Correlates of age at diagnosis of autism spectrum disorders in six Canadian regions.

INTRODUCTION: Early identification of autism spectrum disorders (ASD) is important, since earlier exposure to behavioural intervention programs may result in better outcomes for the child. Moreover, it allows families timely access to other treatments and supports. METHODS: Using generalized linear modeling, we examined the association between child and family characteristics and the age at which 2180 children were diagnosed with ASD between 1997 and 2005 in six Canadian regions. RESULTS: A diagnosis of pervasive developmental disorder-not otherwise specified (PDD-NOS) or Asperger syndrome, rural residence, diagnosis in more recent years, and foreign birthplace were associated with a later age at diagnosis. Children who are visible minorities or who have siblings with ASD were more likely to be diagnosed earlier. Collectively, these factors explained little of the variation in age at diagnosis, however. CONCLUSION: While it is encouraging that ethnocultural identity, neighbourhood income, urban or rural residence, and sex of the child were not major contributors to disparities in the age when children were identified with ASD, more work is needed to determine what does account for the differences observed. Regional variations in the impact of several factors suggest that aggregating data may not be an optimal strategy if the findings are meant to inform policy and clinical practice at the local level.

Alternaria alternata NADP-dependent mannitol dehydrogenase is an important fungal allergen.

BACKGROUND: Alternaria alternata is one of the most important allergenic fungi worldwide. Mannitol dehydrogenase (MtDH) has previously been shown to be a major allergen of Cladosporium herbarum and cross-reactivity has been demonstrated for several fungal allergens. OBJECTIVE: The present study's objective was to clone the MtDH from an A. alternata cDNA library, express and purify the recombinant non-fusion protein and test its IgE-binding properties. Methods A cDNA library prepared from A. alternata hyphae and spores was screened for mannitol dehydrogenase by DNA hybridization with the radioactively labelled C. herbarum homologue as a probe. The resulting clone was sequenced and heterologously expressed in Escherichia coli as a recombinant non-fusion protein, which was purified to homogeneity and analysed for its IgE-binding capacity. RESULTS: The coding sequence of the full-length cDNA clone comprises 798 bp encoding a protein with a molecular mass of 28.6 kDa and a predicted pI of 5.88. Protein sequence analysis revealed an identity of 75% and a homology of 86% between the MtDHs of A. alternata and C. herbarum. The functional mannitol dehydrogenase was expressed in the E. coli strain BL21(DE3) transformed with the vector pMW172 and purified to homogeneity. The enzyme catalyses the NADPH-dependent conversion of d-fructose to d-mannitol. In IgE-ELISA and immunoblots, MtDH is recognized by 41% of A. alternata-allergic patients. In vivo immunoreactivity of the recombinant MtDH was verified by skin prick testing. Finally, inhibition-ELISA experiments confirmed cross-reactivity between the MtDHs of A. alternata and C. herbarum. CONCLUSION: Mannitol dehydrogenase (Alt a 8) represents an important new allergen of the ascomycete A. alternata that might be suitable for improving diagnostic and therapeutic procedures.

GermOnline, a cross-species community knowledgebase on germ cell differentiation.

GermOnline provides information and microarray expression data for genes involved in mitosis and meiosis, gamete formation and germ line development across species. The database has been developed, and is being curated and updated, by life scientists in cooperation with bioinformaticists. Information is contributed through an online form using free text, images and the controlled vocabulary developed by the GeneOntology Consortium. Authors provide up to three references in support of their contribution. The database is governed by an international board of scientists to ensure a standardized data format and the highest quality of GermOnline's information content. Release 2.0 provides exclusive access to microarray expression data from Saccharomyces cerevisiae and Rattus norvegicus, as well as curated information on approximately 700 genes from various organisms. The locus report pages include links to external databases that contain relevant annotation, microarray expression and proteome data. Conversely, the Saccharomyces Genome Database (SGD), S.cerevisiae GeneDB and Swiss-Prot link to the budding yeast section of GermOnline from their respective locus pages. GermOnline, a fully operational prototype subject-oriented knowledgebase designed for community annotation and array data visualization, is accessible at http://www.germonline.org. The target audience includes researchers who work on mitotic cell division, meiosis, gametogenesis, germ line development, human reproductive health and comparative genomics.

Sensitization to fungi: epidemiology, comparative skin tests, and IgE reactivity of fungal extracts.

BACKGROUND: Several fungal species are known to cause severe respiratory and cutaneous allergic diseases. Extracts from several allergenic fungi are used for in vivo and in vitro tests, as standard preparations are still not available. OBJECTIVE: The aims are to define the pattern of in vivo and in vitro IgE reactivity to fungal species in an allergic population with respiratory symptoms; to determine the influence of different extract preparations on diagnostic results; and to evaluate whether there exists a relationship between the diagnostic pattern of reactivity and the pattern of specific IgE reactivity in immunoblots. METHODS: Skin prick tests were applied to a cohort of 4962 respiratory subjects, aged 3-80 years. Fungal extracts from Alternaria, Aspergillus, Candida, Cladosporium, Penicillium, Saccharomyces, and Trichophyton were used, along with extracts from pollens, mites, and animal dander. Demographical and diagnostic data were recorded. IgE detection was carried out with the same allergenic extracts plus Malassezia. Comparative skin tests and IgE detection were carried out using extracts from three commercial suppliers. IgE immunoblots were carried out with the same panel of commercial fungal extracts and were compared with in-house extracts. Data analysis was carried out by grouping the population on the basis of their reactivity to a single, to two or to more than two, mould species. RESULTS: Nineteen percent of the allergic population reacted to at least one fungal extract by means of the skin test. Alternaria and Candida accounted for the largest number of positive tests, and along with Trichophyton they were the main sensitizers in the subset of patients with an isolated sensitization. The prevalence of skin test reactivity increased for these three fungi in the subsets with two associated reactivities and, furthermore, in the subset showing reactivity to more than two mould species. In the latter group, a steady increase of the skin test reactivity was recorded for all the other fungal sources, suggesting a clustered reactivity. Comparative skin and IgE testing with different groups of subjects with a simple pattern of skin reactivity resulted in sensitivity differences between in vivo and in vitro tests, whereas discrepant results were recorded in the subsets of patients with multiple fungi sensitization. Although hampered by the limited reliability of fungal extracts, IgE immunoblots revealed differing patterns of reactivity when sera from the three subsets were used. This suggests a link between the diagnostic reactivity pattern and the IgE sensitization to extracts' components. Age and gender distribution differed among the Alternaria-, Candida-, and Trichophyton-sensitized subjects, but not in the subset with more than two fungi sensitizations. CONCLUSIONS: The preliminary assessment of a new classification of the mould-sensitized population has been reached. The limiting quality of fungal extracts requires future studies using an allergenic molecule-based approach. The diagnostic process and the definition of the reactivity pattern would thus be easy, and it could lead to a novel specific immunotherapy approach.

Nuclear transport factor 2 represents a novel cross-reactive fungal allergen.

BACKGROUND: Ubiquitously occuring moulds are important allergenic sources known to elicit IgE-mediated allergic diseases and to share cross-reactive allergens. Limited information is available about the molecular structures involved in cross-reactivity. We aimed to clone and characterize cross-reactive mould allergens. METHODS: Phage surface-displayed Alternaria alternata and Cladosporium herbarum cDNA libraries were screened using sera from Aspergillus fumigatus-sensitized patients. Inserts encoding putative allergens were sequenced, and recombinant proteins used to demonstrate cross-reactivity by inhibition experiments and skin test. Three-dimensional homology models of cloned putative nuclear transport factor 2 (NTF2) were constructed based on known NTF2 structure to corroborate the functional and structural properties of the novel allergens. RESULTS: After six rounds of affinity selection, the libraries were enriched for clones displaying allergens. Sequencing of inserts showed that some clones derived from Alternaria alternata and Cladosporium herbarum contain open reading frames predicting proteins of 124 and 125 amino acids corresponding to NTF2. The recombinant proteins were able to bind and cross-inhibit IgE binding and to elicit type I skin reactions in mould-sensitized individuals, demonstrating the allergenicity of the proteins. CONCLUSIONS: NTF2 represents a novel cross-reactive fungal allergen as demonstrated by sequence homology, three-dimensional modelling, inhibition experiments and skin test reactivity.

Is the infertility in hypothyroidism mainly due to ovarian or pituitary functional changes?

The objective of the present study was to examine whether hypothyroidism affects the reproductive system of adult female rats by evaluating ovarian morphology, uterus weight and the changes in serum and pituitary concentrations of prolactin and gonadotropins. Three-month-old female rats were divided into three groups: control (N = 10), hypothyroid (N = 10), treated with 0.05 percent 6-propyl-2-thiouracil (PTU) in drinking water for 60 days, and T4-treated group (N = 10), receiving daily sc injections of L-thyroxine (0.8 æg/100 g body weight) during the last 10 days of the experiment. At the end of 50 days of hypothyroidism no hypothyroid animal showed a regular cycle, while 71 percent of controls as well as the T4-treated rats showed regular cycles. Corpora lutea, growing follicles and mature Graafian follicles were found in all ovaries studied. The corpora lutea were smaller in both the hypothyroid and T4-replaced rats. Graafian follicles were found in 72 percent of controls and only in 34 percent of hypothyroid and 43 percent of T4-treated animals. Serum LH, FSH, progesterone and estradiol concentrations did not differ among the three groups. Serum prolactin concentration and the pituitary content of the three hormones studied were higher in the hypothyroid animals compared to control. T4 treatment restored serum prolactin concentration to the level found in controls, but only partially normalized the pituitary content of gonadotropins and prolactin. In conclusion, the morphological changes caused by hypothyroidism can be a consequence of higher prolactin production that can block the secretion and action of gonadotropins, being the main cause of the changes observed

Is the infertility in hypothyroidism mainly due to ovarian or pituitary functional changes?

The objective of the present study was to examine whether hypothyroidism affects the reproductive system of adult female rats by evaluating ovarian morphology, uterus weight and the changes in serum and pituitary concentrations of prolactin and gonadotropins. Three-month-old female rats were divided into three groups: control (N = 10), hypothyroid (N = 10), treated with 0.05% 6-propyl-2-thiouracil (PTU) in drinking water for 60 days, and T4-treated group (N = 10), receiving daily sc injections of L-thyroxine (0.8 microg/100 g body weight) during the last 10 days of the experiment. At the end of 50 days of hypothyroidism no hypothyroid animal showed a regular cycle, while 71% of controls as well as the T4-treated rats showed regular cycles. Corpora lutea, growing follicles and mature Graafian follicles were found in all ovaries studied. The corpora lutea were smaller in both the hypothyroid and T4-replaced rats. Graafian follicles were found in 72% of controls and only in 34% of hypothyroid and 43% of T4-treated animals. Serum LH, FSH, progesterone and estradiol concentrations did not differ among the three groups. Serum prolactin concentration and the pituitary content of the three hormones studied were higher in the hypothyroid animals compared to control. T4 treatment restored serum prolactin concentration to the level found in controls, but only partially normalized the pituitary content of gonadotropins and prolactin. In conclusion, the morphological changes caused by hypothyroidism can be a consequence of higher prolactin production that can block the secretion and action of gonadotropins, being the main cause of the changes observed.

Somatic diversity of the immunoglobulin repertoire is controlled in an isotype-specific manner.

We have studied two aspects of the IgE immune response. First, we have compared the kinetics of the IgE response to the T cell-dependent antigen ph-Ox coupled to ovalbumin with that of the IgG1 response and we have assessed the quality of the IgE response. Second, we have studied the generation of somatic diversity, understood as the combined effect of somatic mutation and the selection of D(iversity) and J(oining) elements, in germinal center B cells at the molecular level, using the germ-line sequence of the prototype anti-ph-Ox heavy chain variable element V(H)Ox1 as reference. We evaluated sequences derived from mu-, gamma 1- and epsilon-variable elements and showed that somatic diversification was different for all isotypes studied. We further compared the IgE responses of wild-type mice with those of mice expressing a truncated cytoplasmic IgE tail (IgE(KVK Delta tail)). IgE(KVK Delta tail) mice showed a more diverse sequence pattern. We corroborated previous results suggesting that short CDR3 regions are indicative for high-affinity antibodies by measuring relative affinities of phage-expressed Fab fragments with prototype long and short CDR3 regions. Therefore, the composition of the antigen-receptor is responsible for the selection process and the expansion of antigen-specific cells, leading to an isotype-specific antibody repertoire.

Menstrual irregularity in the first postmenarchal years: an early clinical sign of polycystic ovary syndrome in adolescence.

We determined the hormonal, metabolic and ultrasonographic pattern of adolescents with menstrual irregularity since menarche but without clinical signs of hyperandrogenism with the aim of evaluating whether this condition represents an early stage of polycystic ovary syndrome (PCOS). These adolescents were divided in two groups: 13 adolescents with irregular cycles (IC) within the first 3 postmenarchal years (IC < or = 3) and 15 adolescents having persistent irregular cycles for more than three postmenarchal years (IC > 3). These adolescents were compared with 15 adolescents with PCOS and 18 normal adolescents. The values of free testosterone, free androgen index, luteinizing hormone (LH) and LH/follicle-stimulating hormone (FSH) ratio were similar in IC < or = 3, IC > 3 and PCOS, and higher than in the normal group (p < 0.005). The total testosterone and androstenedione levels were higher and sex hormone-binding globulin (SHBG) lower in PCOS only when compared with the normal group (p < 0.05). The ovarian volume was similar in IC < or = 3, IC > 3 and PCOS, and higher than in the normal group (p < 0.005). A higher incidence of polycystic structure was found in IC < or = 3, IC > 3 and PCOS, whereas normal structure was more common in normal adolescents (p < 0.0005). There were no significant differences in glucose and insulin parameters between groups. These results indicate that menstrual irregularity within the first postmenarchal years can be an early clinical sign of PCOS.

The IgE antigen receptor: a key regulator for the production of IgE antibodies.

Immunoglobulins in general form a substantial component of serum proteins, and play a role in homeostatic mechanisms, a first line of defense against pathogenic organisms and in immunological memory. In the secreted form, immunoglobulins represent the effector arm of the humoral immune system. However, immunoglobulins are not only secreted, but can also be expressed on the surface of a B lymphocyte (membrane immunoglobulin), and, in this physical state, most likely convey signals to steer the B cell along its differentiation pathway. A step forward in the understanding of the role of membrane immunoglobulins other than membrane IgM or IgD was achieved with two mouse lines with mutations in the epsilon heavy chain gene. In IgE(DeltaM1M2) mice serum IgE is reduced to less than 10% of normal mice, while IgE(KVKDeltatail) mice show a reduction of 50%, reflecting a serious impairment of the IgE-mediated immune response. We think that the cytoplasmic tail of IgE is involved in a signal transduction which leads to the expression of high quantities and qualities of secreted IgE immunoglobulins.

Quantitation of the major fungal allergens, Alt a 1 and Asp f 1, in commercial allergenic products.

BACKGROUND: Alternaria is one of the most important fungi associated with allergic disease, whereas Aspergillus fumigatus is involved in a broad spectrum of pulmonary diseases. Currently, fungal extracts used for diagnosis in the United States are unstandardized, and their allergenic content cannot be compared directly. OBJECTIVE: The goal of this study was to compare the variability of major allergen levels among US allergenic products derived from fungi: specifically, Alt a 1 levels in Alternaria alternata extracts, and Asp f 1 levels in A fumigatus extracts. METHODS: A novel 2-site monoclonal antibody ELISA was used for measuring Alt a 1 using recombinant Alt a 1 as a standard. Asp f 1 was also measured by ELISA. Allergenic products produced by 8 US manufacturers over a 2-year period were compared, as were multiple lots produced by a single company. RESULTS: Alt a 1 levels in Alternaria extracts from 8 companies produced in 1998 and 1999 ranged from less than 0.01 to 6.09 microg/mL (mean 1.4 +/- 1.6 microg/mL, n = 15). In general, Alt a 1 levels were consistent within and between companies (1.4 +/- 1.1 microg/mL, n = 27), with 21 of 32 (66%) of all extracts tested containing 0.7 to 2 microg/mL Alt a 1. Aspergillus extracts showed much greater variability in Asp f 1 levels, with extracts from 8 companies containing from less than 0.1 to 64 microg/mL Asp f 1 (mean 16.3 +/- 23.9 microg/mL, n = 15). Overall variability was greater for Aspergillus products within and between manufacturers (22 +/- 22 microg/mL Asp f 1, n = 20). CONCLUSIONS: ELISA-based assays for specific allergens showed greater consistency among allergenic products derived from Alternaria than from Aspergillus. These assays should facilitate improved quality control and standardization of fungal allergen extracts and lead to the development of more consistent products for clinical use.

Aged mother cells of Saccharomyces cerevisiae show markers of oxidative stress and apoptosis.

Recently, we and others have shown that genetic and environmental changes that increase the load of yeast cells with reactive oxygen species (ROS) lead to a shortening of the life span of yeast mother cells. Deletions of yeast genes coding for the superoxide dismutases or the catalases, as well as changes in atmospheric oxygen concentration, considerably shortened the life span. The presence of the physiological antioxidant glutathione, on the other hand, increased the life span of yeast cells. Taken together, these results pointed to a role for oxygen in the yeast ageing process. Here, we show by staining with dihydrorhodamine that old yeast mother cells isolated by elutriation, but not young cells, contain ROS that are localized in the mitochondria. A relatively large proportion of the old mother cells shows phenotypic markers of yeast apoptosis, i.e. TUNEL (TdT-mediated dUTP nick end labelling) and annexin V staining. Although it has been shown previously that apoptosis in yeast can be induced by a cdc48 allele, by expressing pro-apoptotic human cDNAs or by stressing the cells with hydrogen peroxide, we are now showing a physiological role for apoptosis in unstressed but aged wild-type yeast mother cells.

The nucle(ol)ar Tif6p and Efl1p are required for a late cytoplasmic step of ribosome synthesis.

Deletion of elongation factor-like 1 (Efl1p), a cytoplasmic GTPase homologous to the ribosomal translocases EF-G/EF-2, results in nucle(ol)ar pre-rRNA processing and pre-60S subunits export defects. Efl1p interacts genetically with Tif6p, a nucle(ol)ar protein stably associated with pre-60S subunits and required for their synthesis and nuclear exit. In the absence of Efl1p, 50% of Tif6p is relocated to the cytoplasm. In vitro, the GTPase activity of Efl1p is stimulated by 60S, and Efl1p promotes the dissociation of Tif6p-60S complexes. We propose that Tif6p binds to the pre-60S subunits in the nucle(ol)us and escorts them to the cytoplasm where the GTPase activity of Efl1p triggers a late structural rearrangement, which facilitates the release of Tif6p and its recycling to the nucle(ol)us.

Brix from xenopus laevis and brx1p from yeast define a new family of proteins involved in the biogenesis of large ribosomal subunits.

A clone was isolated from a cDNA library from early embryos of Xenopus laevis that codes for a highly charged protein containing 339 amino acids. Two putative nuclear localization signals could be identified in its sequence, but no other known motifs or domains. Closely related ORFs are present in the genomes of man, C. elegans, yeast and Arabidopsis. A fusion protein with GFP expressed in HeLa cells or Xenopus oocytes was found to be localized in the nucleolus and coiled (Cajal) bodies. Moreover, immunoprecipitation experiments demonstrated that the new Xenopus protein interacts with 5S, 5.8S and 28S RNAs of large ribosomal subunits. The name Brix (biogenesis of ribosomes in Xenopus) is proposed for this protein and the corresponding gene. In Saccharomyces cerevisiae, the essential gene YOL077c, now named BRX1, codes for the Brix homolog, which is also localized in the nucleolus. Depletion of Brx1 p in a conditional yeast mutant leads to defects in rRNA processing, and a block in the assembly of large ribosomal subunits.

IgE-binding epitopes of enolases, a class of highly conserved fungal allergens.

BACKGROUND: Cladosporium herbarum and Alternaria alternata are two of the most prominent fungal species inducing type I allergy. Previously, we have demonstrated that enolase (Cla h 6) is the second most important allergen of C herbarum in terms of frequency of sensitization. OBJECTIVE: IgE-reactive B-cell epitopes of C herbarum enolase were analyzed, and cross-reactivity between fungal enolases was investigated. METHODS: Cla h 6 glutathione-S-transferase fusion peptides were constructed by means of PCR cloning. A alternata enolase (Alt a 5) was isolated by screening a complementary (c)DNA expression library with a C herbarum enolase DNA probe. RESULTS: Mapping of Cla h 6 IgE-binding epitopes identified a peptide with a length of 69 amino acids (peptide 9), which bound IgE from 8 of 8 patients. Analysis of the conformation of peptide 9 revealed that it does not form a compact structure but rather spans the whole length of the protein, with side chains exposed to solvent at 3 locations. Peptide 9 in the context of Escherichia coli glutathione-S-transferase not only binds IgE but also competitively inhibits IgE binding to Alt a 5. This result indicates that the epitope or epitopes on peptide 9 constitute a major cross-reacting epitope or epitopes on the enolases from C herbarum and A alternata in the case of the one patient tested. CONCLUSIONS: We demonstrated that the glycolytic enzyme enolase is an allergen not only in C herbarum but also in A alternata. Additionally, enolase was shown to exhibit high cross-reactivity to other fungal enolases. On the basis of the results presented here, we propose the use of recombinant Cla h 6 or maybe even peptide 9 of Cla h 6 for diagnosis and possibly therapy of mold allergy.

The influence of oxygen toxicity on yeast mother cell-specific aging.

The effect of deleting both catalase genes and of increased oxygen as well as paraquat (a pro-oxidant) on the replicative life span of yeast mother cells has been investigated to test the so-called oxygen theory of aging. This is well established in higher organisms, but has not been extensively tested in the unicellular yeast model system. Life span determinations were performed in ambient air or in a controlled atmosphere (55% oxygen) and an isogenic series of strains deleted for one or both yeast catalases was used and compared with wild type. In the absence of cellular catalase, increased oxygen caused a marked decrease in life span that could be completely reversed by adding 1 mM GSH, a physiological antioxidant, to the yeast growth medium. In a second unrelated strain, the effects were similar although even the wild type showed a decrease in life span when oxygen was increased. The effect could again be compensated by addition of extracellular GSH. Our results show that manipulating the detoxification of reactive oxygen species has a profound effect on yeast aging. These findings are discussed in the light of recent results relating to oxygen toxicity in the aging process of higher organisms.

Ultrasonic transmission in viscoelastic substances.

PURPOSE: To study the propagation of ultrasonic shock waves in viscoelastic agents and the resulting corneal load. SETTING: University Siegen, Institute for Mechanics and Control Engineering, Siegen, Germany. METHODS: The anterior chamber of a manufactured artificial eye was constructed according to anatomic dimensions. Three openings were drilled--for the phaco tip, for the exchange of a viscoelastic agent or water, and for the shock-wave sensor. The sensor was fixed to the area corresponding to the corneal apex. The sensor signal was analyzed using a direct oscilloscope that measured the amplitude reaching the corneal apex. Shock-wave propagation in several viscoelastic agents was compared with that in balanced salt solution. RESULTS: In hydroxypropyl methylcellulose, the shock wave was amplified or influenced slightly. In hyaluronic-acid preparations, acoustic dampening occurred. CONCLUSION: Removal of hyaluronic-acid derivatives prior to phacoemulsification is not necessary.

Reduced in vivo allergenicity of Bet v 1d isoform, a natural component of birch pollen.

BACKGROUND: The major allergen of birch pollen, Bet v 1, is present in structurally slightly different isoforms. It has been postulated that certain isoforms show a distinct ability to bind birch pollen-specific IgE, although the T-cell response remains similar. OBJECTIVE: We verified the hypothesis of a distinct allergenicity but similar T-cell immunogenicity of 2 isoforms in birch pollen-allergic subjects by in vivo tests and an in vitro assay for T-cell stimulation. METHODS: Forty-eight birch pollen-allergic, 11 grass pollen-allergic, and 10 nonatopic control individuals were tested with 10-fold increasing concentrations (0.01 to 10.0 microg/mL) of recombinant (r) Bet v 1a and rBet v 1d by skin prick test (SPT), intradermal test (IDT), and conjunctival provocation test (CPT). An allergen-specific proliferation assay was performed on 21 patients with the 2 recombinant and the natural birch pollen allergens. RESULTS: In each test system only birch pollen-allergic subjects but no controls reacted to the recombinant allergens. A positive in vivo response to 10 microg/mL of rBet v 1a was observed in 21 of 48 by SPT, in 48 of 48 by IDT, and in 33 of 48 by CPT. In contrast, the IDT response to 10 microg/mL of rBet v 1d was reduced by a factor of 100 because it was equivalent to the response to 0.1 microg/mL of rBet v 1a. rBet v 1d failed to elicit a positive reaction in SPT and CPT. The proliferative response of T cells was similar for both recombinant isoforms because 8 of 21 individuals reacted to rBet v 1a and 6 of 21 to rBet v 1d. Only 1 subject had a positive reaction to rBet v 1d alone. CONCLUSION: The natural isoforms rBet v 1a and rBet v 1d differ in their ability to bind IgE but are similar in their immunogenicity for T cells. Thus rBet v 1d might be a promising candidate for use in immunotherapy of birch pollen-allergic individuals.