Clinical trial to assess tolerability and subrogate efficacy effects of an abbreviated schedule with house dust mites mixture subcutaneous immunotherapy.

Summary: Objective. To evaluate the tolerability and efficacy of Dermatophagoides pteronyssinus/Dermatophagoides farinae mixture subcutaneous immunotherapy (SCIT). Methods. Patients received an abbreviated build-up schedule. The aims were: number, percentage, and severity of adverse reactions. Secondary outcomes included: changes in immunoglobulin titers and changes in dose-response skin prick tests. Results. Out of 289 administrations, 17% elicited any clinically relevant adverse reaction. Most of them were local reactions (LR) (9.4%) and the rest (7.6%) were systemic. Significant increases in sIgG and sIgG4 were detected in serum samples. Cutaneous reactivity decreased significantly. Conclusions. SCIT with house dust mites mixture of ROXALL Medicina España S.A. seems to have an acceptable tolerability profile, induces blocking IgG and decreases skin reactivity.

Clinical assessment of tolerability, immunological and cutaneous reactivity effects of an abbreviated schedule with Olea europaeanative extract of subcutaneous immunotherapy.

Summary: Objectives. To evaluate the tolerability and efficacy of Olea europaea subcutaneous immunotherapy (SCIT) on patients with rhinoconjunctivitis. Methods. In this open clinical trial patients were assigned to an abbreviated build-up scheme. The outcomes were: number, percentage, and severity of adverse reactions. Secondary outcomes included: changes in immunoglobulin titers and changes in dose-response skin prick tests. Results. Only 8 systemic reactions were registered, which represented 7/47 (14.9%) of patients and 8/429 (1.9%) of administered doses. Regarding immunological parameters the significant increases of sIgG and sIgG4 evidenced the changes in the patient immune system. Cutaneous reactivity decreased significantly. Conclusions. Olea europaea SCIT (Allergovac® depot ROXALL Medicina España S.A.) showed a good safety and tolerability profile. Immunological changes with induction of blocking IgG and decreases in cutaneous reactivity were detected in the patients.

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.

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.

In vivo and in vitro testing with rAni s 1 can facilitate diagnosis of anisakis simplex allergy

Background: Traditional diagnostic tests such as skin prick tests (SPT) and specific IgE (sIgE) against whole Anisakis simplex extract have low specificity. Consequently, allergy to A simplex is overdiagnosed. Objective: Our aim was to compare tests used in component-resolved diagnosis. Methods: We evaluated 34 patients with allergy to A simplex, 15 patients with acute urticaria who were sensitized to A simplex but had no clinical history of allergy to A simplex, and 10 patients allergic to seafood. SPT, sIgE (ELISA and ISAC-112), and the basophil activation test (BAT) were performed with A simplex whole extract and the molecular components rAni s 1, rAni s 3, and nPen m 1. Sensitivity and specificity were calculated and compared with different cutoffs. Results: With the A simplex whole extract, SPT, sIgE, and BAT yielded specificity values of 72%, 68%, and 70%, respectively, with a cutoff (wheal size) of 11.2 mm, an sIgE value of 7.9 kUA/L, and a stimulation index of 1.9. Specificity increased to 100% using the molecular component rAni s 1 with SPT, sIgE by ELISA, and ISAC-112. Neither rAni s 3 sensitization nor cross-reactivity with Pen m 1 was observed in patients sensitized to A simplex. Conclusion: rAni s 1 is recognized by 100% of our patients and is able to distinguish between patients allergic to A simplex and patients with acute urticaria who are sensitized to A simplex but have no clinical history of allergy to this parasite (AU)

Introducción: Las pruebas diagnósticas tradicionales como pruebas cutáneas (PC) e IgE específica (sIgE) con el extracto completo de Anisakis simplex tienen una baja especificidad. Esto conlleva a un sobrediagnóstico de alergia a A simplex. Objetivo: Nuestro objetivo fue comparar diferentes pruebas de diagnóstico basado en componentes moleculares. Métodos: Se estudiaron 34 pacientes con alergia a A simplex, 15 con urticaria aguda sensibilizados a A simplex pero sin historia clínica compatible con alergia a A simplex y 10 alérgicos a mariscos. A todos ellos se les realizaron PC, sIgE mediante ELISA e ISAC-112 y TAB con el extracto completo de A simplex y los componentes moleculares rAni s 1, rAni s 3 y nPen m 1. Se calculó y se comparó la sensibilidad y especificidad de cada prueba con diferentes puntos de corte. Resultados: Las PC, la sIgE y el TAB con el extracto completo de A simplex mostraron una especificidad del 72%, 68% y 70% con un punto de corte de 11,2 mm de tamaño de pápula, 7,9 kUA/L de sIgE y un índice de estimulación de 1,9, respectivamente. La especificidad incrementó al 100% utilizando el componente rAni s 1en PC e sIgE mediante ELISA e ISAC-112. No se observó sensibilización a rAni s 3 ni reactividad cruzada con nPen m 1 en los pacientes sensibilizados a A simplex. Conclusión: el alérgeno rAni s 1 es reconocido por el 100% de nuestros pacientes y nos permite distinguir entre pacientes alérgicos a A simplex y pacientes con urticaria aguda sensibilizados a A simplex sin historia clínica de alergia a éste parásito (AU)

In vivo and in vitro testing with rAni s 1 can facilitate diagnosis of Anisakis simplex allergy.

BACKGROUND: Traditional diagnostic tests such as skin prick tests (SPT) and specific IgE (slgE) against whole Anisakis simplex extract have low specificity. Consequently, allergy to A simplex is overdiagnosed. OBJECTIVE: Our aim was to compare tests used in component-resolved diagnosis. METHODS: We evaluated 34 patients with allergy to A simplex, 15 patients with acute urticaria who were sensitized to A simplex but had no clinical history of allergy to A simplex, and 10 patients allergic to seafood. SPT, slgE (ELISA and ISAC-I 12), and the basophil activation test (BAT) were performed with A simplex whole extract and the molecular components rAni s 1, rAni s 3, and nPen m 1. Sensitivity and specificity were calculated and compared with different cutoffs. RESULTS: With the A simplex whole extract, SPT, slgE, and BAT yielded specificity values of 72%, 68%, and 70%, respectively, with a cutoff (wheal size) of 11.2 mm, an slgE value of 7.9 kUAIL, and a stimulation index of 1.9. Specificity increased to 100% using the molecular component rAni s 1 with SPT, slgE by ELISA, and ISAC-112. Neither rAni s 3 sensitization nor cross-reactivity with Pen m 1 was observed in patients sensitized to A simplex. CONCLUSION: rAni s 1 is recognized by 100% of our patients and is able to distinguish between patients allergic to A simplex and patients with acute urticaria who are sensitized to A simplex but have no clinical history of allergy to this parasite.

Diagnostic utility of components in allergy to Anisakis simplex.

BACKGROUND: In our region, Anisakis allergy is responsible for 8% of acute urticarial reactions, 25% of which progress to anaphylactic shock. The poor specificity of skin tests and in vitro specific immunoglobulin (Ig) E means that Anisakis allergy is frequently overdiagnosed. OBJECTIVE: We studied the diagnostic value of 2 Anisakis allergens: rAni s 1 and rAni s 3. METHODS: Skin tests, the basophil activation test (BAT), and specific IgE determination were performed with rAni s 1 and 3 in 25 patients allergic to Anisakis, 17 atopic controls, and 10 controls with acute urticaria and positive skin test and sIgE results for Anisakis, but no allergy to Anisakis. RESULTS: For rAni s1, skin tests had a sensitivity and specificity of 100% and specific IgE had a sensitivity and specificity of 100% in the atopic control group and 90% in the urticaria control group. BAT had a sensitivity of 96.8% and a specificity of 100% in the atopic control group and 66.7% in the urticaria control group. For rAni s 3, only 1 patient had positive specific IgE results to rAni s 3. All other techniques gave negative results in patients and controls CONCLUSIONS: rAni s 1 is the major allergen of Anisakis and the target allergen when diagnosing allergy to Anisakis, rAni s 3 is not relevant when attempting to explain false-positive results.

Neutrophil defensins: their possible role in allergic asthma.

BACKGROUND: Neutrophil defensins, originally identified as broad-spectrum antimicrobial peptides, have been implicated in the regulation of inflammatory and immunological processes. OBJECTIVES: To investigate whether the in vitro challenge of neutrophils from patients with bronchial asthma with allergens stimulated the release of alpha-defensins and whether levels released were dependent on lung infections. METHOD: The neutrophils were cultivated with different agonists and the concentration of alpha-defensin in cell-free supernatant was measured with enzyme-linked immunosorbent assay (ELISA). RESULTS: Neutrophils from allergic patients released alpha-defensins via an allergen-dependent mechanism. Our results indicate that the in vitro activation of neutrophils is highly allergen-specific. In this context, allergens other than those which produced clinical symptoms did not elicit alpha-defensin release, and allergens had no effect on neutrophils from healthy donors. However, neutrophils from both allergic patients and healthy controls were able to release alpha-defensins upon treatment with PMA. In the allergen-stimulated neutrophils, cells from asthmatic patients stimulated with a sensitizing allergen showed a significantly higher production of alpha-defensin under respiratory tract infection than cells from the same patients without such an infection. CONCLUSION: Neutrophils from allergic patients release alpha-defensins via an allergen-dependent mechanism.

Cupressus arizonica pollen: a new pollen involved in the lipid transfer protein syndrome?

BACKGROUND: Lipid transfer proteins (LTP) are responsible for systemic manifestations in food allergy. Their relationship with pollinosis is not clear. In our area, many patients allergic to multiple LTP-containing foods present pollinosis due to Cupressus arizonica. METHODS: We selected 6 patients with cypress pollinosis and food allergy to peach. Skin prick tests (SPT) were performed for pollens (grass, cypress, wall pellitory, plane tree, and olive tree) and plant foods (hazelnut, kiwifruit, peach peel, maize, wheat, peanut, lettuce, apple, mustard, and melon). In vitro assays included specific immunoglobulin (Ig) E to C arizonica and peach LTP (Pru p 3), enzyme allergosorbent test (EAST) inhibition, immunoblotting, immunoblotting-inhibition, and immunocytochemical techniques for the detection of Pru p 3-like LTP in cypress pollen grains. RESULTS: SPT were positive for C arizonica, peach, lettuce, mustard, and hazelnut in all patients. Specific IgE to C arizonica and Pru p 3 was positive in all but 1 patient, whose Pru p 3 IgE was negative. Immunoblotting under nonreducing conditions with C arizonica extract and patients' sera showed a band at 14-15 kDa that was inhibited by Pru p 3. Pru p 3 partially inhibited the C arizonica pollen extract in EAST-inhibition. Pru p 3-like LTP was localized in the cytoplasm and walls of C arizonica pollen grains. CONCLUSION: A 15-kDa allergen in C arizonica pollen was found in a group of patients presenting peach allergy and respiratory symptoms to cypress. In vitro tests and immunocytochemical techniques indicate that this protein is an LTP.

The Russian Thistle (Salsola kali) pollen major allergen, Sal k 1, can be quantified in allergenic extracts and airborne pollen.

BACKGROUND: Russian thistle (Salsola kali) pollen is an important cause of pollinosis in areas where rainfall is not abundant. Our aim was to develop an ELISA for quantification of the major allergen of S. kali extracts, Sal k 1, and to assess the correlation of this allergen content with the allergenic activity of extracts. METHODS: Sal k 1 was purified by ion exchange and gel permeation chromatography and identified by mass spectrometry. Monoclonal antibody 4C11 was used for capture at 5 microg/ml and biotin-labeled specific antiserum at 0.25 microg/ml served for detection. The allergenic activity of the pollen extracts was measured by enzyme allergosorbent test inhibition. RESULTS: Sal k 1 reacted to 85% of sera from 40 S. kali-allergic patients and was able to inhibit 92% of the IgE-binding capacity of patients' serum pool to the whole extract. The ELISA had a lineal range between 1.25 and 20 ng/ml of purified Sal k 1. The intra- and interassay coefficients of variation were lower than 5 and 10%, respectively. The assay was very sensitive since it had a detection limit of 0.08 ng/ml. No reactivity was found outside the Amaranthaceae family where only Kochia and Salicornia sp. gave significant reactivity. A good correlation (Spearman's rho = 0.92) was obtained between Sal k 1 content of different S. kali extracts and their IgE-binding activity. CONCLUSIONS: The results proved the usefulness of the two-site sandwich ELISA for aeroallergen control and for the standardization of S. kali pollen extracts intended for clinical use.

Engineering of major house dust mite allergens Der p 1 and Der p 2 for allergen-specific immunotherapy.

BACKGROUND: Specifically designed recombinant allergens with reduced IgE reactivity are promising candidates for a more defined, effective, and safer specific immunotherapy (SIT). OBJECTIVE: We sought to obtain hypoallergenic hybrid molecules which could potentially be applied to house dust mite (HDM) allergy treatment. METHODS: Two hybrid molecules (QM1 and QM2) derived from the two major Dermatophagoides pteronyssinus allergens, Der p 1 and Der p 2, were engineered by PCR, produced in Escherichia coli, and purified. The overall IgE-binding capacity of the hybrids was compared with their single components by Western blot, specific IgE, skin prick test (SPT), and IgE-inhibition assays. T cell proliferation assay were performed to confirm their retention of T cell reactivity. Immune responses to the hybrid molecules were studied in BALB/c mice. RESULTS: The IgE reactivity of both hybrid proteins was strongly reduced as evaluated by in vitro methods. Furthermore, in vivo SPTs performed on 106 HDM-allergic patients showed that the hybrid proteins had a significantly lower potency to induce cutaneous reactions than the individual components. Hybrid molecules induced higher T cell proliferation responses than those produced by an equimolecular mixture of Der p 1 and Der p 2. Immunization of mice with the hybrid proteins induced Der p 1- and Der p 2-specific IgG, which inhibited the binding of allergic patients' IgE to these natural allergens. CONCLUSION: QM1 and QM2 hybrids exhibited less IgE-binding activity but preserved immunogenicity and fulfilled the basic requirements for hypoallergenic molecules suitable for a future SIT of HDM allergy.

Guidelines on the clinical usefulness of determination of specific immunoglobulin E to foods.

The diagnostic gold standard for food allergy is challenge with the culprit food, particularly in double-blind placebo-controlled challenge. This approach involves risks and consumes both time and resources. A more efficient system would be desirable. The detection of serum specific immunoglobulin E (sIgE) against the culprit food enables us to establish sensitization, although this is not always accompanied by clinical reactivity. Age, symptoms (immediate/late reaction, local/systemic reaction), concomitant condition (eg, atopic dermatitis, pollinosis) and selection sample criteria (eg, presence of symptoms related to ingestion, positive skin prick test result) can influence the detection and concentration of IgE against foods. We analyze the clinical usefulness of sIgE determination in light of studies in which oral food challenge is used as the diagnostic method. We review clinical usefulness at diagnosis and in the decision to reintroduce the food, as well as the prognostic value of the determination of IgE to foods.

Expression of a recombinant protein immunochemically equivalent to the major Anisakis simplex allergen Ani s 1.

BACKGROUND: Anisakis simplex is a nematode which can parasitize humans, producing anisakiasis and can induce immunoglobulin-(Ig)-E-mediated allergic symptoms. Parasite recombinant proteins, such as the major allergen Ani s 1, may be useful tools to avoid misdiagnosis of A simplex allergy due to cross-reactivity when whole parasite extracts are used. OBJECTIVE: To obtain Ani s 1 allergen as a recombinant protein with IgE-binding properties similar to its natural counterpart. METHODS: Ani s 1-encoding cDNA was amplified by polymerase chain reaction and cloned. The allergen was expressed in Escherichia coli as a nonfusion protein. Natural and recombinant Ani s 1 were investigated by means of Western blotting, enzyme allergosorbent test, enzyme-linked immunosorbent assay (ELISA), and ELISA inhibition using sera from 53 patients with A simplex allergy. RESULTS: Residues of the amino acid sequence of the encoded protein were 99.4% identical to the reported one. Purified rAni s 1 was obtained with a yield of 2 mg/L of culture while the yield of the natural counterpart was only 50 micro/g of larvae. rAni s 1 reactivity was not significantly different from that of the natural allergen; the correlation was excellent (p = 0.92, P < .001). ELISA-inhibition experiments showed that the dose-response inhibition curve obtained with rAni s 1 overlapped with that of nAni s 1. In an enzyme allergosorbent analysis, 86.8% of the A simplex-allergic patient sera reacted to rAni s 1. CONCLUSION: Recombinant Ani s 1 is immunochemically equivalent to its natural counterpart and therefore might be useful for the in vitro diagnosis of anisakiasis and A simplex-mediated allergy.

An antibody-based ELISA for quantification of Ani s 1, a major allergen from Anisakis simplex.

Anisakis simplex is a nematode parasite that can infect humans who have eaten raw or undercooked seafood. Larvae invading the gastrointestinal mucosa excrete/secrete proteins that are implicated in the pathogenesis of anisakiasis and can induce IgE-mediated symptoms. Since Ani s 1 is a potent secreted allergen with important clinical relevance, its measurement could assess the quality of allergenic products used in diagnosis/immunotherapy of Anisakis allergy and track the presence of A. simplex parasites in fish foodstuffs. An antibody-based ELISA for quantification of Ani s 1 has been developed based on monoclonal antibody 4F2 as capture antibody and biotin-labelled polyclonal antibodies against Ani s 1 as detection reagent. The dose-response standard curves, obtained with natural and recombinant antigens, ranged from 4 to 2000 ng/ml and were identical and parallel to that of the A. simplex extract. The linear portion of the dose-response curve with nAni s 1 was between 15 and 250 ng/ml with inter-assay and intra-assays coefficients of variation less than 20% and 10%, respectively. The assay was specific since there was no cross-reaction with other extracts (except Ascaris extracts) and was highly sensitive (detection limit of 1.8 ng/ml), being able to detect Ani s 1 in fish extracts from codfish and monkfish.

Cloning, expression and characterization of mugwort pollen allergen Art v 2, a pathogenesis-related protein from family group 1.

Mugwort (Artemisia vulgaris) belongs to the Compositae family, and is one of the main causes of allergy in late summer and autumn. The aim of the study was to characterize the allergen Art v 2 from mugwort pollen. Skin prick tests, performed in 19 patients allergic to mugwort and 10 control patients, showed an Art v 2 sensitization prevalence of 58%, whereas none false-positives were detected among control patients. Art v 2 was purified by standard chromatography and binding to Concanavalin A column and had an apparent molecular mass of 33 and 20 kDa, calculated by gel permeation and SDS-PAGE under denaturing conditions, respectively, showing that the allergen is composed of two identical subunits. Art v 2-encoding cDNA was amplified by PCR using degenerate primers based on reported partial amino acid sequences. Cloned cDNA encoding Art v 2 contains 140 bp that codify for a polypeptide of 15.8 kDa, with a predicted pI value of 5.2, and one potential N-glycosylation site. Protein homology search demonstrated that Art v 2 share 55-42% identical residues with pathogenesis-related protein PR-1 of tomato, potato, rape, wheat and rice. Homology was also found to Ves v 5 (41% identical residues). Bacterial-expressed recombinant Art v 2 was recognized only by 21% of mugwort-allergic patients. In conclusion, Art v 2 from mugwort is the first weed pollen allergen that belongs to the pathogenesis-related protein PR-1 and its recombinant form could help molecular diagnosis of mugwort associated allergy.

Tolerance and immunological changes of chemically modified allergen vaccine of Parietaria judaica in accelerated schedules.

The physicochemical modification of allergen vaccines provides a chance for administering higher doses in a shorter period of time. We sought to assess the safety and immunological changes of using a biologically standardized and modified Parietaria judaica pollen extract in accelerated schedules. Two accelerated schedules were tested in 45 P. judaica-allergic patients: 20 patients reached the maximum dose after two visits using two different concentrations and 25 patients reached the maximum dose after only one visit with two injections of the maximum concentration vial. The tolerance was assessed by recording all side effects related with immunotherapy. Specific antibody levels against native extract and rPar j 2 allergen were evaluated at the beginning and the end of the study. Allergenic potency determined by enzyme allergosorbent test (EAST) inhibition and skin prick test showed that modified P. judaica pollen had a 99.9% less allergenicity than native extract. After 650 doses administered, two clinically irrelevant local reactions (diameter<0 x 5 cm) and no systemic reactions were registered. Significant increases in allergen-specific IgG4 and IgG against P. judaica extract and rPar j 2 and significant decrease of specific IgE against Par j 2 were observed. The modified extract of P. judaica is safe to treat sensitive patients, even at accelerated regimens, and induces significant immunological changes.

Diagnosis of Parietaria judaica pollen allergy using natural and recombinant Par j 1 and Par j 2 allergens.

BACKGROUND: Parietaria judaica pollen is one of the main causes of allergic diseases in the Mediterranean area and contains two major allergens, called Par j 1 and Par j 2. OBJECTIVE: To evaluate the diagnostic potential of natural and recombinant forms of Par j 1 and Par j 2 in comparison with standardized P. judaica pollen extract. METHODS: Thirty patients allergic to P. judaica pollen and 15 control patients were investigated. Skin prick tests and determination of specific IgE levels were performed with commercial P. judaica extract, natural Par j 1 and Par j 2, and recombinant forms of both allergens expressed in P. pastoris. RESULTS: The whole group of patients with allergy to P. judaica had a positive skin test reaction to purified nPar j 1-Par j 2 and rPar j 2 at 5 microg/mL, and no false-positive reactions were detected. Natural and recombinant Par j 1 and Par j 2 showed no significantly different responses in skin tests compared with P. judaica extract. A high correlation was found between the serum-specific IgE levels to P. judaica extract vs. natural (R=0.996; P<0.001) and recombinant allergens (R=0.887 and 0.982 for rPar j 1 and rPar j 2, respectively; P<0.001). rPar j 2 displayed a 100% sensitivity and specificity among P. judaica-allergic patients. CONCLUSIONS: In vivo and in vitro diagnosis of P. judaica pollen allergy could be simplified using rPar j 2. This protein showed comparable IgE response and skin prick reactivity with those produced by P. judaica pollen extract.

Purified allergens vs. complete extract in the diagnosis of plane tree pollen allergy.

BACKGROUND: Plane tree pollen allergy is a clinical disorder affecting human population in cities of Europe, North America, South Africa, and Australia. OBJECTIVE: To compare IgE-reactivity of the natural and recombinant forms of two major plane allergens, Pla a 1 and Pla a 2, with the reactivity of Platanus acerifolia pollen extract. METHODS: Forty-seven patients with P. acerifolia allergy, 15 of them monosensitized, and 24 control subjects were included in the study. Natural Pla a 1 and Pla a 2 were purified by standard chromatographic methods and recombinant proteins were expressed in Escherichia coli. Skin prick test and determination of specific IgE were performed with commercial P. acerifolia extract and natural and recombinant purified allergens. RESULTS: Pla a 1 and Pla a 2 were responsible for 79% of the IgE-binding capacity against P. acerifolia pollen extract. A high correlation has been found between the IgE response to nPla a 1 (R = 0.80; P < 0.001) or nPla a 2 (R = 0.79; P < 0.001) vs. P. acerifolia extract as well as between natural and recombinant Pla a 1 (R = 0.89; P < 0.001). Skin testing showed no significant differences between extract and nPla a 2, whereas a higher reactivity was found with nPla a 1. In contrast, rPla a 1 revealed markedly reduced sensitivity in comparison with extract by skin prick test and specific IgE. The sensitivity of the mix Pla a 1+Pla a 2 was 100% and 87.5% for monosensitized and polysensitized patients, respectively, with no false-positive reactions detected. Conclusion Pla a 1 and Pla 2 are sufficient for a reliable diagnosis of P. acerifolia in most patients and induce comparable skin test reactivity as a whole extract.

Biological standardization and maximum tolerated dose estimation of an Alternaria alternata allergenic extract.

BACKGROUND: The manufacture of allergenic extracts from the mold Alternaria alternata is influenced by factors such as strain variability, allergenic origin, culturing conditions and extraction process, which affect the reproducibility of the preparations intended for diagnostic and therapeutic use. OBJECTIVES: To select the most adequate antigenic source of A. alternata extracts and determine its maximum tolerated dose (MTD) to be used in a subsequent immunotherapy efficacy clinical trial. METHODS: Twenty-one patients monosensitized to A. alternata were involved in a biological standardization process of A. alternata extracts. Four different mold strains were cultured and used to produce extracts by three different methods, each incorporating proteins from different origins: culture filtrate, buffer extractable fraction and cellular antigens. The selected extract, characterized as in-house reference (IHR) preparation was used in a MTD finding immunotherapy study. Serum IgE, IgG, IgG1 and IgG4 specific of complete extract and purified natural and recombinant forms of Alt a 1 were determined by different EIA methods. RESULTS: Culture filtrate extract containing the allergens secreted to the spent medium was shown to be the most adequate option for establishing an IHR preparation for A. alternata extract manufacturing. A maximum dose of 1670 UBE, equivalent to 0.1 microg Alt a 1, was determined as MTD for immunotherapy. One year of administration of such a dose at monthly intervals elicited pronounced immunological changes with statistically significant decreases in IgE and increases in IgG4, both estimated with whole extract or purified Alt a 1. CONCLUSION: A high quality natural A. alternata extract has been developed and preliminarily tested to define its MTD for subsequent determination of the optimal dose in an immunotherapy efficacy clinical trial.

The major allergen of Alternaria alternata (Alt a 1) is expressed in other members of the Pleosporaceae family.

There is general consensus regarding the scarce cross-reactivity existing between Alternaria alternata and other allergenic moulds such as Aspergillus fumigatus, Penicillium notatum or Cladosporium herbarum. However, A. alternata has been shown to have a very significant level of allergenic cross-reactivity with other fungi belonging to the Pleosporaceae family. To date, no biological identity or homologies with other proteins have been described for Alt a 1, and it remains unclear whether the major allergen Alt a 1 contributes to the cross-reactivity shown for these moulds. Specific quantification of Alt a 1 in culture filtrates of Stemphylium botryosum, Ulocladium botrytis, Curvularia lunata, Alternaria tenuissima, C. herbarum, Penicillium chrysogenum and Asp. fumigatus, and immunoblotting using culture filtrate extracts from the above-mentioned moulds and rabbit serum anti-recombinant Alt a 1 have shown significant amounts of Alt a 1 in culture filtrates as well as antigenic components ranging from 14 to 20 kDa that strongly react with the specific serum for all taxonomically related species (Pleosporaceae family). No reactions were revealed in culture filtrates of Cladosporium, Penicillium and Aspergillus. These results restrict the cross-reactivity phenomenon due to Alt a 1 to the scope of the taxonomical term of family.