Minor allergen patterns in birch pollen allergen products-A question of pollen?

BACKGROUND: Contrary to the scientific differentiation between major and minor allergens, the regulatory framework controlling allergen products in the EU distinguishes relevant and non-relevant allergens. Given the lack of knowledge on their clinical relevance, minor allergens are usually not controlled by allergen product specifications. Especially, in birch pollen (BP) allergen products, minor allergens are commonly disregarded. OBJECTIVES: To quantify three minor allergens in BP allergen products from different manufacturers and to assess the influence of the utilized BP on minor allergen patterns. METHODS: Apart from common quality parameters such as Bet v 1 content, Bet v 4, Bet v 6 and Bet v 7 were quantified in 70 BP allergen product batches from six manufacturers, using ELISA systems developed in-house. Batch-to-batch variability was checked for agreement with a variability margin of 50%-200% from mean of the given batches for individual allergen content. Subsequently, minor allergen patterns were generated via multidimensional scaling and related to information on the pollen lots used in production of the respective product batches. RESULTS: Like the already established Bet v 4 ELISA, the ELISA systems for quantification of Bet v 6 and Bet v 7 were successfully validated. Differences in minor allergen content between products and batch-to-batch consistency were observed. Correlations between minor and major allergen content were low to moderate. About 20% of batches exceeded the variability margin for at least one minor allergen. Interestingly, these fluctuations could not in all cases be linked to the use of certain BP lots. CONCLUSIONS AND CLINICAL RELEVANCE: The impact of the observed minor allergen variability on safety and efficacy of BP allergen products can currently not be estimated. As the described differences could only in few cases be related to the used pollen lots, it is evident that additional factors influence minor allergens in BP allergen products.

Standardization of allergen products: 3. Validation of candidate European Pharmacopoeia standard methods for quantification of major birch allergen Bet v 1.

BACKGROUND: The BSP090 project aims at establishing European Pharmacopoeia Reference Substances in combination with the corresponding ELISA methods for the quantification of major allergens in allergen products. Two sandwich ELISAs proved suitable for quantification of Bet v 1, the major birch pollen allergen, in preceding phases of BSP090. METHODS: Two Bet v 1-specific ELISA systems were compared with respect to accuracy and precision in a ring trial including 13 laboratories. Model samples containing recombinant rBet v 1.0101 as well as native birch pollen extracts were measured independently at least three times in each facility. The assessment was completed with a comparative quantification of Bet v 1 in 30 marketed birch allergen products in one laboratory, simulating the future use as reference method. RESULTS: In the collaborative study, both candidate ELISAs confirmed their suitability to quantify recombinant and native Bet v 1. ELISA-A showed higher precision and lower interlaboratory variability, yet ELISA-B exhibited slightly higher accuracy. Subsequent parallel measurement of Bet v 1 in a panel of 'real-life' birch allergen products indicated better repeatability of ELISA-B. Both systems detected substantial differences in Bet v 1 content between allergen products, but the effect was more pronounced using ELISA-B due to persistently higher values compared to ELISA-A. CONCLUSIONS: In the collaborative study, no deciding differences were observed between the two candidate ELISAs. Further comparison under conditions simulating the intended use combined with the criterion of long-term availability enabled the selection of one Bet v 1-specific ELISA for proposal as European Pharmacopoeia standard method.

[Governmental batch sample testing of allergen products]. / Staatliche Chargenprüfung von Allergenpräparaten.

Allergen products for specific immunotherapy of type I allergies were first authorized for the German market in the 1970s. In addition to finished products manufactured in advance and in batches, so-called named patient products have recently been defined as Medicinal Products by the German Medicinal Products Act ("Arzneimittelgesetz", AMG 14th Revision 2005). Some allergen products previously marketed as named patient products are now required to obtain marketing authorization according to the German ordinance for therapy allergens. Products have to be batch released by the competent German Federal Agency, the Paul-Ehrlich-Institut (PEI). Samples of product batches are delivered to the PEI in order to perform experimental quality controls. With regard to named patient products, PEI tests batch samples of the bulk extract preparations used for manufacturing of the respective, named patient products. The institute releases approximately 2,800 allergen product batches annually.

Validation of ELISA methods for quantification of the major birch allergen Bet v 1 (BSP090).

To date, the potency of allergen products in Europe is expressed in manufacturer-specific units relative to a product-specific in-house reference. Consequently, cross-product comparability of allergen products from different manufacturers with respect to strength and efficacy is impossible. The Biological Standardisation Programme (BSP) project BSP090 addresses this issue via the establishment of reference standards in conjunction with ELISA methods for the quantification of major allergens in allergen products. Since the initiation of BSP090, the recombinant major allergen Bet v 1 has been adopted by the European Pharmacopoeia Commission as a Chemical Reference Substance (CRS). In parallel, two sandwich ELISA systems for quantification of Bet v 1 were found suitable in preliminary phases of BSP090 to be validated in a large collaborative study. In this study, the candidate ELISA systems were compared with respect to accuracy, precision and variability. Thirteen participating laboratories tested model samples containing the CRS as well as spiked and unspiked birch pollen extracts. Both in pre-testing and in the collaborative study, the 2 candidate ELISA systems confirmed their suitability to quantify recombinant and native Bet v 1. As no clear-cut decision for one of the ELISA systems could be made based on the results of the collaborative study, a post-study testing was performed. Bet v 1 content of 30 birch pollen allergen products was determined in parallel in both ELISA systems. Consequently, 1 candidate ELISA system was selected to be proposed as the future European Pharmacopoeia standard method for Bet v 1 quantification.

Genetic analysis of a Rhodobacter capsulatus gene region involved in utilization of taurine as a sulfur source.

Rhodobacter capsulatus was shown to grow efficiently with taurine as sole source of sulfur. We identified a gene region exhibiting similarity to the Escherichia coli tauABC genes coding for a taurine-specific ABC transporter. The R. capsulatus tauABC genes were flanked by two putative operons (orf459-484-590 and cysE-srpI-nifS2) both reading in opposite direction relative to tauABC. Orf459 shows strong similarity to taurine:pyruvate aminotransferase (Tpa) from Bilophila wadsworthia catalyzing the initial transamination during anaerobic taurine degradation, and Orf590 exhibits clear similarity to sulfoacetaldehyde sulfo-lyase from Desulfonispora thiosulfatigenes probably catalyzing the step following the taurine:pyruvate aminotransferase (Tpa) reaction, whereas nifS2 might code for a putative cysteine desulfurase. Expression of R. capsulatus tauABC and nifS2 was inhibited by sulfate, suggesting that tauABC and nifS2 might belong to the same regulon. In contrast, transcription of orf459 was not inhibited by sulfate but was induced by taurine. A tauAB deletion mutant showed significantly reduced growth compared to the wild-type with taurine as sole sulfur source in the presence of serine as a nitrogen source, whereas normal growth was observed in the presence of taurine and ammonium. Deletion of orf459-484-590 completely abolished growth with taurine/serine. Single mutations in any of the three genes resulted in the same phenotype, indicating that all three genes of this putative operon are essential for taurine sulfur utilization in the presence of serine. A model for anaerobic taurine sulfur assimilation in R. capsulatus is discussed.

In vitro fertilization and cleavage of bovine oocytes matured in medium supplemented with estrous cow serum.

Bovine follicular oocytes were matured in TCM 199 supplemented with: (1) fetal calf serum (FCS, 20% v/v), Luteinizing Hormone (LH, 10 ug/ml), and Estradiol-17-beta (E(2), 1 ug/ml) in Experiment 1; (2) 20% cow serum recovered at standing estrus (Experiment 2); or (3) 20% FCS (Experiment 3). Maturation, fertilization, and initial cleavage development were evaluated at 16 and 48 h after in vitro insemination. The proportions of oocytes fertilized after maturation in the presence of added hormones (78.5%, Experiment 1) or estrous serum (71.3%, Experiment 2) were significantly higher (p < 0.01) than after use of FCS alone (39.3%, Experiment 3). Cleavage of zygotes within 48 h post-insemination differed significantly (p < 0.01) between maturation treatments, 27.3%, 75.5% and 6.6% for Experiments 1, 2, and 3, respectively. Results demonstrate a beneficial influence of estrous cow serum, characterized by an elevated concentration of LH, on bovine oocyte maturation in vitro.

Maturation competence and chromatin behaviour in growing and fully grown cattle oocytes.

Chromatin configurations of cattle oocytes from different follicle sizes were classified in three chromatin categories. Early diplotenes, dictyate-like structures, more progressed diplotenes and early diakinesis and metaphases were found. In follicle group A (0.4-0.9 mm) immature diplotenes predominated (82.3%), and a rather low maturation competence was observed (12.1%). With increasing follicle size the maturation competence was improved to 47.8% metaphases II in follicle group C (3-8 mm). Impressive CMA3 positive blocks were visualized especially in early diplotenes. Heterochromatic CMA3 positive clusters surrounded the nucleolus.

Evaluation of seven microsatellite loci in Simmental cattle.

Seven microsatellite loci were evaluated for their suitability for parentage control. The polymerase chain reaction (PCR) was used to amplify the short tandem repeat (STR) loci in separate reactions. The microsatellite polymorphisms were visualized by radioisotopic autoradiographic detection. The microsatellite loci showed extensive polymorphism with allele numbers ranging from 4-23 and polymorphism information content (PIC) values in the range of 0.57-0.87. The analysis of these loci also revealed that they have a 99.9% combined probability of exclusion (PE) of erroneous parentage. The results of this study revealed that a very high probability of exclusion could be reached with only four microsatellite loci.

Sindrome de ovario poliquistico: presentacion clinica, bioquimica y ultrasonografica

En forma retrospectiva se efectúa una revisión de 115 historias clínicas de pacientes con Síndrome de Ovario Poliquístico (SOP) que consultan en la Unidad de Endocrinología del Servicio de Ginecología del Hospital San José en el periodo comprendido entre los años 1996 y 2002. Se determinó la prevalencia y se estableció una caracterización demográfica, hormonal y ultrasonográfica de estas pacientes. Destaca la presencia de un alto porcentaje de obesidad que alcanzó el 63% y una insulinoresistencia del orden del 76%. La LH se encontró elevada en el 47% de nuestras pacientes y la testosterona total y libre mostraron un bajo porcentaje de incremento (11% y 27% respectivamente). La Ultrasonografía mostró patrones característicos de SOP, de acuerdo a los criterios estandarizados actuales, en alrededor del 70% de los casos. Estos hallazgos nos inducen a priorizar el estudio de estas pacientes en base a LH y a insulinoresistencia por sobre los niveles de andrógenos. Desde el punto de vista metabólico se encontró un bajo porcentaje de Hipertensión Arterial y Diabetes Mellitus II (2,6 y 6,1% respectivamente).

Efecto de la bromocriptina en el síndrome de ovario poliquístico / Effect of the bromocriptine on the polycystic ovarian syndrome

Se estudió los efectos de la administración crónica de la Bromocriptina en las pacientes, con síndrome ovarico poliquístico normoprolactinémico. Se administró bromocriptina 5 mg diarios por hasta 4 meses y se evaluó las respuestas clínicas y bioquímicas a intervalos mensuales. No se observó variaciones en los niveles de gonadotrofinas ni andrógenos y sí se registró una significativa reducción de los niveles circulantes de prolactina. Concomitantemente con ello hubo mejoría importante de la función menstrual, al mes, 5 de 10 normalizaron las menstruaciones y en otras 3 hubo aumento de la frecuencia de las menstruaciones hacia lo normal, lográndose embarazo en dos pacientes. Ninguna medición o respuesta hormonal, predijo la respuesta clínica al tratamiento. Concluimos que la bromocriptina puede tener rol como inductor de ovulación en síndrome ovarico poliquístico normoprolactinémico, en aquellas pacientes que han fracasado en ovular con clomifeno y que requerían otro tipo de terapia

Desarrollo puberal espontáneo en una mujer con disgenesia gonadal pura 46, XY / Spontaneous puberal development in a woman with pure gonadal dysgenesis 46, XY

Presentamos el caso de una mujer de 26 años que tuvo su menarquia a los 16 seguida de dos ciclos menstruales espontáneos y luego amenorrea. Su examen físico mostró una estatura normal, proporciones corporales eunucoides, desarrollo mamario adulto, distribución pilosa normal, genitales externos femeninos normales sin virilización, útero hipoplásico y anexos no palpables, lo que fue confirmado por ecografía. Su cariotipo fue 46, XY. Tenía niveles plasmáticos elevados de FSH y normales de testosterona. Con el diagnóstico de disgenesia gonadal pura XY en su forma completa, se realizó gonadectomía bilateral. El estudio microscópico, demostró presencia de tejido fibroso tipo estroma ovárico con múltiples calcificaciones nodulares, sin estructuras foliculares, ni células de Leydig. Sertoli o carácter maligno. El cariotipo de ambas gónadas fue 46, XY. El estudio molecular de su ADN genoma mostró ser SRY positivo. Se discuten las bases del diagnóstico de disgenesia gonadal completa XY en este caso y los posibles mecanismos etiopatogénicos involucrados