Validation of an ELISA method for quantification of the major Timothy grass pollen allergen Phl p 5a (BSP090).

Progress towards standardisation of allergen products has been made in recent years. Nevertheless, no standardised test method to quantify the allergen content of grass pollen allergen products is available at present. One aim of the BSP090 project was to validate a quantitative assay for a major Timothy grass (Phleum pratense) pollen allergen, Phl p 5. Qualification of a candidate ELISA system was performed with regard to range, robustness and cross-reactivity in preliminary studies. The assay specifically detected Phl p 5 with a quantification range from 3.9 ng/mL to 62.5 ng/mL. Suitability to quantify recombinant and natural Phl p 5 was further assessed in a collaborative study including 14 laboratories in Europe and the USA. Precision and accuracy of the assay was satisfactory with 93% of calculated Phl p 5 concentrations and 100% of total recoveries being within the ± 30% acceptance range. Similar results were obtained for spike recoveries, with exclusion of the lowest concentration spike, showing spike recoveries exceeding the acceptance range for six laboratories. Inter-assay (repeatability) and inter-laboratory (reproducibility) variability were satisfactory, in the format used in the present study. Robustness towards different statistical methods for data analysis was demonstrated. In conclusion, the assay can easily be established in routine testing and results of the preliminary testing and collaborative study support the proposal of the assessed Phl p 5-specific ELISA as a European Pharmacopoeia general method.

False-Positive Results of Serological Tests for Allergy in Alcoholic Patients.

BACKGROUND AND OBJECTIVE: Alcohol consumption is associated with enhanced TH2 immune responses. Objective: To investigate the frequency of false-positive results in serological tests for allergy in alcoholic patients. METHODS: A total of 138 alcoholic patients consecutively admitted to hospital underwent a panel of allergy tests that included serum total IgE, a multiallergen IgE test (UniCAP Phadiatop), and skin prick tests to relevant aeroallergens in the area, which were considered the standard reference for atopy. In selected cases with positive specific IgE (sIgE) to cross-reactive carbohydrate determinants (CCDs) on ImmunoCAP, we determined sIgE to hymenoptera venom components (ADVIA Centaur) and a microarray of 103 allergen components (ISAC). RESULTS: Increased serum total IgE (>170 IU/mL) was observed in 59/110 (54%) of nonatopic (skin prick test-negative) patients. The result of the multiallergen IgE test was positive in 46 nonatopic patients (42%). This finding was closely associated with high serum concentrations of total IgE and sIgE to CCDs. The vast majority of patients with positive CCD-sIgE showed positivity to glycosylated plant and hymenoptera allergen components on ISAC and ADVIA Centaur. Only 1 out of 26 patients with positive sIgE to CCD and hymenoptera venom developed honeybee venom allergy after a median follow-up of 166 months. Correlations between measurements of sIgE to CCD markers on ImmunoCAP, ADVIA Centaur, and ISAC were imperfect. CONCLUSIONS: Serological tests for allergy should be interpreted with caution in alcoholic patients, who frequently have increased levels of total IgE and CCD-sIgE and subsequent positivity of sIgE to glycosylated allergen components, irrespective of the method used.

Saturated salt solution: a further step to a formaldehyde-free embalming method for veterinary gross anatomy.

In the field of veterinary anatomy, most of the specimens used in practical sessions are perfused with fixatives. Thus, they can be used for a longer time, reducing the number of animals for educational purposes. Formalin is the most commonly used fixative, consisting of a 37% formaldehyde solution. However, formaldehyde is a powerful irritant of the eyes and airways and is considered carcinogenic, causing nasopharyngeal cancer in exposed workers and professionals. In the present study, we explored an alternative method to avoid the use of formaldehyde in specimens used for gross anatomy practical sessions. We propose an inexpensive, non-toxic fixative that is available worldwide, such as sea salt. This method consists of a continuous perfusion of saturated salt solution for a period of 6-8 h, enabling drainage of the solution to avoid a weight increase of the specimen, and allowing salt to be retained in the tissue. The method is based on recirculation of the saturated salt solution instead of maceration. Perfused specimens retained their natural consistency and joint mobility, with no blood, resembling a piece of meat from the slaughterhouse. They could be used immediately without a maceration period, or stored in the fridge until use and then kept in a bath of saturated salt solution for future conservation. In the case of the former, no refrigeration was needed. The specimens did not have an irritating or offensive smell, and could be used for long sessions (several hours per day) and stored for long periods. However, the blood vessels used for perfusion determine the results: a less invasive approach (through common carotid arteries) gave good preservation of the musculoskeletal system, whereas more invasive access to cannulate the abdominal aorta and vena cava caudalis was required to achieve better preservation of the viscera. In conclusion, we propose that perfusion followed by immersion in a saturated salt solution is a good alternative method for the preservation of specimens used in the practical teaching of gross veterinary anatomy. It is a very simple and inexpensive technique, and is much healthier for users than traditional formalin. Moreover, specimens can be preserved for prolonged periods, and maintain a similar appearance and consistency to fresh material.

Determination of hepatitis B phenotype using biochemical and serological markers.

The aim of this study was to assess the validity of categorization of chronic hepatitis B viral infection into stages or phases based upon measures of disease activity and viral load, assuming these phenotypes will be useful for prognostication and determining the need for antiviral therapy. We assessed the phenotype of hepatitis B of 1,390 adult participants enrolled in the Hepatitis B Research Network Cohort Study, using a computer algorithm. Only 4% were immune tolerant, while 35% had chronic hepatitis B (18% e antigen positive and 17% e antigen negative) while 23% were inactive carriers. Strikingly, 38% of participants did not fit clearly into any one of these groups and were considered indeterminant. The largest subset of indeterminants had elevated serum aminotransferases with low levels of HBV DNA (less than 10,000 iu/mL). Subsequent determination of hepatitis B phenotype on the next available laboratory tests showed that 64% remained indeterminant. These findings call into question the validity of conventional staging of hepatitis B, in large part because of the substantial proportion of patients who do not fit readily into one of the usual stages or phases. Further studies are needed of the indeterminant category of chronic hepatitis B viral infection, including assessments of whether patients in this group are perhaps in transition to another phase or if they are a distinct phenotype with a need to assess liver disease severity and need for antiviral therapy. (ClinicalTrials.gov identifier NCT01263587).

Masting behaviour in a Mediterranean pine tree alters seed predator selection on reproductive output.

Context-dependency in species interactions is widespread and can produce concomitant patterns of context-dependent selection. Masting (synchronous production of large seed crops at irregular intervals by a plant population) has been shown to reduce seed predation through satiation (reduction in rates of seed predation with increasing seed cone output) and thus represents an important source of context-dependency in plant-animal interactions. However, the evolutionary consequences of such dynamics are not well understood. Here we describe masting behaviour in a Mediterranean model pine species (Pinus pinaster) and present a test of the effects of masting on selection by seed predators on reproductive output. We predicted that masting, by enhancing seed predator satiation, could in turn strengthen positive selection by seed predators for larger cone output. For this we collected six-year data (spanning one mast year and five non-mast years) on seed cone production and seed cone predation rates in a forest genetic trial composed by 116 P. pinaster genotypes. Following our prediction, we found stronger seed predator satiation during the masting year, which in turn led to stronger seed predator selection for increased cone production relative to non-masting years. These findings provide evidence that masting can alter the evolutionary outcome of plant-seed predator interactions. More broadly, our findings highlight that changes in consumer responses to resource abundance represent a widespread mechanism for predicting and understanding context dependency in plant-consumer evolutionary dynamics.

Population biology of the European woodwasp, Sirex noctilio, in Galicia, Spain.

Sirex noctilio Fabricius (Hymenoptera, Siricidae) is rare and rarely studied where it is native in Eurasia, but is a widespread pest of pines in the Southern Hemisphere. Here we report on the abundance, basic biology, host use patterns and natural enemies of native S. noctilio in Galicia, Spain. Most trees attacked by S. noctilio failed to produce any adult progeny: >90% of emergences came from <20% of the attacked trees. The highest reproduction was in Pinus pinaster, followed by Pinus sylvestris and Pinus radiata. The proportions of S. noctilio requiring 1, 2 or 3 years for development were 0.72: 0.24: 0.04. Delayed development could be an adaptation to avoid parasitic nematodes, which sterilized 41.5% adults with one year generation time but only 19% of adults with 2 years generation time. Hymenoptera parasitoids accounted for 20% mortality. Sex ratios were male biased at 1: 2.9. Body size and fecundity were highly variable and lower than previously reported from the Southern Hemisphere. On attacked trees, there were 5-20 attacks per standard log (18 dm2), with usually 1-3 drills per attack. Attack densities and drills per attack were higher in trees that subsequently died. The production of S. noctilio per log was positively related to total attacks, and negatively related to: (1) attack density, (2) incidence of blue stain from Ophiostoma fungi and (3) frequency of lesions in plant tissue around points of attack. A preliminary life table for S. noctilio in Galicia estimated effects on potential population growth rate from (in decreasing order of importance) host suitability, unequal sex ratio, parasitic nematodes and Hymenoptera parasitoids.

Allergen expression in the European house dust mite Dermatophagoides pteronyssinus throughout development and response to environmental conditions.

House dust mites are a major source of allergy worldwide. While diagnosis and treatment based on mite extracts have remarkably advanced, little information exists on the expression of allergens in mites. We have studied gene expression of eight Dermatophagoides pteronyssinus (Trouessart) (Acari: Pyroglyphidae) allergens (Der p 1, 2, 3, 4, 5, 7, 10 and 21). All allergens showed higher transcription in nymphs compared with larvae or adults, with the only exception of Der p 10. The transcription of Der p 4 and Der p 10, together with the transcription and protein ratios Der p 1 to Der p 2, were higher in males than in females. One-week exposure of mite cultures to 16 or 35 °C (versus 24 °C) or low RH (44% versus 76%) significantly influenced the allergen gene transcription profile. Our results demonstrate that allergen expression is quantitatively and/or qualitatively influenced by mite development and sex, as well as by the environment. We suggest that monitoring allergen gene expression may be a useful tool to assist the optimization of mite cultures in the production of standardized allergenic extracts for clinical use.

Mite species identification in the production of allergenic extracts for clinical use and in environmental samples by ribosomal DNA amplification.

The identification of allergy-causing mites is conventionally based on morphological characters. However, molecular taxonomy using ribosomal DNA (rDNA) may be particularly useful in the analysis of mite cultures and purified mite fractions in the production of allergenic extracts. Full-length internal transcribed spacers (ITS1 and ITS2) were obtained from Dermatophagoides farinae, Dermatophagoides pteronyssinus, Dermatophagoides microceras and Euroglyphus maynei (Astigmata: Pyroglyphidae), Glycyphagus domesticus and Lepidoglyphus destructor (Astigmata: Glycyphagidae), Tyrophagus fanetzhangorum, Tyrophagus putrescentiae, Tyrophagus longior, Tyrophagus neiswanderi, Acarus farris and Acarus siro (Astigmata: Acaridae), and Blomia tropicalis (Astigmata: Echymopodidae), using mite-specific primers. Polymerase chain reaction (PCR) products were digested with HpaII and RsaI restriction enzymes in order to produce species-specific PCR restricted fragment length polymorphism (RFLP) profiles. A semi-nested re-amplification step was introduced before the RFLP in order to apply the method to environmental samples. Results demonstrate that rDNA sequences can be used for the unambiguous identification of mite species. The PCR-RFLP system allows the identification of species in purified mite fractions when the availability of intact adult mite bodies for morphological identification is limited. This reliable and straightforward PCR-RFLP system and the rDNA sequences obtained can be of use in the identification of allergy-causing mite species.

Analysis of mite allergic patients in a diverse territory by improved diagnostic tools.

BACKGROUND: There are few studies comparing the sensitization with mite allergens from different mite species which could potentially be the cause of allergy. OBJECTIVE: To improve the diagnosis of mite allergic patients from a diverse territory in which D. pteronyssinus/D. farinae mites together with storage mites could be present in the environment. METHODS: Four hundred and seventy-seven patients (both children and adults) from different regions, covering the main mite prevalent areas of Spain, were recruited. sIgE to eight allergens was measured together with SPT to whole mite extracts, level of mite allergen exposure, and specific IgG(4) . BAT and CAST was performed in a subgroup of patients. RESULTS: D. pteronyssinus and L. destructor were more prevalent in Atlantic areas, whereas D. farinae predominate in Mediterranean areas. About 90% of patients were sensitized to group 1 and/or group 2 allergens. Group 2 was the most prevalent, and the IgE response/intensity of sensitization in BAT was higher. sIgE to Der p 2/Der f 2 was almost fully cross-reactive, but no cross-reactivity was detected with Lep d 2. Group 1 allergens were also cross-reactive, but in some patients a species-specific response was observed. sIgE to Lep d 2 was associated with SPT results to storage mites. Sensitization to Der p 1 was more frequent in children, whereas Lep d 2 sensitization was more frequent in adults. A higher ratio IgE/IgG(4) to Der p 2 was associated with the presence of allergic asthma. CONCLUSION: An improved diagnosis algorithm has been established. Group 2 allergens seem to have a leading role in mite allergy, but as group 1 sensitization could be species-specific in some patients and its prevalence is higher in children, an adequate balance on major mite species and major allergens must be consider in the design of mite allergy vaccines.

Profilin as an aeroallergen by means of conjunctival allergen challenge with purified date palm profilin.

BACKGROUND: The role of profilin as a food allergen is well established, but little research has been done about its ability to elicit respiratory disease. Profilin is considered more of a confounding allergen on skin testing with whole pollen extracts than other airborne allergens. Our aim was to find out whether or not profilin can cause symptoms in sensitized individuals, which would be compatible with its role as an airborne allergen. METHODS: We performed conjunctival allergen challenges with date palm profilin in a series of consecutive pollen-allergic patients with rhino-conjunctivitis, divided in two groups: profilin sensitized (n = 17) and not sensitized (n = 14), who served as controls. We investigated the possible association between profilin sensitization and profilin allergy in these groups of patients. RESULTS: None of the patients from the not profilin-sensitized group had a positive result in conjunctival allergen challenges. In contrast, 65% of profilin sensitized patients had a positive conjunctival allergen challenge and were considered allergic to profilin. We found a significant statistical association between being profilin allergic and being profilin sensitized (χ(2) = 10.39, p < 0.005). CONCLUSIONS: Profilin seems to work as an aeroallergen in a significant proportion of profilin-sensitized patients. This might explain the uselessness of conjunctival challenges with whole pollen extracts to disclose genuine sensitization. In the future, the possibility of quantifying this allergen in pollen immunotherapy vaccines should be considered.

Follow-up of venom immunotherapy (VIT) based on conventional techniques and monitoring of immunoglobulin E to individual venom allergens.

OBJECTIVES: To assess the efficacy of venom immunotherapy (VIT) and monitor changes in in vivo and in vitro test results after 5 years of treatment and subsequent follow-up. To study the profile of immunoglobulin (Ig) E to individual allergens prior to treatment and 1 year afterwards. METHODS: We studied 562 patients with hymenoptera venom allergy (438 to bee, 124 to wasp), all of whom underwent immunotherapy with Apis or Vespula extract. The patients were followed up using conventional in vivo and in vitro tests, and in 51 cases, specific IgE against the main hymenoptera allergens was measured before starting and after 1 year of treatment. RESULTS: Of the 387 patients who completed VLT, 130 sensitized to Apis and 68 to Vespula suffered spontaneous re-stings during treatment. Of these, 123 (94.6%) did not suffer any reaction and 64 (94.1%) suffered only a local reaction. Sixty-two patients sensitized to Apis and 14 sensitized to Vespula suffered spontaneous re-stings after stopping treatment. Only 3 patients suffered a systemic reaction (grade I Müller). At the end of treatment, the results of skin tests and specific IgE to whole extract improved significantly. Reductions in IgE to the main allergens were observed after 1 year of treatment (median differences in Ves v 5, -238.0, P = .0425; and in Api m 1, -183.0, P = .0024). CONCLUSION: The high rate of spontaneous re-stings shows that efficacy is maintained for years after completing treatment in a real-world setting. Determination of IgE to individual venom allergens may offer new perspectives in the diagnosis and follow-up of these patients.

Component-resolved diagnosis of pollen allergy based on skin testing with profilin, polcalcin and lipid transfer protein pan-allergens.

BACKGROUND: Allergy diagnosis needs to be improved in patients suffering from pollen polysensitization due to the existence of possible confounding factors in this type of patients. OBJECTIVE: To evaluate new diagnostic strategies by comparing skin responses to pan-allergens and conventional allergenic extracts with specific IgE (sIgE) to purified allergen molecules. METHODS: One thousand three hundred and twenty-nine pollen-allergic patients were diagnosed by a combination of an in vitro method with a panel of 13 purified allergens, including major allergens and pan-allergens, using a high-capacity screening technology (ADVIA-Centaur) and skin prick test (SPT) to pan-allergens and conventional extracts. RESULTS: There was a high concordance (kappa index) between in vitro (sIgE to major allergens) and in vivo (SPT to conventional extracts) methods in patients who were not sensitized to pan-allergens, but SPT with conventional extracts failed to diagnose patients with sensitization to pan-allergens. In patients who were simultaneously sensitized to polcalcins and profilins, there was a duplication both in the number of sensitizations to major allergens and in the years of disease evolution. There was a statistical association between sensitization to profilins and/or lipid transfer proteins and food allergy (P<0.0001). CONCLUSION: The novel diagnostic strategy has proven to be a valuable tool in daily clinical practice. Introduction of routine SPT to pan-allergens is a simple and feasible way of improving diagnostic efficacy. Patients sensitized to pan-allergens should be tested by an adequate panel of allergenic molecules in order to identify the allergens that are responsible for the allergic disease.

Allergenicity and cross-reactivity of pine pollen.

BACKGROUND: Pine pollen has long been considered a non-allergenic pollen. The large size of the grain and its low levels of proteins are the main reasons invoked to explain this low allergenicity. The aim of this study was to describe the main allergenic bands of Pinus radiata (PR) and its cross-reactivity with other pine species, other conifers and grass pollen. METHODS: Sixty-five pine-pollen-allergic patients (51% also sensitized to grass pollen) were studied. Skin prick tests (SPT) to a battery of allergens including PR, Pinus pinea, Pinus sylvestris, Pinus nigra and Cupressus sempervirens pollens and specific IgE determination to PR and Pinus strobus were performed. IgE-immunoblotting to a PR extract and other pine pollens was also carried out. UniCAP inhibition and immunoblotting inhibition studies were performed to assess the cross-reactivity between different pollens. RESULTS: The SPTs were positive with all the pine pollen extracts tested in 69% of the patients. Specific IgE was positive to PR or P. strobus in 77% of the patients, and to Lolium perenne in 51%. Nine different allergenic bands were detected. The two main allergens were a 42 kDa band recognized by 85% of the patients and a band of approximately 6-8 kDa recognized by 40%. A high degree of cross-reactivity was observed between different pine pollen species, but not between pines and C. sempervirens pollen. A partial cross-reactivity could be seen between pine and grass pollens only in patients also sensitized to L. perenne. CONCLUSIONS: Pine pollen should be considered as a potential allergenic pollen especially where this pollen is abundant. The detection of a high number of patients that were monosensitized to pine pollen suggests the possibility of treating these patients with specific immunotherapy.

Component-resolved in vitro diagnosis in peach-allergic patients.

BACKGROUND: The in vitro diagnosis of pollen-related food allergy presents low specificity and reproducibility with many conventional extracts. This can be improved using natural purified allergens, recombinant purified allergens, or both. OBJECTIVE: We compared specific immunoglobulin (Ig) E determination (slgE), the basophil activation test (BAT), the histamine release test (HRT), and the cellular allergen stimulation test (CAST) using natural and recombinant allergens in the diagnosis of peach allergy. METHODS: Thirty-two peach allergic patients were studied. Skin prick tests were performed with commercial peach and extract with Mal d 1, nPru p 3, and profilin (nPho d 2). slgE, BAT, CAST, and HRT were determined using rPru p 3, rMal d 3, rBet v 1, rMal d 1, and rMal d 4. RESULTS: Agreement between the techniques was good with all the allergens, except HRT with rMal d 1 and rMal d 4. With rPru p 3, slgE, CAST, BAT, and HRT showed sensitivity values of 88%, 81%, 72%, and 69% and specificity values of 100%, 93%, 97%, and 83%, respectively. In patients with systemic symptoms or contact urticaria, the values were 100%, 85%, 81%, and 81%. In patients with oral allergy syndrome, sensitivity to profilins or homologues of Bet v 1 was detected in 100% of the cases by all the techniques, except by HRT with rMal d 1, which detected 66% of the cases. CONCLUSIONS: The use of single allergens in the in vitro diagnosis of peach allergy by specific IgE determination, BAT, and CAST offers high specificity and sensitivity, with better results than the HRT.

Allergy to quail's egg without allergy to chicken's egg. case report.

INTRODUCTION: We present a case of quail's egg allergy without allergy to chicken's egg. CASE: Girl of 10.5 years old who presents anaphylactic reaction after she ate an uncooked quail's egg. She had eaten boiled quail's egg before. She eats chicken's eggs without clinical symptoms. METHODS: We made a prick to chicken's egg and prick-by-prick to uncooked quail's and raw chicken's egg. We determined specific IgE to chicken's egg; electrophoresis and IgE by immunoblot to eggs from chicken, duck, goose, and quail. RESULTS: We obtained negative results to prick, prick-by-prick and specific IgE to chicken's egg. Prick-by-prick to quail's egg was positive. By immunoblot we recognised a protein in quail's egg white, which is ovotransferrin without any similar bands in other species' eggs. CONCLUSIONS: The protein that we recognised is a specific protein of quail's egg. These proteins did not cross-react with proteins of chicken's egg. Cooking may degrade such proteins.

Understanding patient sensitization profiles in complex pollen areas: a molecular epidemiological study.

BACKGROUND: Allergy diagnosis in patients exposed to multiple pollen species is complex and misdiagnosis is often a cause for unsuccessful specific immunotherapy. OBJECTIVE: We studied the sensitization profile of individual allergens (major, minor and pan-allergens) in pollen-sensitized patients in a region with high exposure to olive pollen by investigating the influence of minor allergens on allergic disease and the association between pan- and minor allergen sensitizations. METHODS: A panel of 13 purified allergens, which included the most relevant allergens in the area, as well as minor olive allergens and pan-allergens, were screened using a high-capacity technology (ADVIA-Centaur) in 891 patients. RESULTS: Olive allergy as measured by specific IgE to Ole e 1 was the leading pollinosis in the area. The minor olive allergens Ole e 7 and Ole e 9 were markers of more severe allergic illness. Profilin sensitization was associated mainly with grass allergy, the second most prevalent pollinosis. Salsola kali pollen allergy was the third most common cause of pollinosis in the area. The prevalence of sensitization to the peach allergen Pru p 3, a nonspecific lipid-transfer protein, was notable. CONCLUSION: Epidemiological analysis by component-resolved diagnosis is a new method, which elucidates the interaction between allergen exposure gradient and patient sensitization. High exposure leads to differential sensitization profiles some of which are associated with more severe allergic conditions. Profilin sensitization, related mainly to grass pollinosis, was a marker of more severe grass pollen sensitization.