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.

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.

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.

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.

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.

[Consensus food hypersensitivity]. / Consensus voedselovergevoeligheid.

Approximately 500 allergists, dermatologists, ENT specialists, paediatricians, general practitioners, pulmonologists and dietitians attended a consensus meeting about food hypersensitivity in September 1990 in Utrecht. At this meeting the clinical manifestations, mechanisms, diagnosis and treatment were discussed. Consensus was reached about the use of terminology such as food allergy (defined as immunologically mediated), intolerance (non-immunological) and aversion (psychological). However consensus was not reached on all points; there was much discussion on the role of food allergy in atopic dermatitis. During the meeting the opinions of the different health care professionals appeared to concur.

[Measurement of selective intestinal permeability using a new, simple sugar absorption test]. / Meting van de selectieve darmpermeabiliteit met een nieuwe, eenvoudige suikerabsorptietest.

OBJECTIVE: To study the clinical value of the sugar absorption test (SAT) as a function test of the selective permeability of the small intestine in various intestinal diseases. DESIGN: Inventory of the results of the SAT in a number of patient groups and controls. SETTING: Beatrix Children's Hospital, Groningen and Rijnstate Hospital, Arnhem. METHODS: The SAT was performed in 51 controls (25 children, 26 adults) and in a number of diseases: (suspected) allergy to cow's milk albumin (30 children), (suspected) coeliac disease (86 children and 35 adults), Crohn's disease (25 patients) with ulcerative colitis (9), and pancreatic insufficiency (31) due to cystic fibrosis or chronic pancreatitis. For the SAT, the fasting patient is given a solution of mannitol (M) and lactulose (L) following which the L/M ratio, as an indicator of the selective intestinal permeability, is determined in 5-hour urine by means of gas chromatography. RESULTS: In cow's milk protein allergy, the L/M ratio showed a statistically significant increase in clinically positive cow's milk provocation results, unlike that in clinically negative results. After pretreatment with cromoglycate this difference decreased to non-significant values. The L/M ratio was increased in active coeliac disease (with villous atrophy), first-degree relatives of coeliac disease patients, Crohn's disease, clinically active ulcerative colitis and pancreatic insufficiency. The L/M ratio was not increased in inactive coeliac disease (with normal villi), suspicion of coeliac disease because of short stature, dystrophy and/or aspecific gastrointestinal symptoms with normal villi, and in clinically inactive ulcerative colitis. CONCLUSION: The selective intestinal permeability can be determined by means of the SAT. This could be an important tool for diagnosis and evaluation of therapy in gastrointestinal disorders.

[Standardized multidisciplinary diagnosis of cow's milk protein allergy in children. Work Group Cow's Milk Protein Allergy of the Groningen Academic Hospital]. / Gestandaardiseerde multidisciplinaire diagnostiek van koemelkeiwitallergie bij kinderen. Werkgroep Koemelkeiwitallergie van het Academisch Ziekenhuis Groningen.

OBJECTIVE: Evaluation of standardized multidisciplinary diagnostic procedures for cow's milk allergy (CMA) in children. DESIGN: Prospective standardized study. SETTING: Beatrix Children's Hospital, Department of Allergology, Dermatology and Dietetics, University Hospital Groningen, the Netherlands. METHODS: From August 1991 until May 1993, 114 children suspected of CMA for the first time were investigated according to the protocol for diagnosis of CMA, together with 23 children, previously diagnosed as CMA, in whom CMA was re-evaluated. Of 114 children with first suspicion of CMA, 66 improved on a cow's milk-free diet. The remaining 48 were excluded from the study because of no improvement on a cow's milk free diet, no diet given, insufficient data or no follow-up. The protocol was evaluated by questionnaire sent to 10 representatives of the departments involved. RESULTS: In 26/66 (39%) children, the diagnosis of CMA was confirmed by cow's milk challenge. The eosinophilic granulocytes were higher (p = 0.04), both IgE RAST and Skin Prick Test (SPT) for cow's milk were more often positive (both p = 0.01) in CMA than in non-CMA. The sensitivity and specificity were 50%-82% for IgE RAST and 60%-84% for the SPT, respectively. Four of the 23 children still had CMA at re-evaluation. In three of them, a SPT was performed, which was positive in all. In 12 of the 19 children, without CMA at re-evaluation, a SPT was performed, which was negative in all. At 1, 2, 3 and 4 years of age 13%, 48%, 74% and 78%, respectively, of the re-evaluation CMA patients had developed tolerance for cow's milk. The use of the protocol was found important by the representatives involved, although some practical difficulties remain. CONCLUSION: A multidisciplinary approach of CMA is possible. Improvement on a cow's milk-free diet by itself is not sufficient to diagnose CMA. Cow's milk challenge is obligatory. Laboratory investigations are of limited value. Re-evaluation of CMA after one year of age is necessary in view of the temporary character of CMA. When the SPT for cow's milk is positive, postponement of re-evaluation may be considered.

Eosinophil-related respiratory disease in aluminium workers.

We carried out a nested case-control study in an aluminium producing plant, using the results of pre-employment examination to identify workers with an increased risk of developing work-related obstructive respiratory symptoms. Cases (n=49) are those who had become unable to work because of work-related respiratory disease. They were compared with 49 matched controls. Pre-employment eosinophil count was strongly related to the occurrence of work-related obstructive respiratory disease. This finding is interesting in the light of a possible pathogenesis.