Combining genomewide association study and lung eQTL analysis provides evidence for novel genes associated with asthma.

BACKGROUND: Genomewide association studies (GWASs) of asthma have identified single-nucleotide polymorphisms (SNPs) that modestly increase the risk for asthma. This could be due to phenotypic heterogeneity of asthma. Bronchial hyperresponsiveness (BHR) is a phenotypic hallmark of asthma. We aim to identify susceptibility genes for asthma combined with BHR and analyse the presence of cis-eQTLs among replicated SNPs. Secondly, we compare the genetic association of SNPs previously associated with (doctor's diagnosed) asthma to our GWAS of asthma with BHR. METHODS: A GWAS was performed in 920 asthmatics with BHR and 980 controls. Top SNPs of our GWAS were analysed in four replication cohorts, and lung cis-eQTL analysis was performed on replicated SNPs. We investigated association of SNPs previously associated with asthma in our data. RESULTS: A total of 368 SNPs were followed up for replication. Six SNPs in genes encoding ABI3BP, NAF1, MICA and the 17q21 locus replicated in one or more cohorts, with one locus (17q21) achieving genomewide significance after meta-analysis. Five of 6 replicated SNPs regulated 35 gene transcripts in whole lung. Eight of 20 asthma-associated SNPs from previous GWAS were significantly associated with asthma and BHR. Three SNPs, in IL-33 and GSDMB, showed larger effect sizes in our data compared to published literature. CONCLUSIONS: Combining GWAS with subsequent lung eQTL analysis revealed disease-associated SNPs regulating lung mRNA expression levels of potential new asthma genes. Adding BHR to the asthma definition does not lead to an overall larger genetic effect size than analysing (doctor's diagnosed) asthma.

Allergology in Europe, the blueprint.

The number of patients with allergic diseases in Europe, and thus relevant demand for health care, is continuously increasing. In this EAACI-UEMS position paper, a rationale is given for the medical specialty of allergology. General practitioners and general paediatricians usually cannot elucidate and address all causative factors. Throughout Europe, therefore, the expertise of allergologists (allergists) is required. In collaboration with other medical professionals, they take care of allergic patients, in private practices or in specialized public centres. A well-structured collaboration between allergists and allergy centres offers the possibility of rapid signalling of new trends developing in the population of allergic patients (e.g., in food and drug allergy). Allergy centres also can perform clinical (and basic) research, teach medical students, future allergists and provide postgraduate training. To prevent that the quality of care in one or several countries within Europe lags behind developments in other countries, the UEMS Section and Board on Allergology together with the European Academy of Allergy and Clinical Immunology advocates the status of a full specialty of allergology in each European country, with a further intention to align their activities (blueprint, curriculum and centre visitation) with the UEMS Section of Paediatrics.

Treatment of hereditary angioedema with nanofiltered C1-esterase inhibitor concentrate (Cetor®): multi-center phase II and III studies to assess pharmacokinetics, clinical efficacy and safety.

From 1997, plasma-derived C1-inhibitor concentrate (Cetor®) has been available to HAE and AAE patients. Recently, a virus reducing 15 nm nanofiltration step has been introduced in the production process. A randomized, double-blind controlled cross-over study was performed to compare the pharmacokinetics (PK) of nanofiltered (C1-INH-NF) with conventional C1-inhibitor (C1-INH). Efficacy and safety were investigated in an open-label, on-demand and a prophylactic study. No differences in pharmacokinetic parameters between C1-INH and C1-INH-NF were found (13 non-symptomatic HAE patients). Both C1-inhibitor products equally increased plasma C4 levels. In the on-demand study, 14 acute angioedema attacks in 8 patients were analyzed. In the prophylactic study, 1 AAE and 5 HAE patients experienced in total 31 attacks during 748 observation days. In total 180,000 units of C1-INH-NF were administered. No product-related adverse events occurred, and no anti-C1-antibodies were induced. Nanofiltration in the production process of C1-inhibitor did not affect the pharmacokinetics, efficacy, and safety.

High prevalence of fractures and osteoporosis in patients with indolent systemic mastocytosis.

BACKGROUND: Indolent systemic mastocytosis (ISM) is a rare disease characterized by accumulation of abnormal mast cells in various tissues, including bone marrow. Symptoms are usually related to release of mast cell mediators. The aims are to establish the prevalence of osteoporotic fractures in ISM and to investigate the association with serum tryptase and the urinary histamine metabolites, methylhistamine (MH), and methylimidazole acetic acid. METHODS: The fracture prevalence in 157 patients (65 men; 92 women), mean age 54 ± 12 years, was assessed by vertebral morphometry and data from patient records, supplemented by a questionnaire. Bone mineral density (BMD) of lumbar spine and femoral neck was measured, and tryptase and histamine metabolites were analysed. RESULTS: We registered 235 lifetime fractures in 154 patients, including 140 osteoporotic (low-energy trauma) fractures, of which 62% were vertebral, 1% hip and 36% other nonvertebral fractures. Osteoporotic fractures and osteoporosis were found in 37% and 28% of the patients, respectively. In men, the prevalence of these osteoporotic manifestations (46% <50 years; 73% ≥50 years) was much higher compared with women (18% <50 years; 58% ≥50 years). Older age, male gender, and higher urinary MH were independently related to the osteoporotic manifestations. CONCLUSIONS: This first publication about prevalence of fractures and osteoporosis in patients with ISM shows that the risk of osteoporotic fractures is high, especially in men. Higher urinary MH levels are associated with a higher risk of osteoporotic manifestations. Routine measurements of BMD and vertebral morphometry are warranted in these patients for early detection of osteoporosis.

Tryptase and histamine metabolites as diagnostic indicators of indolent systemic mastocytosis without skin lesions.

BACKGROUND: Risk indicators of indolent systemic mastocytosis (ISM) in adults with clinical suspicion of ISM without accompanying skin lesions [urticaria pigmentosa (UP)] are lacking. This study aimed at creating a decision tree using clinical characteristics, serum tryptase, and the urinary histamine metabolites methylimidazole acetic acid (MIMA) and methylhistamine (MH) to select patients for bone marrow investigations to diagnose ISM. METHODS: Retrospective data analysis of all adults, in whom bone marrow investigations were performed to diagnose ISM, was carried out. RESULTS: In total, 142 patients were included. SM was absent in all 44 patients with tryptase <10 µg/l, in 45 of 98 (46%) patients with tryptase ≥10 µg/l and in 18 of 52 patients (35%) with tryptase >20 µg/l. Above 43 µg/l, all patients had ISM (n = 11). Male gender, insect venom anaphylaxis as presenting symptom, tryptase, MIMA, and MH were independent ISM predictors. If tryptase was ≥10 µg/l, the diagnostic accuracy of MIMA and MH was high (areas under the ROC curve 0.92). CONCLUSIONS: In suspected patients without UP, the ISM risk is very low (if present at all) if tryptase is <10 µg/l. If tryptase is ≥10 µg/l, this risk depends on MIMA and MH, being low if these are normal, but high if these are elevated. Male gender and insect venom anaphylaxis are additional risk indicators. We recommend refraining from bone marrow examinations in suspected patients without UP if tryptase is <10 µg/l. Our results question the reliability of the minor diagnostic World Health Organization criterion of tryptase >20 µg/l.

Allergy, living and learning: diagnosis and treatment of allergic respiratory diseases in Europe.

BACKGROUND: Allergy Living and Learning (ALL) is a European initiative designed to increase knowledge and understanding of people living with allergies in order to improve respiratory allergy care. OBJECTIVES: To investigate diagnostic and treatment patterns associated with respiratory allergies, patients' perception of their treatment, and restrictions on daily activities. METHODS: Using a telephone-based randomized screening method, we recruited and analyzed 7004 patients (aged 16-60 years) with self-reported respiratory allergic disease from 10 European countries. Patients answered questions assessing their knowledge, experience, and perception of their condition and its treatment. Data analyses were descriptive. RESULTS: The most prevalent conditions were allergic rhinitis (66%) and asthma (26%), and the average duration of the symptoms of respiratory allergy was 14.5 years. Over 30% of patients had never had a specific diagnostic test. About 80% of patients used medication for their respiratory allergy, and 10% of those not receiving treatment had severe symptoms. One-third of patients were not satisfied with their treatment, and two-thirds experienced restrictions in daily activities. Medication was most commonlytaken in the form of tablets and nasal spray. Allergy-specific immunotherapy was received by 16% of patients. Knowledge of specific immunotherapy was low overall and varied widely by country: 30% of patients (country range, 10%-52%) had never heard of this treatment option. CONCLUSIONS: A notable proportion of individuals with respiratory allergy in Europe are underdiagnosed, undertreated, and dissatisfied with their treatment. Addressing these shortcomings may help to optimize respiratory allergy care and, ultimately, quality of life.

Gene expression analysis predicts insect venom anaphylaxis in indolent systemic mastocytosis.

BACKGROUND: Anaphylaxis to insect venom (Hymenoptera) is most severe in patients with mastocytosis and may even lead to death. However, not all patients with mastocytosis suffer from anaphylaxis. The aim of the study was to analyze differences in gene expression between patients with indolent systemic mastocytosis (ISM) and a history of insect venom anaphylaxis (IVA) compared to those patients without a history of anaphylaxis, and to determine the predictive use of gene expression profiling. METHODS: Whole-genome gene expression analysis was performed in peripheral blood cells. RESULTS: Twenty-two adults with ISM were included: 12 with a history of IVA and 10 without a history of anaphylaxis of any kind. Significant differences in single gene expression corrected for multiple testing were found for 104 transcripts (P < 0.05). Gene ontology analysis revealed that the differentially expressed genes were involved in pathways responsible for the development of cancer and focal and cell adhesion suggesting that the expression of genes related to the differentiation state of cells is higher in patients with a history of anaphylaxis. Based on the gene expression profiles, a naïve Bayes prediction model was built identifying patients with IVA. CONCLUSIONS: In ISM, gene expression profiles are different between patients with a history of IVA and those without. These findings might reflect a more pronounced mast cells dysfunction in patients without a history of anaphylaxis. Gene expression profiling might be a useful tool to predict the risk of anaphylaxis on insect venom in patients with ISM. Prospective studies are needed to substantiate any conclusions.

Gene expression profile, pathways, and transcriptional system regulation in indolent systemic mastocytosis.

BACKGROUND: Mastocytosis is an uncommon disease resulting from proliferation of abnormal mast cells infiltrating skin, bone marrow, liver, and other tissues. The aim of this study was to find differences in gene expression in peripheral blood cells of patients with indolent systemic mastocytosis compared to healthy controls. The second aim was to define a specific gene expression profile in patients with mastocytosis. METHODS: Twenty-two patients with indolent systemic mastocytosis and 43 healthy controls were studied. Whole genome gene expression analysis was performed on RNA samples isolated from the peripheral blood. For amplification and labelling of the RNA, the Illumina TotalPrep 96 RNA Amplification Kit was used. Human HT-12_V3_expression arrays were processed. Data analysis was performed using GeneSpring, Genecodis, and Transcriptional System Regulators. RESULTS: Comparison of gene expression between patients and controls revealed a significant difference (P < 0.05 corrected for multiple testing) and the fold change difference >2 in gene expression in 2303 of the 48.794 analysed transcripts. Functional annotation indicated that the main pathways in which the differently expressed genes were involved are ubiquitin-mediated proteolysis, MAPK signalling pathway, pathways in cancer, and Jak-STAT signalling. The expression distributions for both groups did not overlap at all, indicating that many genes are highly differentially expressed in both groups. CONCLUSION: We were able to find abnormalities in gene expression in peripheral blood cells of patients with indolent systemic mastocytosis and to construct a gene expression profile which may be useful in clinical practice to predict the presence of mastocytosis and in further research of novel drugs.

Intravenous voriconazole after toxic oral administration.

In a male patient with rhinocerebral invasive aspergillosis, prolonged high-dosage oral administration of voriconazole led to hepatotoxicity combined with a severe cutaneous reaction while intravenous administration in the same patient did not. High concentrations in the portal blood precipitate liver enzyme abnormalities, and therefore, oral administration of voriconazole may have a hepatotoxicity profile different from that of intravenous (i.v.) administration. Intravenously administered voriconazole might still be an option after oral-voriconazole-induced toxicity has resolved.

Application of a prediction model for work-related sensitisation in bakery workers.

Identification of work-related allergy, particularly work-related asthma, in a (nationwide) medical surveillance programme among bakery workers requires an effective and efficient strategy. Bakers at high risk of having work-related allergy were indentified by use of a questionnaire-based prediction model for work-related sensitisation. The questionnaire was applied among 5,325 participating bakers. Sequential diagnostic investigations were performed only in those with an elevated risk. Performance of the model was evaluated in 674 randomly selected bakers who participated in the medical surveillance programme and the validation study. Clinical investigations were evaluated in the first 73 bakers referred at high risk. Overall 90% of bakers at risk of having asthma could be identified. Individuals at low risk showed 0.3-3.8% work-related respiratory symptoms, medication use or absenteeism. Predicting flour sensitisation by a simple questionnaire and score chart seems more effective at detecting work-related allergy than serology testing followed by clinical investigation in all immunoglobulin E class II-positive individuals. This prediction based stratification procedure appeared effective in detecting work-related allergy among bakers and can accurately be used for periodic examination, especially in small enterprises where delivery of adequate care is difficult. This approach may contribute to cost reduction.

Mastocytosis and insect venom allergy: diagnosis, safety and efficacy of venom immunotherapy.

The most important causative factor for anaphylaxis in mastocytosis are insect stings. The purpose of this review is to analyse the available data concerning prevalence, diagnosis, safety and effectiveness of venom immunotherapy (VIT) in mastocytosis patients. If data were unclear, authors were contacted personally for further information. Quality of evidence (A: high, B: moderate, C: low and D: very low) and strength of recommendation (strong 1 and weak 2) concerning VIT in mastocytosis patients are assessed according to the Grading of Recommendations Assessment, Development and Evaluation and are marked in square brackets. Results of VIT were described in 117 patients to date. The mean rate of side-effects during treatment in studies published so far is 23.9% (7.6% requiring adrenaline) with an overall protection rate of 72%. Based on the review we conclude that (1) mastocytosis patients have a high risk of severe sting reactions in particular to yellow jacket, (2) VIT could be suggested [2] in mastocytosis, (3) probably should be done life long [2], (4) VIT in mastocytosis is accompanied by a higher frequency of side-effects, so (5) special precautions should be taken into account notably during the built up phase of the therapy [2], (6) VIT is able to reduce systemic reactions, but to a lesser extent compared to the general insect venom allergic population [2], so (7) patients should be warned that the efficacy of VIT might be less than optimal and they should continue carrying two adrenaline auto injectors [2].

Polyneuropathy after massive exposure to parathion.

Many organophosphorus compounds, including the organophosphate insecticides, may cause polyneuropathy of delayed onset. An exception is parathion, which has been considered the prototype of nonneurotoxic cholinesterase inhibitors. Nevertheless, we describe a patient with delayed polyneuropathy after suicidal ingestion of parathion.

Induction of low density and up-regulation of CD11b expression of neutrophils and eosinophils by dextran sedimentation and centrifugation.

Neutrophils and eosinophils circulating in an activated state are of low density. However, purification procedures such as dextran sedimentation and centrifugation may influence the density and function of cells. In the present study we have evaluated the effect of dextran sedimentation and subsequent centrifugation on the density and CD11b expression of neutrophils and eosinophils. Direct density separation of whole blood resulted in 17.7 +/- 9.0% low density neutrophils (< 1.090 g/ml) and 8.7 +/- 3.5% low density eosinophils (< 1.093 g/ml). Dextran sedimentation at room temperature prior to density separation yielded 57.8 +/- 14.7% low density neutrophils and 43.2 +/- 8.0% of low density eosinophils. Additional centrifugation after dextran sedimentation resulted in an increase of these numbers to 91.7 +/- 3.1 and 69.8 +/- 11.7% respectively. The density shifts were found with hypertonic as well as isotonic Percoll. Furthermore, it was shown that dextran sedimentation resulted in an increased CD11b expression on neutrophils as well as eosinophils. During subsequent washing by centrifugation, a further increase in CD11b expression was observed together with lactoferrin release. The effects of dextran sedimentation on density and CD11b expression were independent of extracellular calcium. These results indicate that dextran sedimentation induces the release of specific granule compartments with subsequent expression of CD11b, resulting in changes in granulocyte density.

Potentiation of adenylyl cyclase in human peripheral blood mononuclear cells by cell-activating stimuli.

The isoprenaline-induced production of cAMP in human peripheral blood mononuclear cells (PBMC) was potentiated significantly by incubating PBMC with isoprenaline in the presence of phytohaemagglutinin (PHA), Concanavalin A (Con A) or A23187. This potentiation, that proved to be dependent on the concentration of PHA, Con A or A23187, increased the maximal response but did not cause a change in the potency of isoprenaline. Potentiation could not be induced by the phorbol ester phorbol-myristate acetate, suggesting that protein kinase C-dependent pathways are not likely to be involved in potentiation of adenylyl cyclase. Potentiation could be inhibited by chelating extracellular Ca2+ with EGTA and also by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamine, an inhibitor of calmodulin. Potentiation could not be induced by preincubation of PBMC with PHA, suggesting that transient biochemical changes are involved. It was concluded from these results that potentiation in PBMC probably involves the activation of Ca2+/calmodulin-dependent adenylyl cyclase subtypes. Potentiation of the adenylyl cyclase activity could be an important physiological mechanism in vivo preventing cells from becoming "over stimulated".

Allergen-induced recruitment of inflammatory cells in lavage 3 and 24 h after challenge in allergic asthmatic lungs.

To determine whether a link exists between the recruitment of inflammatory cells in the airways on a bronchial and bronchoalveolar level and the development of allergen-induced increase in bronchial hyperresponsiveness after allergen challenge, we used bronchial lavage and bronchoalveolar lavage to assess the airway responses to allergen. Twelve symptomatic atopic asthmatics were studied. In all patients bronchial and bronchoalveolar lavage was performed before, 3 h, and 24 h after allergen challenge. Monoclonal antibodies were used directed against T cells (CD3, CD4, CD8) and the eosinophil cationic protein (EG2). Eight patients showed a dual asthmatic response; four patients showed only an early asthmatic reaction after allergen challenge. Clear differences were found between bronchial and bronchoalveolar lavage. Activated eosinophils (EG2) were significantly increased both at 3 h (p = 0.01) and 24 h (p = 0.005). The number of activated eosinophils was significantly higher in the dual responders. A correlation was observed between the severity of the late asthmatic reaction (LAR) and the number of epithelial cells in the bronchial recovery at 3 h, but not at 24 h, in patients who clinically developed a LAR. No significant changes in the number of CD3, CD4, and CD8 cells 3 and 24 h after the challenge both in the bronchial and bronchoalveolar recovery were observed. We conclude that the number of activated eosinophils in bronchial lavage is associated with the development of the LAR and allergen-induced increase in bronchial hyperresponsiveness.

Determination of N tau-methylimidazoleacetic acid (a histamine metabolite) in urine by gas chromatography using nitrogen-phosphorus detection.

N tau-Methylimidazoleacetic acid, the quantitatively most important metabolite of histamine, was isolated from urine by ion exchange chromatography. After esterification with 2-propanol and extraction, N tao-methylimidazoleacetic acid was analyzed by capillary gas chromatography with nitrogen-phosphorus detection, using N tao-ethylimidazoleacetic acid as internal standard. The synthesis of this internal standard is described. In contrast to the methods hitherto described, this method is appropriate for use in clinical chemical laboratories. Normal 24-h excretion ranged from 8.3 to 18.5 mumol (n = 20). Five patients with mastocytosis, a patient with chronic myelocytic leukemia and a patient after an anaphylactoid reaction on acetylsalicylic acid showed highly elevated values.

National prevalence of respiratory allergic disorders.

BACKGROUND: Many epidemiological studies have assessed the prevalence of respiratory allergic disorders in confined geographical locations. However, no study has yet established nationally prevalence data in a uniform manner representing whole countries and, thus, enabling cross-national comparisons. METHODS: In 10 European countries, screening of random, representative samples of telephone numbers identified the target population aged 16-60. The inclusion criteria were a positive reporting of respiratory allergy to named allergens and, concomitantly, an unassisted description of appropriate symptoms. To obtain a truly representative, national prevalence of each country, the data were weighted against the actual sex and age composition. RESULTS: 31,065 screening interviews were performed. The nationally balanced prevalence varied significantly among the 10 countries (P<0.001) from 11.7% in Spain to 33.6% in Italy. The overall weighted prevalence for Europe was 24.4%. Comparing males and females, overall, the odds-ratio was 0.874 (P<0.001). For age intervals of 16-29, 30-49, and 50-60 years, the odds-ratios for males were 1.104 (P<0.088), 0.827 (P<0.001), and 0.658 (P<0.001), respectively. The prevalence correlated inversely with age. CONCLUSIONS: Respiratory allergic disorders constitute a huge health problem in Europe, and the impact may be increasing as the prevalence is highest among young people.

Living & learning with allergy: a European perception study on respiratory allergic disorders.

BACKGROUND: Knowledge of allergy patients' perception of own disease is inadequate, and understanding of the impact of local environment, including family and health-care system, on patients' management of disease is insufficient. We examined the potential of telephone-based survey techniques for establishing this knowledge in 10 European countries. METHODS: A two-phased questionnaire developed by use of focus groups in seven countries was translated into 10 languages. To ensure that the true values of the populations were restored in randomly selected populations, 75,343 telephone numbers selected for screening represented balanced national distributions of households. RESULTS: Eight thousand two hundred and sixty-eight respiratory allergy sufferers were identified by the telephone screening process. 85.4% accepted to participate in the survey and 89.6% completed both phases comprising 34 questions and rating of 49 statements. Data for each country were weighted in terms of age, sex and the recorded allergy prevalence within age intervals. CONCLUSIONS: The telephone survey technique allowed for establishment of random representative samples, and application of mathematical weighting procedures assured that the true national values were restored in the data set. As all interviews were performed in a standardised manner we conclude that the telephone-based survey methodology enables national representative data set to be established and compared.