Diagnosis of cypress pollen allergy: in vivo and in vitro standardization of a Juniperus ashei pollen extract.

BACKGROUND: Cypress pollen allergy is a major cause of rhinoconjunctivitis and asthma in the Mediterranean area. The nonstandardized cypress allergen extracts currently available for the diagnosis of cypress allergy have a low level of activity. The search for an active material has led to the selection of Juniperus ashei (Ja) pollen because of its very high cross-reactivity with cypress extracts and its superior allergenic activity. The aim of this study was to characterize in vitro and calibrate in vivo an in-house reference extract (IHRS) of J. ashei pollen and determine the specificity and sensitivity of a standardized Ja extract for the prick test diagnosis of cypress allergy. METHODS: Juniperus ashei pollen extract was analysed by 2-D electrophoresis. The IHRS Ja extract was calibrated by skin prick testing in 28 cypress-allergic patients. The sensitivity and specificity of cypress allergy diagnosis using a standardized Ja extract was studied by skin prick test in 42 cypress-allergic patients and 53 nonallergic patients. Jun a 1 content of the IHRS was determined by a monoclonal antibody-based electrophoretic technique. RESULTS: The Jun a 1 content of the 100 IR/ml Ja IHRS extract was 180 microg/ml. For in vivo diagnosis of cypress allergy, Ja pollen extract demonstrated a sensitivity of 95%, a specificity of 100%, a negative predictive value of 96%, and a positive predictive value of 100%. CONCLUSION: Standardized Ja pollen extract is therefore a very appropriate tool for the in vivo diagnosis of cypress pollen allergy and good candidate for specific immunotherapy.

[Juniperus ashei: the gold standard of the Cuppressaceae]. / Juniperus ashei: le golden standard des cupressacées.

The non-standardized Cupressus sempervirens allergen extract currently available for the diagnosis of cypress allergy has a low level of activity. The search for an active material consisted of in vitro and in vivo comparison of three Cupressaceae pollen extracts: Cupressus sempervirens (Cs), Cupressus arizonica (Ca) and Juniperus ashei (Ja) (synonyms: Juniperus sabinoides and Mountain Cedar). These 3 trees belong to the same botanical family of Cupressaceae. While Cs and Ca are commonly encountered in Mediterranean regions, Ja is only present in Europe in the Balkans, but is a major cause of allergy in the USA. In vitro, with a similar protein content, the allergenic properties of Ja extract are 20-Fold higher than those of Cs and 11-fold higher than those of Ca. IgE immunoblotting revealed 14, 42 and 70 kDa allergens common to all 3 extracts. The inhibition curves of the 3 extracts were more than 88% parallel. A significant correlation was observed between serum specific IgE titres for Ja and Cs in 23 patients (r = 0.916; p < 0.001). In vivo, in 23 patients with cypress allergy, the mean diameter of the prick test papule at 1/20 W/V of Ja (8.3 mm) was greater than that of the Cs papule (6.3 mm) (p = 0.001) and the Ca papule (6.7 mm) (p < 0.001). Correlations between cutaneous responses to Cs and Ja (r = 0.629; p = 0.002), and to Cs and Ca (r = 0.75; p = 0.001) were significant. These results demonstrate the intense cross-reactivity between Cs, Ca and Ja. The allergenic potency of the Ja extract is superior to that of Cs and Ca extracts, both in vitro and in vivo. This superiority is correlated with a high concentration of the major allergen, Jun a 1. The non-standardized The now standardized extract of in vitro ashei pollen therefore represents an effective and documented solution for identification, and probably for treatment, of Cupressaceae pollen allergy.

Latex allergy diagnosis: in vivo and in vitro standardization of a natural rubber latex extract.

For the diagnosis of IgE-mediated (immediate) hypersensitivity to natural rubber latex (NRL), skin prick testing with extracts of latex gloves has been widely used, but such extracts are difficult to standardize. The present study aimed to produce on an industrial scale an NRL extract from freshly collected NRL and to evaluate, calibrate, and standardize the extract by both in vivo and in vitro testing. The source material, latex of the rubber tree, Hevea brasiliensis (clone RRIM 600), was frozen immediately after collection in Malaysia and shipped in dry ice to Stallergènes SA, France. Protein and allergen profiles were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, isoelectric focusing (IEF), crossed immunoelectrophoresis (CIE), and crossed radioimmunoelectrophoresis (CRIE). Allergen quantification was effected by RAST inhibition. The capacity of the preparation to elicit immediate hypersensitivity reactions in vivo was measured by skin prick testing in 46 latex-allergic patients and 76 nonallergic control subjects. SDS-PAGE and immunoblot profiles of the extract and an NRL standard (E8) provided by the US Food and Drug Administration were almost identical, disclosing several distinct IgE-binding proteins with apparent molecular weights of 14, 20, 27, 30, and 45 kDa, conforming to reported molecular weights of several significant NRL allergens. An arbitrary index of reactivity (IR) of 100 was assigned to the extract at 1:200 dilution (w/v), having a protein content of 22 micrograms/ml. Skin prick testing of latex-allergic patients and controls using the extract at 100 IR revealed 93% sensitivity, 100% specificity, 100% negative predictive value, and 96% positive predictive value. In conclusion, a skin prick test reagent for diagnosis of type I NRL allergy was successfully standardized. The reagent was demonstrated to contain most, if not all, of the currently known clinically significant NRL allergens, and it showed high sensitivity and specificity.

A double-blind, placebo-controlled trial by the sublingual route of immunotherapy with a standardized grass pollen extract.

Fifty-eight patients with well-documented history of seasonal rhinoconjunctivitis caused by grass pollens were allocated randomly on a double-blind basis to receive either sublingual therapy with a solution of purified, standardized allergen preparation (Stallergènes) or a matched placebo for 17 weeks. The assessment of the effect of oral immunotherapy, done with drops of five-grass allergen extract, was on the clinical symptoms and on the medication score of the authorized rescue treatments. The actively treated patients had significantly (P < 0.05 to P < 0.01) fewer symptoms of rhinitis (sneezing and rhinorrhea) and of conjunctivitis (redness and tears) during the pollen season than the placebo group. Consumption of nasal solution of sodium cromoglycate and of betamethasone and dexchlorpheniramine was significantly less in the desensitized group (P < 0.01). Side-effects were negligible. This study concludes that perlingual immunotherapy with grass pollen extract in grass-pollen-sensitive seasonal hay fever and conjunctivitis patients is effective, easy to perform, inexpensive, and safe.

[Sublingual specific immunotherapy for rhinoconjunctivitis caused by grass pollens]. / Immunothérapie spécifique par voie sublinguale de la rhinoconjonctivite aux pollens de graminées.

Fifty eight patients with rhino-conjunctivitis caused by grass pollen were included in a double-blind study in which they received, by the sub-lingual route over 5 months, either a solution of purified and standardised allergen or a placebo. Assessment of the effect of this immunotherapy, which was done with drops of Stallergenes "5-grass pollen" was by clinical symptoms and the use of authorized drugs and treatments. Compared with the placebo group, the patients on active treatment showed significantly less (P = 0.05 to P = 0.01) rhinitis symptoms (sneezing and rhinorrhea) and conjunctivitis (reddening and tears) during the pollen season. Consumption of nasal cromoglycate solution, of betamethasone and dexchlorpheniramine was significantly lower (p = 0.01) in the desensitised group. Secondary effects were negligible. From this study, it can be concluded that immunotherapy with grass pollen extract, by the sub-lingual route, of patients with rhino-conjunctivitis who were sensitive to these allergens, is efficacious, easy to do, economic and sure.