Sensitization to nut allergens in children with food allergy and other atopic diseases - just a coexistence?

Introduction: Allergy to nuts, the most common food allergy in childhood, is considered as a significant health problem. Aim: To investigate sensitization to selected nuts in children with or without atopic allergy. Material and methods: Retrospective analysis involved records of 598 children, diagnosed with food allergy. Laboratory data concerned screening for sensitization to major allergens of hazelnut, peanut and walnut. Results: Approximately 77.8% of children with food allergy presented at least one concomitant atopic disease: allergic rhinitis (52.9%), atopic dermatitis (48%) or asthma (31.4%). Nearly one-third experienced at least one episode of anaphylaxis. The nut-specific antibodies were found in 67% of children. Among them, 56% were sensitized to hazelnut, and 54% to peanut. Sensitization to other nuts was less frequent (< 30%). Only 27% of patients were mono-sensitized, the remaining 73% were co-sensitized to two or three of tested nuts. Noteworthy, the occurrence of sensitization varied among age-related groups, and also depended on clinical diagnosis. In patients with sole food allergy the frequency of sensitization was highest in youngest children, whereas, when accompanied by other atopic disease, it was highest in schoolchildren. In children without food allergy, but with another atopic disease, the prevalence of sensitization was relatively low, without any specific pattern. Conclusions: The analysis of sensitization patterns may help to identify patients with an increased risk, and gives the opportunity to introduce more effective prophylaxis. However, since even the first exposure to nuts may be sufficient to trigger the anaphylaxis, this risk should be considered as a serious issue at any age.

Prevalence of nut allergen sensitization among children in central Poland.

Introduction: The diagnostics of plant-derived food allergy may be challenging. However, the recognition of sensitization patterns in defined populations, especially in children, is clinically relevant as it enables the use of secondary prophylaxis to prevent life-threatening complications. Aim: To investigate the rates and sensitization patterns to nut allergens in children from central Poland. Material and methods: The retrospective assessment concerned data of 598 children diagnosed in a single centre due to suspected food allergy. The analysis included the results of component-based multiparametric assay Allergy Explorer2 (ALEX2). Results: The sensitization to particular nut allergens varied among patients, depending on their age and nut type. The sensitization to any nut was found in 67% of children, whereas sensitization to hazelnut and peanut was the most common (56% and 55% of all children, respectively). Hazelnut sensitization was predominant in every age, and its prevalence increased with age, while peanut sensitization was detected in more than half of individuals from all groups, except for teenagers (44% of cases). Among hazelnut molecules sensitization to Cor a 1.04 was the most prevalent (74% of sensitized children), and for peanut allergens - Ara h 1 (65% of sensitized patients). The simultaneous sensitization to hazelnut, peanut and walnut (two or all of them) was found in almost half of the entire group. Conclusions: Component-based diagnostics enables differentiation between primary and cross-reactive sensitization to nut allergens and detects co-sensitization. The clinical relevance of the latter observation is remarkable as co-sensitization increases the risk of life-threatening reactions even in trace nut contamination.

Diagnostic Value of IP-10 Level in Plasma and Bronchoalveolar Lavage Fluid in Children with Tuberculosis and Other Lung Diseases.

OBJECTIVES: IP-10 has been proposed as a new diagnostic biomarker for Mycobacterium tuberculosis infection (MTBI). However, data on IP-10 concentration in bronchoalveolar lavage fluid (BALF) for pediatric tuberculosis are lacking. AIM: To determine IP-10 levels in unstimulated BALF and plasma in children with and without MTBI. METHODS: IP-10 concentrations in BALF and plasma were measured in children hospitalized with suspected tuberculosis or other respiratory disease and scheduled for bronchoscopy. Thirty-five children were enrolled: 13 with suspected tuberculosis and 22 controls. The association between IP-10 and age was examined. RESULTS: The IP-10 expression was increased in BALF compared to plasma (p = 0.008). We noticed higher BALF IP-10 levels in children with asthma, interstitial lung disease, and lung anomaly than in children with MTBI and other respiratory tract infections, but the differences were statistically insignificant. There was a moderate correlation between plasma and BALF IP-10 concentrations (rs = 0.46, p = 0.018). No correlation between IP-10 level and age was detected. CONCLUSIONS: IP-10 is detectable in unstimulated BALF in children with respiratory diseases, reaches higher concentrations in unstimulated BALF vs plasma, and does not correlate with age. However, it could not discriminate MTBI from other respiratory diseases.

Exhaled breath condensates from healthy children induce cell death of in vitro cultured cells by activation of apoptosis.

INTRODUCTION: Exhaled breath condensate (EBC) is a liquefied air, containing a mixture of non-volatile compounds, reflecting pathophysiological status of the bronchopulmonary system. Therefore, EBC analysis may be useful in diagnostics and monitoring of various respiratory diseases. In previous studies it was found that EBC from asthmatic children contained several regulators of angiogenesis. In vitro experiments with EBCs from children with asthma revealed their weak influence on proliferation of various cells. Surprisingly, EBCs from healthy children led to apoptosis of all tested cells. AIM: To assess the expression of selected apoptosis-related proteins in human and murine cells exposed to EBC from healthy children. MATERIAL AND METHODS: EBCs from healthy children were added to cultures of murine endothelial cells (C166) or human lung fibroblasts (HLF) to induce their apoptosis. For proteome analysis the apoptosis pathway-specific protein microarrays were used. RESULTS: The homogenates from EBC-treated C166 cells contained low amounts of Hsp27, which correlated with their fast death. Contrary to C166, the lysates from EBC-treated fibroblasts displayed increased amounts of Hsp27, which correlated with delayed HLF response to the induction of apoptosis. Except for increased caspase-3 in EBC-treated HLF, none of the other apoptosis regulators revealed any significant changes in that analysis. CONCLUSIONS: The screening of apoptosis pathways with microarray technology allowed identification of two molecules, Hsp27 and caspase-3, involved in cellular response to EBC. However, the factor responsible for induction of the cytotoxic effect of EBC from healthy children still remains unknown.

Influence of exhaled breath condensates from children with asthma on endothelial cells cultured in vitro. Do we really know everything about our breath condensate?

INTRODUCTION: Asthma-associated remodelling involves subepithelial fibrosis and increased vascularization of the bronchial wall. The latter may be associated with excessive production of several angiogenesis regulators which may be found in exhaled breath condensates (EBCs) collected from children with asthma. AIM: To assess the influence of EBC samples of asthmatic children and healthy controls on in vitro cultures of normal human lung microvascular endothelial cells (HLMVEC) and murine endothelial cell line (C-166). Moreover, the proteomic profile of cytokines in EBC samples was analysed. MATERIAL AND METHODS: Breath condensates collected from children with mild asthma (n = 10) and from healthy controls (n = 10) were used for experiments. Colorimetric tetrazolium salt reduction assay was used to evaluate the effect of EBCs on HLMVEC and C-166 cell lines. Furthermore, influence of EBCs on C-166 cell line was assessed using Annexin V-binding assay. The cytokine screening of EBC samples was performed using a proteome microarray system. RESULTS: The EBCs from patients with asthma revealed a weak inhibitory influence on human and murine endothelial cells. Surprisingly, EBCs from healthy children led to cell death, mainly by the induction of apoptosis. There were no statistically significant differences in the cytokine profile between EBC samples from children with asthma and healthy controls. CONCLUSIONS: Our preliminary report shows for the first time that the incubation of EBCs from healthy controls induced apoptosis in endothelial cells. The detailed mechanism responsible for this action remains unknown and requires further research.

Polymorphic Variants 279R and 668Q Augment Activity of Matrix Metalloproteinase-9 in Breath Condensates of Children with Asthma.

Matrix metalloproteinase (MMP)-9 is involved in pathophysiology of asthma, mainly asthma-associated airway remodeling. Exhaled breath condensates (EBC) of asthmatics contain increased amounts of MMP-9 with activity higher, than in healthy controls. The increased activity of MMP-9 may originate from its excessive production and activation, but may also result from variations in MMP-9 structure, which are determined by single nucleotide polymorphisms (SNPs). In this pilot study we aimed to assess the possible influence of two functional MMP-9 polymorphisms, Q279R and R668Q, on enzymatic activity of MMP-9, measured in EBC of asthmatic children. The concentration and activity of MMP-9 were analyzed in EBC of 20 children with allergic asthma using specific standard ELISA and novel immunoenzymatic activity assay. The SNPs of MMP-9 were assessed using real-time PCR-based genotyping test. We have found that MMP-9 concentration in breath condensates of children with stable asthma was slightly higher in ELISA, than in the activity assay. Moreover, these results and activity-to-amount ratio have revealed some relationship with a presence of specific 279R and/or 668Q MMP-9 gene variants. Our observation suggests that at least in some patients MMP-9 hyperactivity may result from genetic predisposition, determined by polymorphic variants of MMP-9 gene. Moreover, it supports previous reports postulating significance of MMP-9 in pathogenesis of asthma. However, this issue still requires further studies.

Increased angiogenic factors in exhaled breath condensate of children with severe asthma - New markers of disease progression?

Asthma progression is associated with airway remodeling and neo-vascularization. However, role of angiogenesis in these changes remains unclear and available data still incomplete. In this pilot study we verify usefulness of proteome profiler assay in screening of angiogenesis-related factors in exhaled breath condensates (EBC) collected from children with asthma. EBC samples from patients with mild or severe asthma and healthy controls were tested using protein array. In EBC samples from patients with severe asthma we have found large quantities of several angiogenesis regulators, including thrombospondin (TSP)-1, angiogenin, dipeptidyl peptidase (DPP) IV, matrix metalloproteinase (MMP)-9 and its inhibitor TIMP-1. Small amounts of angiopoietin (Ang)-2 and vascular endothelial growth factor (VEGF) were also present. In contrast to them, in EBC from mild asthma group we have detected TSP-1 and small quantities of Ang-2. EBC samples from healthy controls contained only TSP-1. Our preliminary report suggests that, since increased amounts of angiogenesis-related factors in EBC seem to correlate with asthma severity, they may be considered as convenient non-invasive markers of disease progression. However, further research is necessary.

Inhaled corticosteroids do not reduce initial high activity of matrix metalloproteinase (MMP)-9 in exhaled breath condensates of children with asthma exacerbation: a proof of concept study.

Inhaled corticosteroids (ICS) are the key component of asthma treatment. However, it is unclear whether they could control the activity and level of matrix metalloproteinase (MMP)-9, which is an important factor in asthma-associated inflammation and airway remodeling. Therefore, the aim of this proof of concept study was to analyze the influence of increased doses of ICS on MMP-9 in exhaled breath condensates (EBC) of patients with allergic asthma exacerbation. Apart from MMP-9, the assessment concerned selected inflammation markers - exhaled nitric oxide (eNO) and cytokines (IL-8 and TNF). The study involved a small group (n = 4) of individuals with asthma exacerbation. The intervention concerned increased doses of ICS with ß-mimetics for 4 weeks. In addition to clinical evaluation, eNO measurements and EBC collections were done before and after 4 weeks of intense ICS treatment. The biochemical assessment of EBC concerned MMP-9, IL-8 and TNF. The data were compared to results of healthy controls (n = 6). The initial levels of eNO, MMP-9 and TNF in EBC were higher in the asthma group than in controls. In all subjects IL-8 levels were below the detection limit. After 4 weeks of ICS treatment in all patients we observed improvement of clinical and laboratory parameters. Interestingly, despite reduction of eNO and TNF, the activity of MMP-9/EBC remained on the initial level. Practical relevance of our results is limited by a small group. Nevertheless, our data suggest that ICS, although sufficient to control symptoms and inflammatory markers, may be ineffective to reduce MMP-9/EBC activity in asthma exacerbation and, possibly, airway remodeling.

Airway Remodeling in Chronic Obstructive Pulmonary Disease and Asthma: the Role of Matrix Metalloproteinase-9.

Chronic obstructive pulmonary disease (COPD) and asthma are both associated with airflow restriction and progressive remodeling, which affect the respiratory tract. Among various biological factors involved in the pathomechanisms of both diseases, proteolytic enzymes--matrix metalloproteinases (MMPs)--play an important role, especially MMP-9. In this review, the authors discuss the current topics of research concerning the possible role of MMP-9 in both mentioned diseases. They include the analysis of protein levels, nucleotide polymorphisms of MMP-9 gene and their possible correlation with asthma and COPD. Finally, the authors refer to the studies on MMP-9 inhibition as a new perspective for increasing the effectiveness of treatment in asthma and COPD.

Prolonged Treatment with Inhaled Corticosteroids does not Normalize High Activity of Matrix Metalloproteinase-9 in Exhaled Breath Condensates of Children with Asthma.

The airway remodeling in asthma is associated with increased amount of matrix metalloproteinase (MMP)-9. High levels of MMP-9 were found in mucosal biopsies, sputum and in exhaled breath condensates (EBC) of asthma patients. However, there are no data concerning real in vivo activity. Inhaled corticosteroids are effective in asthma control, but it is unclear, whether they only attenuate inflammation, or also protect against progressive remodeling of respiratory tract. Therefore, the aim of the study was to assess the amount and activity of MMP-9 in context of pro-inflammatory cytokines (IL-6, IL-8 and tumor necrosis factor, TNF), measured in EBC of asthma-suffering children, treated with inhaled steroids. The study involved 27 children with asthma, continuously treated with inhaled fluticasone propionate, and 22 healthy controls. In addition to routine clinical screening, the selected cytokines in EBC were analyzed using Ultrasensitive ELISA, whereas activity of MMP-9 was assessed using a novel immunozymography method. Despite chronic treatment with inhaled steroids mean MMP-9/EBC activity in asthma group was significantly higher than in healthy controls. Moreover, high MMP-9/EBC in asthma-suffering children significantly correlated with IgE serum levels. The IL-6 and IL-8 concentration was below the detection limit in all EBC samples. TNF/EBC levels were similar in both, asthma and healthy children. We hypothesize that MMP-9 hyperactivity in asthma may be closely related to high IgE serum levels. Our results suggest that inhaled steroids may be ineffective to prevent asthma-associated airway remodeling. Finally, we emphasize the necessity of further research focused on MMP-9 inhibition in asthma treatment.

Nitric oxide, IL-6 and IL-13 are increased in the exhaled breath condensates of children with allergic rhinitis.

AIM: To evaluate nitric oxide and interleukin (IL)-6, IL-8 and IL-13 in the exhaled breath of children with allergic rhinitis (AR), before and after intranasal allergen exposure. METHODS: A total of 49 children with AR ­ comprising 20 who also had episodic asthma (AR+A) and 29 without asthma (AR) ­ were compared with 34 healthy controls. Nitric oxide concentrations in exhaled air (eNO) and IL-6, IL-8 and IL-13 in exhaled breath condensates (EBC) were measured in winter, outside the natural allergen exposure season, before and after an intranasal allergen challenge. RESULTS: The mean concentrations of eNO, IL-6 and IL-13 were significantly higher in the two AR groups. The concentration of IL-8 was below the assay detection limit in all EBC samples. The intranasal allergen challenge increased IL-13/EBC levels in both AR groups, but did not influence mean concentrations of eNO, IL-6 or IL-8. No challenge-related changes in IL-13/EBC were observed in the allergen-exposed controls or placebo-exposed children. CONCLUSION: Despite local application, the intranasal allergen challenge increased IL-13/EBC concentration in the AR children. As EBC reflects the status of lower airway segments, our observation may support the 'united airways' hypothesis, suggesting a functional link between the upper and lower airways.

Increased cys-leukotrienes in exhaled breath condensate and decrease of PNIF after intranasal allergen challenge support the recognition of allergic rhinitis in children.

Exhaled breath condensate (EBC) contains various mediators of inflammation. Since their concentrations correlate with severity of inflammatory response, EBC assessment allows non-invasive detection of various respiratory tract diseases and enables monitoring of their progression or treatment effectiveness. In this study, authors evaluate the usefulness of cysteinyl leukotrienes (cysLT) measurement in EBC, as non-invasive diagnostic markers of allergic rhinitis in children. It has been found that the assessment of cysLT in EBC, when performed out of the natural allergen exposure, can discriminate between healthy and allergic rhinitis individuals, with sensitivity 87.8% and specificity 76.4%, at the threshold level 39.05 pg/ml. The change of peak nasal inspiratory flow (ΔPNIF), measured before and after intranasal allergen challenge allowed recognition of healthy/allergic rhinitis-suffering individuals with sensitivity 76.8% and specificity 78.6%, at the threshold level of -3.2 l/min. When ΔPNIF assessment was combined with the measurement of cysLT in EBC, the sensitivity of such diagnostic approach reached 100% and its specificity increased up to 84.6%. The proposed algorithm was found to sufficiently discriminate between allergic rhinitis-suffering and healthy children, however, its clinical usefulness especially in young children requires further studies.

[Adaptation of Vespide Allergy Quality of Life Questionnaire for Polish children with venom allergy and for their parents]. / Adaptacja kwestionariuszy jakosci zycia dla dzieci z alergia na jad owadów oraz dla ich rodziców.

Aim of the study was to assess validity and reliability of adaptations of VQLQ for Polish children with Hymenoptera venom allergy and their parents. Sample under study consisted of 73 children aged up to 14 years, who were treated with specific venom immunotherapy (VIT) in 2008 in centres conducting this procedure in Poland, and their parents. Theoretical validity of the scales was assessed with exploratory factor analysis using principal component analysis method. Reliability of the scales was assessed in terms of internal consistency with Cronbach alpha coefficient. Results of analysis showed that both scales measure 4 dimension of quality of life and reliability of scales measuring particular dimensions is at least acceptable in case of scale for children, and high in case of scale for parents. Both adapted scales are valid and reliable tools measuring quality of life in children with Hymenoptera venom allergy and their parents' quality of life in the face of child's allergy.

[Health-related quality of life in children with Hymenoptera venom allergy treated with VIT and quality of life of their parents]. / Jakosc zycia warunkowana stanem zdrowia dzieci z alergia na jad owadów blonkoskrzydlych leczonych VIT oraz jakosc zycia ich rodziców.

UNLABELLED: Hymenoptera venom allergy, although rare in children, by its potential fatalities, leads to many psychosocial consequences, influencing quality of life of children and their parents. Aim of this paper is the estimation of health-related quality of life of venom allergic children treated with specific immunotherapy, and their parents. Assessment of factors influencing health-related quality of life levels was also performed. MATERIAL AND METHODS: Sample under study consisted of 73 children: mean age 10.6, SD 2, 4, treated because of Hymenoptera venom allergy in 5 clinical allergy centers in Poland. Data was collected using VQLQ questionnaire adapted for children and their parents. Determinants of quality of life were assessed with multivariate linear and logistic regression models. Analysis were done with SPSS 15 for Windows package. RESULTS: Girls reported higher level of anxiety than boys (B = 0.47; 95% CI = (0.01; 0.94)). Level of caution in children increased along with increase of their anxiety against re-sting (B = 0.49; 95% CI = (0.27; 0.71)). Level of anxiety of children who were under treatment from 6 months to 2 years was lower than level of anxiety of parents of children treated shorter than 6 months (B = -1.21; 95% CI = (-2.16; -0.25)). The lowest level of caution was reported by parents of children aged 10 year or less (B = -0.86; 95% CI = (-1.67; -0.05)), while the highest was reported by parents of children aged 11 years (B = 0.86; 95% CI = (0.20; 1.53)) in comparison to parents of children aged 12 years or more. Parents' caution increased along with increase of their anxiety (B = 0.61; 95% CI = (0.40; 0.83)). Higher level of limitations was imposed by parents of children treated with rush or ultra rush method, in comparison to parents of children treated with conventional method (B = 1.27; 95% CI = (0.21; 2.33)). Levels of quality of life in children and their parents were strongly dependent in the same dimensions. CONCLUSIONS: 1. Levels of quality of life in particular dimension in children is related to level of the same dimension in parents. 2. Age of children influenced level of caution of their parents. 3. Treatment duration influenced level of anxiety of parents. 4. Safety feeling acquired by parents at the beginning of treatment improves their quality of life in all dimensions.

Risk of allergy development correlates with IL-4 receptor expression on newborns' monocytes and Th lymphocytes.

BACKGROUND: IL-4 receptor (IL-4R) overexpression on immunoregulatory/effector cells was found in allergic patients. However, its role in allergy development remains unclear. The aim of this study was to assess the correlation between IL-4R expression and allergy development within the first year of life. MATERIAL/METHODS: IL-4R expression on monocytes and Th lymphocytes of 43 newborns was analyzed using flow cytometry. Plasma levels of IL-4, -12 and IFN-gamma were also measured using ELISA. The same parameters were assessed one year later. Furthermore, clinical evaluation was performed every three months for one year. RESULTS: Mean IL-4R expression on monocytes and Th lymphocytes did not differ at birth. After one year it increased on Th-lymphocytes and decreased on monocytes. However, among 10 children with severe atopy during the observation period, 8 displayed IL-4R above the mean value for the group on both monocytes and Th cells at birth as well as one year later. No correlation was found between IL-4 or IFN-gamma and IL-4R expression at birth. After one year, significant IL-4 increases and IFN-gamma decreases were observed which correlated with IL-4R expression. IL-4R expression on the newborns' monocytes correlated negatively with IL-12 plasma level; however, it was statistically significant only in the children developing allergy. Moreover, only in these patients was a significant decrease in IL-12 found after one year. CONCLUSIONS: IL-4R-dependent over-signaling in newborns' monocytes and Th lymphocytes could contribute to Th1/Th2 imbalance. IL-4R overexpression on newborns' monocytes and lymphocytes could be an early risk marker of allergy development.