Characterization of Der p 21, a new important allergen derived from the gut of house dust mites.

BACKGROUND: The house dust mite (HDM) Dermatophagoides pteronyssinus is a major allergen source eliciting allergic asthma. The aim of the study was to identify new important HDM allergens associated with allergic asthma. METHODS: A cDNA coding for a new mite allergen, designated Der p 21, was isolated using immunoglobulin E (IgE) antibodies from patients with allergic asthma out of a D. pteronyssinus expression cDNA library and expressed in Escherichia coli. RESULTS: Circular dichroism analysis of the purified allergen showed that rDer p 21 (14 726 Da) is one of the few mite allergens with an alpha-helical secondary structure. The protein exhibited high thermal stability and refolding capacity, and, as determined by small angle X-ray scattering, formed a dimer consisting of two flat triangles. rDer p 21 bound high levels of patients' IgE antibodies and showed high allergenic activity in basophil activation experiments. Rabbit anti-Der p 21 IgG antibodies inhibited mite-allergic patients' IgE binding and allowed the ultrastructural localization of the allergen in the midgut (epithelium, lumen and faeces) of D. pteronyssinus by immunogold electron microscopy. Der p 21 revealed sequence homology with group 5 mite allergens, but IgE and IgG reactivity data and cross-inhibition studies identified it as a new mite allergen. CONCLUSIONS: Der p 21 is a new important mite allergen which is liberated into the environment via faecal particles and hence may be associated with allergic asthma.

House dust mite species and allergen levels in Galicia, Spain: a cross-sectional, multicenter, comparative study.

BACKGROUND AND OBJECTIVES: Mites are important sources of allergens in Galicia, Spain. The objective of this study were to identify the main mite species and to determine allergen levels in mattresses from different locations in Galicia. MATERIAL AND METHODS: Dust samples were collected with a portable vacuum cleaner for 2 minutes from 2 m2 of the surface of the mattress used by mite allergic patients and controls. After collection, samples were immediatel frozen. Mite species were collected, identified, and counted, and the results expressed as mites per gram of dust. Mite allergen levels (Der p 1 and Der f 1) were measured using monoclonal antibodies. All individuals answered a questionnaire about the characteristics of their homes. RESULTS: A total of 332 dust samples were collected (112 from mite allergic patients and 220 from controls). Thirty-two species were identified. The mean age +/-SD of all the participants was 32.4 +/- 20.8 years and of the mattresses 7.6 +/- 5.9 years. The geometric mean of the total mite counts was 910.2 mites per gram (896.2 mites per gram in the mattresses of mite allergic patients and 917.3 in the mattresses of control subjects; P > .05). The main species was Dermatophagoides pteronyssinus, which was present in 97.6% of the samples (geometric mean, 584.7 mites per gram). The geometric mean level of Der p 1 was 13.1 microg/g of dust: 11.9 in the mattresses of mite allergic patients and 13.8 in the mattresses of control subjects. The geometric mean level of Der f1 was 1.1 microg/g: 1.5 in the mattresses of mite allergic patients and 0.8 in the mattresses of control subjects. Environmental risk factors associated with high mite counts included obvious signs of humidity in the bedroom and the age of the mattress. CONCLUSIONS: The mite fauna in Galicia is comprised of many species, several of which are known to be of allergologic importance. The total population is exposed to very high levels of mite allergens.

House dust mite, cat, and cockroach allergen concentrations in daycare centers in Tampa, Florida.

BACKGROUND: Allergen exposure in early childhood is a risk factor for sensitization and the development of asthma. Studies performed in Europe, New Zealand, and Singapore indicated the presence of indoor allergens in childcare centers and schools. However, the importance of indoor allergens in daycare centers in humid and warm regions of the world is not known. OBJECTIVE: To measure total mite counts, Der p 1, Der f 1, Fel d 1, and Per a 1 allergens in dust samples and mite allergen airborne concentrations in daycare centers in Tampa, Florida, United States. METHODS: Twenty daycare centers were surveyed for mite, cat, and cockroach allergens in Tampa, FL. One dust and two air samples (one during the day and one during the night) were collected in each center. Dust samples were extracted and analyzed for mite (Der p 1 and Der f 1), cat (Fel d 1), and cockroach (Per a 1) allergens. Mite airborne allergen concentrations were analyzed by RAST inhibition and expressed in standardized mite allergen units per m3 of air (AU/m3). RESULTS: Mites were identified in 15 samples, and concentrations ranged from 10 to 1,200 mites/g (298 +/- 355.2). The most prevalent mite species was Dermatophagoides pteronyssinus ( Der p 1). Der p 1 and/or Der f 1 were detected in 10 daycare centers. Der p 1 was detected in eight centers and ranged from I to 21.8 microg/g of dust (5.4 +/- 6.9); Der f 1 was detected in 3 centers and ranged from 0.2 to 2.1 microg/g of dust (1.3 +/- 0.9). Per a 1 and Fel d 1 were detected in all centers in small quantities; Per a 1 ranged from 8 to 1,806 ng/g (263.1 +/- 449.7) and Fel d 1 from 0.2 to 120 U/g of dust (16.6 +/- 31.7), respectively. Airborne mite allergen was detected in 18 centers and ranged from 0.01 to 2.7 AU/m3 during the day (0.2 +/- 0.6) and from 0.01 to 0.12 AU/m3 during the night (0.06 +/- 0.03), P = 0.001. CONCLUSIONS: Mite, cat, and cockroach allergens are present in daycare centers in Tampa, FL. Mite allergen concentrations exceeded levels that have been associated with sensitization and symptoms in allergic subjects in 40% of these centers.

Determination of Ole e 1 by enzyme immunoassay and scanning densitometry. Validation by skin-prick testing.

Ole e 1 is an important allergen in Olea europaea pollen extracts. This study describes the development of two new methods that can be used to estimate the Ole e 1 content in olive tree pollen extracts. They are based on (1) an enzyme immunoassay that uses rabbit polyclonal, monospecific antibodies and purified Ole e 1, and (2) scanning densitometry of SDS-PAGE gels. Twelve extracts were evaluated by in vivo and in vitro methods. The in vivo biological potency was estimated by prick skin testing 17 allergic individuals; the in vitro allergenic potency by direct IgE and IgE inhibition assays. The enzyme immunoassay showed an operative range of 0.03-100 microg/ml and demonstrated to be specific for Ole e 1. The Ole e 1 content ranged from 1% to 5% of the total protein in the 12 extracts. The amount of Ole e 1, assessed by gel scanning densitometry significantly correlated with the Ole e 1 content obtained by the immunoassay (r = 0.92; p < 0.001). The Ole e 1 content showed a significant correlation with the total allergenic potency of the extracts, evaluated by direct IgE, specific IgE inhibition and skin-prick testing. These two methods can be used to determine the Ole e 1 content in olive pollen extracts. The content of Ole e 1 can vary from 1% to 5% of the total protein in the extracts.

Determinations of Par j 1 by a competitive enzyme immunoassay using human specific IgE and IgG. Validation by skin prick testing.

Par j 1 is the major allergen of Parietaria judaica. The objectives of this study were the following: 1) to purify Par j 1; 2) to develop an enzyme immunoassay based on the bivalent properties of specific IgE and IgG to determine the Par j 1 content in several batches of P. judaica extracts; and, 3) to study the contribution of Par j 1 to the total allergenicity and antigenicity of P. judaica extracts. P. judaica pollen was extracted and subjected to hydrophobic interaction and gel filtration chromatography for the purification of Par j 1. Inhibition enzyme immunoassays, SDS-PAGE and immunoblotting were used to characterize the allergen content. The in vivo biological potency of the extracts was estimated by skin prick testing 26 P. judaica clinically sensitive patients. The new enzyme immunoassay showed a high degree of specificity and sensitivity, detecting from 2 to 100 ng Par j 1/ml. The range of Par j 1 content in nine batches ranged from 23% to 78% of the total protein in the extracts. The Par j 1 content showed a significant correlation with the allergenic potency of these extracts evaluated by specific IgE inhibition and skin prick testing; the correlation with the specific IgG inhibition capacity was not significant. Purified Par j 1 shows great specific IgE and IgG binding capacity; its content can be determined using this newly developed enzyme immunoassay. Par j 1 levels exhibit a significant correlation with the biological potency of the extracts. This method allows the detection of Par j 1 isoforms.

Identification of the domestic mite fauna of Puerto Rico

This study was conducted to identify the domestic mite fauna of Puerto Rico. A total of 57 dust samples were collected from mattresses in homes of 11 cities on the Island. The analysis of the samples revealed that 73.70 percent of the mattress samples had at least one mite species. The identified species include: Dermatophagoides pteronyssinus (45.6 percent5), Blomia tropicalis (31.6 percent), Cheyletus sp. (19.3 percent), Dermatophagoides farinae (17.5 percent), Euroglyphus maynei (5.3 percent), Dermatophagoides sibonei (1.8 percent, Dermatophagoides sp. (1.8percent), Suidasia melanensis (1.8 percent) and mite species that were not identified (5.3 percent). Differences in the geographical distribution of mites showed that only Blomia tropicalis is more frequently in the northern (43 percent) than in the southern region (19 percent) of Puerto Rico (OR 3.36, p, 0.046). This finding can be explained by the fact that in the northern region the relative humidity is significantly higher that in the southern region (p < 0.001). No significant differences were observed for other species or in the total mite counts between the northern and southern regions. The small sample size of this study may explain the lack significance for some of the differences found. Nevertheless, our results indicate that the domestic mite fauna is composed of several clinically important species, their numbers are high enough to be considered in the sensitizing levels, and the diversity of these species is comparable to other observations in the Caribbean areas, and in the southern states in the continental US. Based upon our results, we recommend that when performing the skin test in Puerto Rico, extracts from the identified local domestic mite species be included in the allergen panel. This may prove useful in the aid for the diagnosis and management of atopic conditions

Serum total and specific IgE levels in children infected with human immunodeficiency virus.

BACKGROUND: Human immunodeficiency virus (HIV) infection is associated with polyclonal B-cell activation, and elevated IgE levels are found in infected adults. Chronic nasal symptoms, sinusitis, and otitis media are particularly common in children infected with HIV. The possible allergic cause of nasal symptoms in children infected with HIV has not been explored. OBJECTIVE: The study was designed to evaluate the relationship between HIV infection and IgE levels in a group of children infected with HIV. METHODS: Total serum IgE was measured by enzyme immunoassay and compared with values for age-matched normal subjects. Specific IgE was determined by RAST test to Dermatophagoides pteronyssinus, American cockroach, Bahia grass, short ragweed, cat, and oak. Lymphocyte subpopulations were determined by standard flow cytometry. RESULTS: Fifty-seven total IgE and 342 specific IgE levels were determined from 29 infected children (19 girls and 10 boys; ages, 7 to 150 months; mean age, 49 months). Initial IgE levels of 29 subjects ranged from 1.4 to 787.6 IU/ml (mean = 123.4 +/- 34.4 IU/ml). Nine of 29 (31%) subjects had initial IgE levels greater than two standard deviations higher than age-matched normal subjects. No patients had detectable specific IgE to any of the six aeroallergens. There was no correlation, as determined by linear regression analysis, between IgE levels and absolute CD4 number or CD4:CD8. CONCLUSION: Elevated total but not specific IgE is common in children infected with HIV and does not correlated with absolute CD4 lymphocyte count, suggesting that the elevated IgE level reflects a somewhat independent polyclonal activation of B-lymphocytes.

Prevalencia de sensibilización a Blomia tropicalis en Rosario: estudio multicéntrico / The prevalence of the sensitivity to Blomia tropicalis in Rosario city: multicenters studies

Se evaluó la prevalencia de la sensibilización a Blomia tropicalis en pacientes con asma y/o rinitis que residen exclusivamente en la ciudad de Rosario,para valorar si existen diferencias entre la respuesta cutánea a cuerpo total y a partículas fecales y relacionar la misma con diferentes parámetros clínicos y de laboratorio. Se estudiaron 314 pacientes, 164 femeninos (52,2 por ciento) con edades entre 5 a 55 años (x 20,8 DS ñ 13,7). 87 pacientes presentaban solamente asma, 91 solamente rinitis y 136 ambas patologías. El estudio se realizó en 8 centros asistenciales de la ciudad de Rosario, 3 públicos y 5 privados, completando todos un cuestionario tipo y realizando la misma técnica de testificación con un extracto de idéntico origen (División de Alergia e Inmunología, University of South Florida, Tampa, U.S.A.). Se llevó a cabo prick test (SPT) a una concentración de 1/50 p/v de cuerpo total (ECT) de Blomia tropicalis y de partículas fecales (EPF) de la misma, comparando la pápula obtenida con cada extracto con la de la histamina y se consideró positivo un índice de histamina > 0,5. Además, cada centro, realizó SPT con una batería de aeroalergenos, considerando como positiva una pápula mayor de 5 mm. Se obtuvo un total de 280 (89,2 por ciento) pruebas cutáneas positivas para al menos un aeroalergenos. De los 314 individuos estudiados 224 (71,3 por ciento) resultaron positivos para el extracto de cuerpo total de B. tropicalis y 207 (66 por ciento) para el extracto de partículas fecales. Veinte (7,1 por ciento) de los 280 SPT "+" respondieron exclusivamente a Blomia tropicalis. La prevalencia de sensibilización a Dermatophagoides pteronyssinus y/o Dermatophagoides farinae fue del 76,1 por ciento (239 pacientes) y la de otros neumoalergenos del 38,5 por ciento (121 pacientes). Confirmando un reporte previo, la prevalencia de sensibilización a Blomia tropicalis posee una relevancia casi tan importante como la de los ácaros Pyroglyphidos en la ciudad de Rosario. El análisis comparativo mostró que la sensibilidad a Blomia tropicalis es significativamente mayor en el grupo etario de 10 a 20 años (x²= 11,1 p<0,01), en los pacientes con una IgE total > 300 kU/l (x²=33,5 p<0,001) y en los pacientes con asma y rinitis (x²=14,4 p<0,001). Fué también significativa la diferencia entre el tamaño de la pápula del extracto de cuerpo de Blomia tropicalis y los distintos grupos etarios (x²=28,6 p<0,001) así como en el nivel de IgE total (x²=34,9 p<0,001).

Respiratory atopy and domestic mite sensitization in Rio de Janeiro

Domestic mites occur in a significant percentage of homens in Rio de Janeiro, a tropical geographic region of Brazil. Local weather conditions (hot and humid) promote the significant mite proliferation. Objective. We are registering the allergic mite sensitization of a population with asthma and/or rhinitis living in this coastal area. Methods. A total of 600 patients with asthma and/or rhinitis aged>=2 years underwent 3,810 allergy skin tests (Multi-Test). Eight different extracts were applied: American house dust (Ahd), Brazilian house dust (Bhd), Dermatophagoides pteronyssinus (Dp), Dermatophagoides farinae (Df), Blomia tropicalis (Bt), Chortoglyhphus arcuatus (Ca), Aleuroglyphus ovatus (Ao) and Lepidoglyphus destructor (Ld). RAST to Dp, Bt, Ca, Ao and Ld were performed with sera of randomized 14 atopic subjects. Results A positive immediate skin test to at least one allergen was detected in 387 patients (64,5 percent). Domestic mite sensitization was documented in about 90 percent of the atopic individuals. Bt has unique antigens since 4.15 percent of the subjects had positive exclusive reactions. RAST analyses showed that both Dp and Bt are the most important local mite sensitizers with cross-reactivity to the other mites. Conclusion. This study confirms that domestic mites are the most prevalent local allergens associated with asthma and/or rhinitis. Sensitization to Bt was very common and it should be checked routinely for the diagnosis of respiratory atopy in Rio de Janeiro

Resistencia del D. pteronyssinus y B. tropicalis a la congelación / Resistance of the D. pteronyssinus and B. tropicalis to congealment

El objetivo fue establecer la resistencia del D. pteronyssinus y B. tropicalis a distintas temperaturas por debajo del punto de congelación. Para ello se separaron del medio de cultivo D. pteronyssinus y B. tropicalis por el método de Tullgren modificado y se colocaron 50 mg de los mismos a -15, -30 y -80º C. El tiempo de congelación fue de 30min., 1, 2, 4, 6 hs. para cada temperatura y especie de ácaro. La descongelación se realizó a temperatura ambiente constante y se cronometró el tiempo desde que se los sacaba del freezer hasta que el primer ácaro presentara un movimiento franco. Se observó la evolución de la vitalidad según movimiento y traslado a los 15, 30, 60 y 120 min. luegode la congelación, dejándolos posteriormente en incubadora a 23ºC y a 70 HRA durante 24 hs. para evaluar el porcentaje de sobrevida. Cada observación fue realizada por triplicado, obteniendo el promedio del tiempo y porcentaje de sobrevida para cada observación. La B. tropicalis no tuvo sobrevida por encima de una hora de congelación a - 15ºC y sólo < del 10 por ciento de sobrevida cuando el tiempo de congelación fue de ó 1 hora; en ningún tiempo de congelación a -30 y - 80ºC se detectó sobrevida, mientras que en el caso del D. pteronyssinus hubo sobrevida ò del 80 por ciento cuando el tiempo de congelación fue de menos de 6 hs. a - 15ºC y menos del 20 por ciento cuando el tiempo fue mayor de 6 hs. a la misma temperatura. A - 30ºC la sobrevida fue de más de 70 por ciento a 30min. de congelación, 50 por ciento a una hora y 0 por ciento a dos horas o más. No existió sobrevida a ningún tiempo de congelación a - 80ºC. Los resultados obtenidos permiten observar que la B. tropicalis tiene una mayor susceptibilidad al frío y que la congelación a - 30ºC por encima de una hora parece ser un medio eficaz para reducir, junto con otras medidas complementarias, el número tanto de B. tropicalis como de D. pteronyssinus en el medioambiente del paciente alérgico

Sensibilidad cutánea a ácaros domésticos y de deposito en niños y adultos asmáticos / Cutaneous sensibility to domestic and of deposit acarus in asthmatics children aqnd adults

La alergia a los ácaros del polvillo doméstico en asociación al asma es, actualmente considerado un problema de diseminación mundial. Entre el 70 al 90% de los pacientes asmáticos tienen test cutáneo positivos a extractos de ácaros, particularmente a D. pteronyssinus y D. farinae. Numerosos estudios han encontrado sensibilidad cutánea a otros Piroglífidos, tal el caso de E. maynei. En los depósitos de granos, alimentos y maderas, entre otros, se han hallado ácaros que se desiganaron "de almacenamiento" y que ahora son llamados "domésticos" ya que también se pueden encontrar en las viviendas. No conocíamos la prevalencia de sensibilidad a extractos de otros ácaros del sub-orden astigmata. En este estudio se presentan la frecuencia de sensibilización cutánea en niños asmáticos de Córdoba y niños y adultos asmáticos de Santa Fe a extractos de D. pteronyssinus, D. farinae, B. tropicalis, A. ovatus, C. arcuatus y L. destructor. Tanto en los pacientes asmáticos de Córdoba como de Santa Fe, la mayor frecuencia de sensibilización cutánea fue a D. pteronyssinus, 63.3% y 72.7% respectivamente. B. tropicalis fue el segundo alergeno hallado en Córdoba (58.3%) y D. farinae en Santa Fe (66.6%). Los extractos alergénicos de los otros ácaros estudiados arrojaron positividades entre el 6 y el 57.5% de los casos estudiados. Los ácaros del sub-orden Astigmata, no Piroglífidos, son capaces de inducir sensibilidad cutánea en niños y adultos asmáticos de nuestro país

Respiratory allergies and skin test reactivity in high school students in Tenerife, Canary Islands, Spain.

The prevalence of skin test reactivity to 22 aeroallergens and of allergic respiratory diseases was determined in 501 high school students on the island of Tenerife, Spain. Two hundred seventy-seven students (55.2%) had at least one positive prick skin test (wheal > or = 2 mm). Two hundred sixteen students (43.1%) had symptoms of upper or lower respiratory tract allergies [24 (4.7%) had asthma with or without rhinitis and 192 (38.3%) seasonal or perennial rhinitis alone]. Two hundred eighty-five students (56.8%) were asymptomatic. One hundred per cent of the students with asthma, 87.5% of the students with rhinitis and 27.7% of the asymptomatic students had at least one positive skin test. The prevalence of positive skin tests was significantly higher in symptomatic than in asymptomatic students (p = 0.0001). One hundred seventy students (33.9%) had a family history of respiratory allergic diseases. The prevalence of positive skin tests among these students was significantly higher than in students without such history (p = 0.0001). Thus, there is a high prevalence of allergic respiratory diseases and skin test reactivity to aeroallergens among high school students in Tenerife. Significant correlations were found between family history, positive skin tests and respiratory allergic symptoms.

Environmental assay for cockroach allergens.

A sandwich ELISA was developed to measure the concentration of cockroach allergen in the environment. The assay was based on a monospecific rabbit antibody preparation reactive with determinants shared by the important allergens, Per a I and Bla g I, from American and German cockroaches. The sensitivity was 0.2 ng Lowry protein of Per a I equivalents per milliliter, corresponding to 1 ng of Per a I equivalents per gram of dust (Per a I eq/gm). The assay did not react with noncockroach-allergen sources. Dust samples from 73 households in a cockroach-infested area were assayed. The concentration in these samples varied from below detection to 200,000 ng of Per a eq/gm of dust. Three commercial cockroach-allergen extracts all contained the allergen. The assay will be valuable for studies of the clinically relevant cockroach-allergen exposure levels and for assessment of efficacy of allergen-avoidance measures. Furthermore, the assay could be used for sanitary documentation in bakeries, restaurants, etc.

Identification and purification of an important cross-reactive allergen from American (Periplaneta americana) and German (Blattella germanica) cockroach.

Aqueous whole body extracts from two major domiciliary cockroaches, the American, Periplaneta americana, and the German, Blattella germanica, were analyzed in crossed immunoelectrophoresis and immunoblotting. Forty-five antigens were found in P. americana and 29 in B. germanica. IgE-binding antigens were identified by crossed radioimmunoelectrophoresis with sera from 30 cockroach-allergic patients. Seven and three precipitates from P. americana and B. germanica bound significant amounts of IgE. A cross-reactive, apparently homologous allergen, from P. americana and B. germanica bound IgE from 100% and 70%, respectively, of the patients. These important allergens were tentatively named Per a I and Bla g I. The allergens were purified by sequential ion exchange, gel filtration, and isoelectric focusing. Both allergens had a molecular size of 33 to 37 kd in Sephadex G-75 gel filtration, and 28 kd in high-performance liquid chromatography gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a minor band at approximately 25 kd, and most of the protein at 6 kd. The isoelectric point of both allergens was found be to 3.5. In amino acid analysis, the allergens were highly similar. Skin test revealed the allergens to be important in vivo sensitizing agents. The allergens may be used for environmental assays for cockroach exposure in the homes of allergic subjects.