A European-Japanese study on peach allergy: IgE to Pru p 7 associates with severity.

BACKGROUND: Pru p 3 and Pru p 7 have been implicated as risk factors for severe peach allergy. This study aimed to establish sensitization patterns to five peach components across Europe and in Japan, to explore their relation to pollen and foods and to predict symptom severity. METHODS: In twelve European (EuroPrevall project) and one Japanese outpatient clinic, a standardized clinical evaluation was conducted in 1231 patients who reported symptoms to peach and/or were sensitized to peach. Specific IgE against Pru p 1, 2, 3, 4 and 7 and against Cup s 7 was measured in 474 of them. Univariable and multivariable Lasso regression was applied to identify combinations of parameters predicting severity. RESULTS: Sensitization to Pru p 3 dominated in Southern Europe but was also quite common in Northern and Central Europe. Sensitization to Pru p 7 was low and variable in the European centers but very dominant in Japan. Severity could be predicted by a model combining age of onset of peach allergy, probable mugwort, Parietaria pollen and latex allergy, and sensitization to Japanese cedar pollen, Pru p 4 and Pru p 7 which resulted in an AUC of 0.73 (95% CI 0.73-0.74). Pru p 3 tended to be a risk factor in South Europe only. CONCLUSIONS: Pru p 7 was confirmed as a significant risk factor for severe peach allergy in Europe and Japan. Combining outcomes from clinical and demographic background with serology resulted in a model that could better predict severity than CRD alone.

Identification of a 62-kDa major allergen from Artemisia pollen as a putative galactose oxidase.

BACKGROUND: Around 20 years ago, a 60- to 70-kDa protein was reported as a major allergen of mugwort (Artemisia vulgaris) pollen. This study was to identify and characterize its molecular properties. METHODS: Sera from 113 Chinese and 20 Dutch Artemisia-allergic/sensitized subjects (and pools thereof) were used to identify the 60- to 70-kDa allergen. Pollen extracts of seven Artemisia species were compared by immunoblotting. Transcriptomics and proteomics (mass spectrometry) of A. annua pollen were used to identify the putative 60- to 70-kDa Artemisia allergen. Both the natural purified and recombinant allergens were evaluated for IgE reactivity by ImmunoCAP. Fourteen Chinese Artemisia-allergic patients were tested intradermally with purified natural allergen. RESULTS: Immunoblots revealed two major bands at 12 and 25 kDa, and a weak band at 70 kDa for all seven Artemisia species. Using a combined transcriptomic and proteomic approach, the high molecular mass allergen in A. annua pollen was shown to be a 62-kDa putative galactose oxidase, with a putative N-glycosylation site. More than 94% of Artemisia pollen-allergic patients had IgE response to this allergen. Although recognition of a nonglycosylated recombinant version was only confirmed in a minority (16%) and at much lower IgE levels, this discrepancy cannot be explained simply by reactivity to the carbohydrate moiety on the natural allergen. Intradermal testing with the natural allergen was positive in five of nine sensitized patients. CONCLUSIONS: The previously reported 60- to 70-kDa allergen of Artemisia pollen is most likely a 62-kDa putative galactose oxidase here designated Art an 7.

Component-resolved diagnosis and beyond: Multivariable regression models to predict severity of hazelnut allergy.

BACKGROUND: Component-resolved diagnosis (CRD) has revealed significant associations between IgE against individual allergens and severity of hazelnut allergy. Less attention has been given to combining them with clinical factors in predicting severity. AIM: To analyze associations between severity and sensitization patterns, patient characteristics and clinical history, and to develop models to improve predictive accuracy. METHODS: Patients reporting hazelnut allergy (n = 423) from 12 European cities were tested for IgE against individual hazelnut allergens. Symptoms (reported and during Double-blind placebo-controlled food challenge [DBPCFC]) were categorized in mild, moderate, and severe. Multiple regression models to predict severity were generated from clinical factors and sensitization patterns (CRD- and extract-based). Odds ratios (ORs) and areas under receiver-operating characteristic (ROC) curves (AUCs) were used to evaluate their predictive value. RESULTS: Cor a 9 and 14 were positively (OR 10.5 and 10.1, respectively), and Cor a 1 negatively (OR 0.14) associated with severe symptoms during DBPCFC, with AUCs of 0.70-073. Combining Cor a 1 and 9 improved this to 0.76. A model using a combination of atopic dermatitis (risk), pollen allergy (protection), IgE against Cor a 14 (risk) and walnut (risk) increased the AUC to 0.91. At 92% sensitivity, the specificity was 76.3%, and the positive and negative predictive values 62.2% and 95.7%, respectively. For reported symptoms, associations and generated models proved to be almost identical but weaker. CONCLUSION: A model combining CRD with clinical background and extract-based serology is superior to CRD alone in assessing the risk of severe reactions to hazelnut, particular in ruling out severe reactions.

Opisthorchis felineus negatively associates with skin test reactivity in Russia-EuroPrevall-International Cooperation study.

BACKGROUND: Most studies on the relationship between helminth infections and atopic disorders have been conducted in (sub)tropical developing countries where exposure to multiple parasites and lifestyle can confound the relationship. We aimed to study the relationship between infection with the fish-borne helminth Opishorchis felineus and specific IgE, skin prick testing, and atopic symptoms in Western Siberia, with lifestyle and hygiene standards of a developed country. METHODS: Schoolchildren aged 7-11 years were sampled from one urban and two rural regions. Skin prick tests (SPT) and specific IgE (sIgE) against food and aeroallergens were measured, and data on allergic symptoms and on demographic and socioeconomic factors were collected by questionnaire. Diagnosis of opisthorchiasis was based on PCR performed on stool samples. RESULTS: Of the 732 children included, 34.9% had opisthorchiasis. The sensitization to any allergen when estimated by positive SPT was 12.8%, while much higher, 24.0%, when measured by sIgE. Atopic symptoms in the past year (flexural eczema and/or rhinoconjunctivitis) were reported in 12.4% of the children. SPT was positively related to flexural eczema and rhinoconjunctivitis, but not to wheezing. Opisthorchiasis showed association with lower SPT response, as well as borderline association with low IgE reactivity to any allergen. However, the effect of opisthorchiasis on SPT response was not mediated by IgE, suggesting that opisthorchiasis influences SPT response through another mechanism. Opisthorchiasis also showed borderline association with lower atopic symptoms. CONCLUSIONS: There is a negative association between a chronic helminth infection and skin prick test reactivity even in a developed country.

A randomized, 5-arm dose finding study with a mite allergoid SCIT in allergic rhinoconjunctivitis patients.

BACKGROUND: The safety and tolerability of a mite allergoid subcutaneous allergen immunotherapy (SCIT) product was previously established. The aim of this study (EudraCT number: 2011-000393-61) was to find the optimally safe and effective allergoid dose by evaluating several dosages in patients with house dust mite (HDM)-induced allergic rhinoconjunctivitis (ARC) using a titrated nasal provocation test (TNPT). METHODS: In total, 290 adult ARC patients (148 females; 142 males) with established HDM allergy and with a positive TNPT were randomized to receive placebo or mite allergoid SCIT 6667, 20 000, 50 000 or 100 000 AUeq/ml for 12 months. Patients were updosed weekly, followed by monthly maintenance dosing. The primary study endpoint comprised the clinical response to TNPT after 12 months of treatment. Secondary endpoints included response to TNPT after 6 months, PNIF measurements, symptom and medication scores during the last 8 weeks of treatment, serum immunoglobulins and safety assessments. RESULTS: After 12 months, a dose-response was observed showing statistically significant improvements in the TNPT with SCIT concentrations of ≥20 000 AUeq/ml, while no significantly different outcomes were reached after 6 months. Specific serum IgG and IgG4 levels were dose dependently increased. In the highest dose group, more treatment-emergent adverse events were observed compared with the lower dose groups. CONCLUSION: In this mite allergoid SCIT dose finding study in HDM-induced ARC, concentrations of ≥20 000 AUeq/ml showed both immunological effects and clinical efficacy in the TNPT compared with placebo. The risk-benefit ratio favours 20 000 AUeq/ml and 50 000 AUeq/ml strengths for further clinical development.

Prevalence of food sensitization and probable food allergy among adults in India: the EuroPrevall INCO study.

BACKGROUND: Data are lacking regarding the prevalence of food sensitization and probable food allergy among general population in India. We report the prevalence of sensitization and probable food allergy to 24 common foods among adults from general population in Karnataka, South India. METHODOLOGY: The study was conducted in two stages: a screening study and a case-control study. A total of 11 791 adults in age group 20-54 were randomly sampled from general population in South India and answered a screening questionnaire. A total of 588 subjects (236 cases and 352 controls) participated in the case-control study involving a detailed questionnaire and specific IgE estimation for 24 common foods. RESULTS: A high level of sensitization (26.5%) was observed for most of the foods in the general population, higher than that observed among adults in Europe, except for those foods that cross-react with birch pollen. Most of the sensitization was observed in subjects who had total IgE above the median IgE level. A high level of cross-reactivity was observed among different pollens and foods and among foods. The prevalence of probable food allergy (self-reports of adverse symptoms after the consumption of food and specific IgE to the same food) was 1.2%, which was mainly accounted for cow's milk (0.5%) and apple (0.5%). CONCLUSION: Very high levels of sensitization were observed for most foods, including those not commonly consumed in the general population. For the levels of sensitization, the prevalence of probable food allergy was low. This disassociation needs to be further explored in future studies.

Peanut allergy is common among hazelnut-sensitized subjects but is not primarily the result of IgE cross-reactivity.

BACKGROUND: Hazelnut and peanut are botanically unrelated foods, but patients are often sensitized and allergic to both, for reasons that are not well understood. METHODS: To investigate molecular cosensitization and cross-reactivity to peanut in hazelnut-sensitized individuals, children (n = 81) and adults (n = 80) were retrospectively selected based on sensitization to hazelnut. IgE to hazelnut extract, Cor a 1, 8, 9 and 14, to peanut extract, Ara h 1, 2, 3, 8 and 9, and to Bet v 1 was determined by ImmunoCAP. Allergy to hazelnut and peanut was established by DBPCFC and/or detailed clinical history. Patients were either tolerant or displayed subjective or objective symptoms to either food. IgE cross-reactivity between hazelnut and peanut storage proteins was assessed by reciprocal ImmunoCAP inhibition experiments. RESULTS: Of the 161 hazelnut-sensitized subjects, 109 (68%) were also sensitized to peanut, and 73 (45%) had clinical expression of allergy to peanut that was not associated with the presence or severity of hazelnut allergy. Instead, it was associated with IgE reactivity to peanut storage proteins, in particular Ara h 2. No cross-reactivity could be detected between Ara h 2 and Cor a 14, and 2 of 13 subjects displayed extensive cross-reactivity between 11S globulins; in plasma of both individuals, Ara h 3 almost completely inhibited IgE binding to Cor a 9. CONCLUSIONS: Peanut allergy is not primarily the result of IgE cross-reactivity to hazelnut storage proteins. IgE to Cor a 14 and Ara h 2 may serve as useful markers of primary sensitization to hazelnut and peanut, respectively.

The prevalence and distribution of food sensitization in European adults.

BACKGROUND: Complaints of 'food allergy' are increasing. Standardized surveys of IgE sensitization to foods are still uncommon and multicountry surveys are rare. We have assessed IgE sensitization to food-associated allergens in different regions of Europe using a common protocol. METHODS: Participants from general populations aged 20-54 years in eight European centres (Zurich, Madrid, Utrecht, Lodz, Sophia, Athens, Reykjavik and Vilnius) were asked whether they had allergic symptoms associated with specific foods. Weighted samples of those with and without allergic symptoms then completed a longer questionnaire and donated serum for IgE analysis by ImmunoCAP for 24 foods, 6 aeroallergens and, by allergen microarray, for 48 individual food proteins. RESULTS: The prevalence of IgE sensitization to foods ranged from 23.6% to 6.6%. The least common IgE sensitizations were to fish (0.2%), milk (0.8%) and egg (0.9%), and the most common were to hazelnut (9.3%), peach (7.9%) and apple (6.5%). The order of prevalence of IgE sensitization against different foods was similar in each centre and correlated with the prevalence of the pollen-associated allergens Bet v 1 and Bet v 2 (r = 0.86). IgE sensitization to plant allergen components unrelated to pollen allergens was more evenly distributed and independent of pollen IgE sensitization (r = -0.10). The most common foods containing allergens not cross-reacting with pollens were sesame, shrimp and hazelnut. DISCUSSION: IgE sensitization to foods is common, but varies widely and is predominantly related to IgE sensitization to pollen allergens. IgE sensitization to food allergens not cross-reacting with pollens is rare and more evenly distributed.

Birch pollen sensitization with cross-reactivity to food allergens predominates in adults with eosinophilic esophagitis.

EoE patients show variable sensitization patterns to food and aeroallergens. The value of allergy testing in adult EoE patients is unclear. Component-resolved diagnosis (CRD) may offer additional insights into sensitization patterns. The aim of this study was to characterize sensitization patterns in adult EoE patients using CRD. Serum from 76 patients (17 female), age 38.6 ± 1.5 years, was analyzed for reactivity to 112 different allergen components using an immuno-solid-phase allergen chip (ISAC). We observed any sensitization in 59 patients (78%), of which 54 patients were polysensitized. Aeroallergen sensitization, mostly against components of grass or tree pollen, or house dust mite, was observed in 74% of the patients. Birch pollen (rBet v 1) sensitization with cross-reactivity to food allergen components was observed in 30 patients (39%). In conclusion, food sensitizations in EoE patients are mainly caused by cross-reactivity to food allergens after primary birch pollen sensitization. Pollen and food sensitizations may cause or maintain esophageal inflammation in EoE patients. CRD provides more insight into sensitization patterns, identifies additional food allergen sensitizations and might be useful to direct dietary therapy in EoE.

The canine sarcoglycan delta gene: BAC clone contig assembly, chromosome assignment and interrogation as a candidate gene for dilated cardiomyopathy in Dobermann dogs.

Dilated cardiomyopathy (DCM) is a common disease of the myocardium recognized in human, dog and experimental animals. Genetic factors are responsible for a large proportion of cases in humans, and 17 genes with DCM causing mutations have been identified. The genetic origin of DCM in the Dobermann dogs has been suggested, but no disease genes have been identified to date. In this paper, we describe the characterization and evaluation of the canine sarcoglycan delta (SGCD), a gene implicated in DCM in human and hamster. Bacterial artificial chromosomes (BACs) containing the canine SGCD gene were isolated with probes for exon 3 and exons 4-8 and were characterized by Southern blot analysis. BAC end sequences were obtained for four BACs. Three of the BACs overlapped and could be ordered relative to each other and the end sequences of all four BACs could be anchored on the preliminary assembly of the dog genome sequence (www. ensembl.org). One of the BACs of the partial contig was localized by fluorescent in situ hybridization to canine chromosome 4q22, in agreement with the dog genome sequence. Two highly informative polymorphic microsatellite markers in intron 7 of the SGCD gene were identified. In 25 DCM-affected and 13 non DCM-affected dogs seven different haplotypes could be distinguished. However, no association between any of the SGCD variants and the disease locus was apparent.

Exclusion of the lim homeodomain gene LHX4 as a candidate gene for pituitary dwarfism in German shepherd dogs.

Pituitary dwarfism in the German shepherd dog is an autosomal recessive inherited abnormality. We tested the hypothesis that a variant of the LIM homeodomain gene LHX4 is responsible for the dwarfism phenotype. To this end, we isolated Bacterial Artificial Chromosome clones for the canine LHX4 gene. Southern blotting experiments showed that the LHX4 gene is a single copy gene in the canine genome. A complex CA-repeat was isolated from the BAC clones and was found to be polymorphic in German shepherd dogs. Genotyping 5 litters in which the dwarfism was segregating showed disconcordance between the inheritance of the dwarfism phenotype and the DNA marker. It is concluded that the LHX4 gene does not play a primary role in the pituitary dwarfism in the German shepherd dogs.

Isolation and characterization of the canine serotonin receptor 1A gene (htr1A).

Although the serotonergic system and htr1A have been studied extensively, little is known about the canine serotonin receptor 1A. We are interested in this receptor in the dog because it is likely to be involved in behavioral disorders such as anxiety. Therefore, we isolated a canine bacterial artificial chromosome (BAC) clone containing htr1A, and, with the help of this clone, the complete canine coding sequence of this gene was determined. Radiation hybrid (RH) mapping showed that htr1A is part of a conserved linkage group also including the survival of motor neuron 1 (smn1) gene. Htr1A is estimated to be located about 7.3 Mb from smn1 on cfa02. In addition, we report a possible breed-specific variant of the gene in four golden retrievers.

A radiation hybrid map of the X-chromosome of the dog (Canis familiaris).

The dog serves as an animal model for several human diseases including X-chromosome diseases. Although the canine X-chromosome is one of the largest chromosomes in the dog, only a few markers have been mapped to it to date. Using a commercially available canine whole genome radiation hybrid (RH) panel we have localized 14 microsatellite markers, 18 genes and 13 STSs on the canine X-chromosome, extending the total number of mapped markers to 45 covering an estimated 830 cR. Out of these 45 markers, seven distinct groups of markers could be established with an average spacing of 18.8 cR(3000) and ten markers remained unlinked. Using FISH analysis, six markers could be mapped physically to the p- or q-arm of the X-chromosome. Combined with the FISH mapping, three RH groups could be assigned to the p-arm and two RH groups to the q-arm. Comparison with the human X-chromosome map revealed conserved synteny up to 234 cR (TIMP1-ALAS2-AR-IL2RG-XIST). We show here that the similarity of the canine and human X-chromosomes is the largest for any mammalian species beyond the primates.

Isolation of DNA markers informative in purebred dog families by genomic representational difference analysis (gRDA).

Genomic Representational Difference Analysis (gRDA) is a subtractive DNA method to clone the differences between two related genomes, called tester and driver. We have evaluated this method to obtain polymorphic DNA markers for pedigree dogs. Amplified size-selected genomic restriction fragments (amplicons) of two dog littermates were repeatedly hybridized to each other in order to remove (subtract) those restriction fragments common to both sibs. Already after two rounds of subtractive hybridization, a clear enrichment of presumably tester-specific restriction fragments was observed, which was even more pronounced after the third round of subtraction. A plasmid library of 3000 recombinant clones was constructed of the second round and of the third round difference product. DNA sequence determination of randomly chosen clones of each difference product showed that approximately 1000 unique clones were obtained in the second-round difference product and approximately 500 in the third-round difference product. About half of the clones identified in the second-round difference product were also present in the third-round difference product. Of the second-round difference product, 39 different gRDA fragments could be identified, of which 21 were tester specific. In the third-round difference product, 22 different gRDA fragments were identified, of which 18 were tester specific. There were 13 fragments in common, resulting in a total of 48 different fragments. In order to establish the localization of these markers, we performed mapping using the dog radiation hybrid panel RHDF5000. Of 39 mapped clones, 29 were mapped to 20 existing RH groups, and 10 remained unlinked. It is concluded that gRDA is suitable to generate DNA markers to track disease genes within lines of pedigree dogs.

Mapping of rabbit chromosome 1 markers generated from a microsatellite-enriched chromosome-specific library.

A genomic DNA library was produced from flow-sorted rabbit chromosome 1 and enriched for fragments containing CA-repeats. Clones containing CA-repeats were identified and primers for amplification of the microsatellite were developed after sequencing the clone. The degree of polymorphism was tested in rabbits from different breeds. This approach identified 12 microsatellite markers which could be used for studying linkage relationships in the progeny of an F(2)-intercross: (AX/JUxIIIVO/JU) F(2), and two backcrosses: (OS/JxX/J)X/J and (WH/JxX/J)X/J. Seven of these markers were mapped on chromosome 1.

Mapping of rabbit microsatellite markers using chromosome-specific libraries.

Recently, rabbit microsatellite markers were developed from a chromosome 1-specific library, and seven new markers were incorporated into the genetic map of the rabbit. We have now developed microsatellite markers from chromosomes 3-, 5-, 6-, 7-, 12-, and 19-specific libraries. Linkage analysis was performed with use of these new markers, five recently physically mapped markers (PMP2, TCRB, ALOX15, MT1, and Sol33), microsatellite markers located in the HBA gene cluster, the MHC region and FABP6 gene, and seven biochemical markers (Es-1, Es-3, Est-2, Est-4, Est-6, Est-X, and HP). This analysis enabled us to verify the specificity of the libraries and to determine the position and orientation of the linkage groups on the chromosomes.