic fibrosis.

Concurrent pulmonary inflammation and neutrophil infiltration are characteristic of children with cystic fibrosis (CF). The production of the major neutrophil chemotactic cytokine IL-8 by alveolar macrophages or other cells could be of great importance in the pathology of acute lung disease, but its role in the persistent lung inflammation characteristic of CF has not been evaluated. In this study, we have measured, by ELISA, the concentration of IL-8 in sputum, bronchoalveolar lavage, and sera specimens obtained from children with CF. For comparison, IL-8 in bronchoalveolar lavage obtained from asthmatic patients and from non-CF children with or without lung infection and in sera from age-matched controls was measured. High levels of IL-8 were measured in sputum (mean = 2952 pM) and in bronchoalveolar lavage (mean = 6624 pM) from CF patients. In both cases, there was a significant correlation between clinical status (Schwachman score) and IL-8 levels. This was not true for IL-8 levels measured in sera, which nevertheless were significantly higher in CF patients (p = 0.0001) than in normal controls in the over-10-y age group.

In vitro allergenicity of cows' milk substitutes.

There are numerous alternatives to cows' milk formula for allergic children. We have investigated the allergenicity of several of these using RAST and RAST inhibition on serum from 16 patients with a known history of cows' milk protein intolerance (CMPI) and 16 atopic controls. A RAST grade of > or = 3 for cows' milk was present in all those with CMPI, whilst all the controls gave RAST of < or = 1. Modified cows' milk formula, goats' infant formula, sheep and goats' milk produced similar results to cows' milk. Only two patients had RAST > or = 3 for soya milk and the soy/beef hydrolysate gave positive results in only three patients. One had positive RAST to Nutramigen and two to Pregestimil. Of the whey hydrolysates investigated, Pepti-junior gave seven positive RASTs whilst we were unable to bind Alfare to the sepharose in sufficient quantities to interpret the results which were negative in all cases. RAST inhibition data on pooled sera from the same patients agreed with the RAST results. The inhibition curves showed high inhibition with goats', sheep, modified cows' milk formula and the casein formula, AL110 (50%). Soy and soy/beef hydrolysate showed a much lower inhibition pattern. Casein hydrolysates showed low inhibition while the whey hydrolysate produced higher inhibition. We have shown that despite claims of low allergenicity, some of these alternative formulae are antigenically recognized in vitro by some cows' milk intolerant patients.