Sensitization to Gibberellin-Regulated Protein (Peamaclein) Among Italian Cypress Pollen-Sensitized Patients.

BACKGROUND AND OBJECTIVES: Peach gibberellin-regulated protein (peamaclein) has recently emerged as a relevant food allergen in cypress pollen-hypersensitive patients. Objective: We investigated monosensitization to peamaclein among Italian cypress pollen-allergic patients. MATERIAL AND METHODS: A total of 835 cypress pollen-hypersensitive patients from 28 Italian allergy centers underwent a thorough work-up to determine food-allergic reactions and performed skin prick testing with a commercial peach extract containing peamaclein. IgE to rPru p 3 was measured in peach reactors, and those with negative results were enrolled as potentially monosensitized to peamaclein. IgE reactivity to rPru p 7 was evaluated using immunoblot and an experimental ImmunoCAP with rPru p 7. RESULTS: Skin prick tests were positive to peach in 163 patients (19.5%); however, 127 (77.9%) were excluded because they reacted to Pru p 3. Twenty-four patients (14.7%) corresponding to 2.8% of the entire study population) were considered potentially monosensitized to peamaclein. No geographic preference was observed. Seventeen of the 24 patients (70.8%) had a history of food allergy, mainly to peach (n=15). Additional offending foods included other Rosaceae, citrus fruits, fig, melon, tree nuts, and kiwi. On peach immunoblot, only 3 of 18 putative peamaclein-allergic patients reacted to a band at about 7 kDa; an additional 4 patients reacted at about 50-60 kDa. Ten of 18 patients (56%) had a positive result for Pru p 7 on ImmunoCAP. CONCLUSION: Allergy and sensitization to peamaclein seem rare in Italy. Most patients react to peach, although other Rosaceae fruits and several citrus fruits may also be offending foods. Peach and cypress pollen probably also share cross-reacting allergens other than peamaclein.

Evaluation of two commercial peach extracts for skin prick testing in the diagnosis of hypersensitivity to lipid transfer protein. A multicenter study.

Summary: The clinical usefulness of two commercial peach extracts for SPT (by Lofarma SpA and ALK-Abellò, respectively) was compared in a multicenter study carried out in Italy. Peach allergic patients were tested with the two extracts in parallel and underwent the detection of IgE specific for all three peach allergens currently available (Pru p1, Pru p3, and Pru p4, respectively). The two extracts were almost identical in terms of sensitivity and specificity, being able to detect virtually all patients sensitized to stable peach allergens (lipid transfer protein (LTP) and, presumably, peamaclein) but scoring negative in patients exclusively sensitive to labile allergens (either PR-10 and/or profilin). Thus, the two extracts represent an excellent tool to carry out a preliminary component-resolved diagnosis of peach allergy at the first patient visit.

The role of allergoids in allergen immunotherapy: from injective to sublingual route.

Summary: Summary Allergen immunotherapy (AIT) is aimed at inducing tolerance to allergens, such as pollens, dust mites or moulds, by administering increasing amounts of the causative allergen through subcutaneous or sublingual route. The evidence of efficacy of AIT is high, but the issue of safety, especially for the subcutaneous route, must be taken into account. The search for safer AIT products aimed at reducing the allergenicity, and thus adverse reactions, while maintaining the immunogenicity, that is essential for effectiveness, gave rise to the introduction of allergoids, which were conceived to fulfill these requirements. In the first allergoids glutaraldehyde or formaldehyde were used as cross-linking agent to polymerize allergens, this resulting in high molecular weight molecules (200,000 to 20,000,000 daltons) which were significantly less allergenic due to a decreased capacity to bridge IgE on its specific receptor, while maintaining the immunogenicity and thus the therapeutic efficacy. In recent years further agents, acting as adjuvants, such as L-tyrosine, monophosphoryl lipid A, aluminium hydroxide, were added to polymerized extracts. Moreover, a carbamylated monomeric allergoid was developed and, once adsorbed on calcium phosphate matrix, used by subcutaneous route. At the same time, in virtue of its peculiarities, such allergoid revealed particularly suitable for sublingual administration. A lot of clinical evidences show that it is well tolerated, largely safer and effective. Importantly, the higher safety of allergoids allows faster treatment schedules that favor patient compliance and, according to pharmaco-economic studies, they might be more cost-effective than other AIT options.

Allergen Immunotherapy in the Era of SARS-CoV-2

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Shrimp Allergy: Analysis of Commercially Available Extracts for In Vivo Diagnosis.

BACKGROUND AND OBJECTIVE: Skin prick testing (SPT) with commercial extracts is the first step in the diagnosis of shrimp allergy, although its clinical efficiency is unknown. Objective: To analyze the clinical usefulness of all commercial crustacean extracts available for SPT in Italy. METHODS: We performed a multicenter study of 157 shrimp-allergic patients who underwent SPT with 5 commercial crustacean extracts and with house dust mite (HDM) extract. Commercial extracts were analyzed using SDS-PAGE and compared with a freshly prepared in-house shrimp extract. IgE to Pen a 1/Pen m 1, Pen m 2, and Pen m 4 was determined, and immunoblot analysis was performed on a large number of sera. RESULTS: The skin reactions caused by commercial crustacean extracts were extremely heterogeneous, resulting in 32 clinical profiles, with marked differences in protein content and missing proteins at molecular weights corresponding to those of major shrimp allergens. Only strong Pen a 1/Pen m 1 reactors reacted to both HDM and all 5 commercial extracts in SPT. Most patients, including those who were tropomyosin-negative, reacted to HDM. Patients reacted to a large and variable array of proteins, and IgE reactivity was common at high molecular weights (>50 kDa). CONCLUSIONS: The in vivo diagnosis of shrimp allergy must continue to be based on SPT with fresh material. Shrimp-allergic patients frequently react to a number of ill-defined high-molecular-weight allergens, thus leaving currently available materials for component-resolved diagnosis largely insufficient. Mites and crustaceans probably share several allergens other than tropomyosin.

Shrimp allergy: analysis of commercially available extracts for in vivo diagnosis

Background: Skin prick testing (SPT) with commercial extracts is the first step in the diagnosis of shrimp allergy, although its clinical efficiency is unknown. Objective: To analyze the clinical usefulness of all commercial crustacean extracts available for SPT in Italy. Methods: We performed a multicenter study of 157 shrimp-allergic patients who underwent SPT with 5 commercial crustacean extracts and with house dust mite (HDM) extract. Commercial extracts were analyzed using SDS-PAGE and compared with a freshly prepared in-house shrimp extract. IgE to Pen a 1/Pen m 1, Pen m 2, and Pen m 4 was determined, and immunoblot analysis was performed on a large number of sera. Results: The skin reactions caused by commercial crustacean extracts were extremely heterogeneous, resulting in 32 clinical profiles, with marked differences in protein content and missing proteins at molecular weights corresponding to those of major shrimp allergens. Only strong Pen a 1/Pen m 1 reactors reacted to both HDM and all 5 commercial extracts in SPT. Most patients, including those who were tropomyosin-negative, reacted to HDM. Patients reacted to a large and variable array of proteins, and IgE reactivity was common at high molecular weights (>50 kDa). Conclusions: The in vivo diagnosis of shrimp allergy must continue to be based on SPT with fresh material. Shrimp-allergic patients frequently react to a number of ill-defined high-molecular-weight allergens, thus leaving currently available materials for componentresolved diagnosis largely insufficient. Mites and crustaceans probably share several allergens other than tropomyosin (AU)

Introducción: Las pruebas cutáneas con extractos comerciales representan el primer paso en el diagnóstico de alergia a gamba, si bien, su eficacia clínica no está bien definida. Objetivos: El objetivo de este estudio fue analizar la utilidad clínica de todos los extractos comerciales disponibles en Italia frente a crustáceos en pruebas cutáneas. Métodos: En un estudio multicéntrico, se incluyeron 157 pacientes alérgicos a gamba a los que se realizaron pruebas cutáneas con cinco extractos comerciales de crustáceos y con ácaros del polvo doméstico. Los extractos comerciales fueron analizados mediante SDS-PAGE y comparados con un extracto de gamba preparado en fresco. Se determinó IgE frente a Pen a 1/Pen m 1; Pen m 2, y Pen m 4; y el análisis mediante inmunoblotting se realizó en un amplio número de sueros. Resultados: Los extractos de gamba comercializados dieron lugar a reacciones cutáneas muy poco homogéneas en 32 perfiles clínicos diferentes; así mismo, mostraron grandes diferencias en contenido proteico y, en algunos casos, a falta de proteína a pesos moleculares correspondientes a alérgenos mayoritarios de gamba. Únicamente los reactores más fuertes a Pen a1 /Pen m 1 reaccionaron tanto a ácaros del polvo de casa como a los cinco extractos comerciales en pruebas cutáneas. La mayoría de los pacientes, incluyendo los negativos a tropomiosina, reaccionaron a los ácaros del polvo. Los pacientes reaccionaron a un amplio y variable array de proteínas y se detectó con frecuencia reactividad de IgE en pesos moleculares altos (>50 kDa). Conclusiones: El diagnóstico in vivo de alergia a gamba todavía debe estar basado en pruebas cutáneas prick con producto fresco. Los pacientes alérgicos a gamba a menudo reaccionan a un número de alérgenos de peso molecular alto poco definido, lo que hace que las moléculas disponibles hoy en día para el diagnóstico por componentes sean muy insuficiente. Ácaros y crustáceos probablemente comparten varios alérgenos además de la tropiomiosina (AU)

IgE Reactivity to Polcalcins Varies According to Pollen Source.

BACKGROUND: Polcalcins are highly cross-reactive pollen panallergens. Less than 10% of allergic patients are sensitized to polcalcins. All pollen species are considered able to sensitize patients to this panallergen. OBJECTIVE: We aimed to assess the presence of polcalcins in various pollen extracts used in allergen immunotherapy. METHODS: ELISA inhibition experiments were performed with sera from patients sensitized to polcalcin and rPhl p 7 and rBet v 4. Recombinant polcalcin was used as the substrate and freshly prepared pollen extracts as inhibitors. RESULTS: All pollen extracts induced significant inhibition of IgE reactivity to rBet v 4, whereas only grass pollen extract induced marked inhibition of IgE reactivity to rPhl p 7. CONCLUSION: Grass polcalcin probably contains more epitopes than polcalcins from other pollen sources. Grass pollen could be responsible for sensitization to polcalcins, and grass pollen immunotherapy is likely to be an option for polcalcin-hypersensitive patients.

Unusual allergy to soy appeared in adult age.

A case of adult onset severe soy allergy is discussed. The allergen protein involved did not correspond to those presently detectable by commercial diagnostic means, but was not identified, possibly due to the insufficient level of specific IgE. Fresh foods and commercial food extracts remain an invaluable tool to support the diagnosis of food allergy, both in-vivo and in-vitro.

IgE reactivity to polcalcins varies according to pollen source

Background: Polcalcins are highly cross-reactive pollen panallergens. Less than 10% of allergic patients are sensitized to polcalcins. All pollen species are considered able to sensitize patients to this panallergen. Objective: We aimed to assess the presence of polcalcins in various pollen extracts used in allergen immunotherapy. Methods: ELISA inhibition experiments were performed with sera from patients sensitized to polcalcin and rPhl p 7 and rBet v 4. Recombinant polcalcin was used as the substrate and freshly prepared pollen extracts as inhibitors. Results: All pollen extracts induced significant inhibition of IgE reactivity to rBet v 4, whereas only grass pollen extract induced marked inhibition of IgE reactivity to rPhl p 7. Conclusion: Grass polcalcin probably contains more epitopes than polcalcins from other pollen sources. Grass pollen could be responsible for sensitization to polcalcins, and grass pollen immunotherapy is likely to be an option for polcalcin-hypersensitive patients (AU)

Antecedentes: Las polcalcinas son panalérgenos de alta reactividad cruzada en pólenes capaces de sensibilizar a un 10% de los pacientes alérgicos. Todas las especies de pólenes se consideran capaces de sensibilizar pacientes mediante este panalérgeno. Objetivo: El objetivo de este trabajo fue analizar la presencia de esta polcalcina en diferentes extractos de pólenes que se utilizan en inmunoterapia. Método: El suero de pacientes reactores a polcalcina, así como frente a rPhl p 7 y a rBet v 4 fue analizado mediante ensayo de ELISA inhibición, utilizando polcalcina recombinante como sustrato y extracto de pólenes como inhibidores. Resultados: En cuanto a los resultados obtenidos, todos los extractos de pólenes indujeron una inhibición significativa de la reactividad de la IgE frente a rBet v 4, mientras que solo el extracto de polen de gramíneas inducía una marcada inhibición de la reactividad de la IgE frente a rPhl p 7. Conclusión: La polcalcina de gramíneas probablemente contiene más epítopes que las polcalcinas de otras fuentes. El polen de gramíneas podría ser responsable de la sensibilización a la polcalcina y la inmunoterapia con polen de gramíneas es probablemente una opción para los pacientes hipersensibles a polcalcina (AU)

SERUM YKL-40 IN CHILDREN WITH ASTHMA.

Asthma is one of the most common chronic diseases in children. To date the diagnosis of asthma is mainly clinical, based on the clinical history, a careful physical examination and lung function tests. However, symptoms are often not specific and lung function tests are not very sensitive. In order to find a solution to this problem new biomarkers of airway inflammation are being developed. YKL-40 is a chitinase-like protein that has a role in the inflammation and tissue remodeling in several human diseases. The aim of this study is to evaluate serum levels of YKL40 in children with intermittent or persistent asthma. We performed a cross-sectional analysis of serum samples from a cohort of patients with asthma and healthy controls. Patients with asthma were stratified according to four levels of asthma severity (mild intermittent, mild persistent, moderate persistent, and severe persistent). The analysis of serum samples was performed with the use of a commercially available enzyme-linked immune-adsorbent assay (ELISA) kit (Quidel). The minimum detection limit of the assay for YKL-40 is 15.6 ng per milliliter (ng/ml). Our data showed that circulating YKL-40 levels are significantly higher in patients with asthma than healthy subjects (36±18.6 vs 14:41±2.88, p= 0.001). Furthermore, we found significantly higher values of YKL-40 in both groups of children with intermittent asthma (p less than 0.001) and persistent asthma (p less than 0.001) than healthy controls. However, no correlation was found with duration and severity of asthmatic disease (r = 0:18, p= 0:33, r = 0.28 P = 0:13, respectively). Our data allow us to suggest that YKL-40 represents a useful biomarker of asthma in children with intermittent or persistent asthma.

Different allergenicity of pollen extracts of three Mediterranean cypress species accounted for cytological observations.

BACKGROUND: Cypresses play an important role in the urban landscape of the Mediterranean region, releasing a huge amount of allergenic airborne pollen which causes a specific pollinosis in exposed people. OBJECTIVE: The aim of this work is to evaluate, in vivo and in vitro, the potential allergenicity of Cupressus macrocarpa pollen, and to compare it with the allergenicity observed for C. arizonica and C. sempervirens. METHODS: Pollen extracts of the three species were prepared, to determine their protein profile through SDS PAGE analysis and to evaluate their allergenic potential through EAST inhibition assays and SPT. Pollen grain composition was evaluated using a cytochemical approach with optical microscopy. RESULTS: SDS PAGE, EAST inhibition and SPT indicated the higher allergenic potential of C. arizonica compared to C. sempervirens and C. macrocarpa. No significant differences in allergenic potential were found between the latter two species. Cytochemical observations reveal higher ß-glucans and protein content in the intine of C. arizonica during hydration. CONCLUSION: The higher protein content found in C. arizonica pollen grains extract may be due to higher enzyme activity leading to the movement of ß-glucans and pectins from the intine to the partially developed pollen cell wall during hydration. This could explain the higher potential allergenicity of C.arizonica in respect to C. macrocarpa and C. sempervirens.

Shrimp allergy beyond Tropomyosin in Italy: clinical relevance of Arginine Kinase, Sarcoplasmic calcium binding protein and Hemocyanin.

BACKGROUND: Little is known about the prevalence and clinical relevance of sensitization to shrimp allergens other than tropomyosin. OBJECTIVE: We detected the prevalence of arginine kinase and sarcoplasmic calcium binding protein sensitization, and identified a high molecular weight allergen that is frequently recognized by Italian shrimp-allergic patients. METHODS: Sera from 40 shrimp-allergic patients underwent the detection of IgE specific for arginine kinase (rPen m 2) and sarcoplasmic calcium-binding protein (rPen m 4) by ISAC 112 Microarray platform and immunoblot analysis. A high molecular weight shrimp allergen was identified by N-terminal amino acid sequencing. RESULTS: IgE to rPen m 2 and rPen m 4 were found in 4/40 (10%) and 6/40 (15%) sera, respectively; two sera reacted to both allergens. Clinically, 6/8 Pen m 2 and/or Pen m 4 reactors experienced severe allergies to shrimp. On immunoblot, 4/6 rPen m 4-positive sera showed IgE reactivity at about 20 kDa, whereas no rPen m 2-positive serum reacted at about 40 kDa. Nineteen (47%) sera showed IgE reactivity at molecular weights > 60 kDa. Such profile was not associated with IgE reactivity to rPen m 2 or rPen m 4. N-terminal amino acid sequencing of the high molecular weight allergen led to the identification of hemocyanin. CONCLUSION: Shrimp arginine kinase and sarcoplasmic calcium-binding protein are minor allergens sensitizing only 10%-15% of Italian shrimp-allergic patients, but are clinically relevant. Hemocyanin is a clinically relevant high molecular weight shrimp allergen possibly cross-reacting to house dust mite.

Detection of 20 kDa and 32 kDa IgE-binding proteins as the major allergens in Italian sesame seed allergic patients.

BACKGROUND: Sesame seed allergy, a potentially very severe food allergy, seems on the rise worldwide but is still uncommon in Italy. The aim of the present study was to investigate the allergenic profile of Italian sesame seed-allergic patients. METHODS: Patients with genuine sesame seed allergy diagnosed over one year in a large number of allergy centers scattered through Italy were considered for the study. Their IgE reactivity to sesame seed allergens was characterized by immunoblot analysis. RESULTS: Eleven sesame seed allergic patients were detected and studied. 10/10 patients showed IgE reactivity against a sesame allergen at about 20 kDa, and 7/10 showed an extremely strong reactivity at about 32 kDa. The same 7 sera reacted also against a 28 kDa allergen, although such reactivity was significantly weaker in 6/7 cases. Eight patients showed IgE reactivity at about 48 kDa, and 5 sera reacted against higher m.w. proteins at about 67 kDa. Two sera showed IgE reactivity at about 43 kDa as well. Only one serum appeared to react to 2S-albumin. CONCLUSIONS: Italian sesame seed-allergic patients react mostly against allergens other than those described so far as major ones. A large number of recombinant sesame allergens will be needed for a comprehensive component- resolved diagnosis of allergy to this food.

Heterogenity of IgE response to walnut and hazelnut in Italian allergic patients.

BACKGROUND: The prevalence of IgE reactivity against genuine walnut and hazelnut allergens is poorly defined. OBJECTIVE: The IgE response to walnut and hazelnut was investigated in Italian patients with primary allergy to these nuts. METHODS: Sera from 36 patients allergic to hazelnut and/or walnut, not reactive to PR-10, profilin, and LTP, underwent immunoblot analysis with extracts of both nuts. RESULTS: Most patients had a history of systemic symptoms following the ingestion of the offending food(s). Twelve patients were sensitized to both walnut and hazelnut, and 13 were sensitized to other nuts and seeds (cashew, peanut, sesame, pine nut, almond, Brazil nut, and pistachio). On walnut immunoblot, the 7 sera which scored positive showed much variability in their IgE profile. Two reacted uniquely at 10 kDa, and the others at 35 , 40, 45, 50, 67, and > 67 kDa. The profiles obtained under reducing and non-reducing conditions showed several differences. The 7 sera positive on hazelnut immunoblot under reducing conditions recognized sera at 10 kDa and at <10 kDa (n=1), 20 kDa (n=4), at about 22, 24, 30, 40, 43, 58, 60, and 90 kDa, and higher m.w. in other cases. Under non-reducing conditions IgE reactivity at 20, 28, 35, 40, 45, 60, 90, and 100 kDa, was detected. Only two sera scored positive under both conditions and showed an IgE profile that partly changed from one assay to another. CONCLUSION: The current list of walnut and hazelnut allergens is far from being complete. Both reducing and non-reducing conditions are needed to detect IgE reactivity in individual patients.

Mugwort-fennel-allergy-syndrome associated with sensitization to an allergen homologous to Api g 5.

BACKGROUND: The cross-reactive allergen responsible for the so called "mugwort-celery-spice-syndrome", a pollen-food allergy that occurs in a minority of mugwort pollen-allergic patients, is still undefined. OBJECTIVE: To identify the allergen responsible for the cross-reactivity between mugwort pollen and plant-derived foods. METHODS: The serum from one index patient with both fennel and mugwort pollen allergy was used to identify IgE-reactive allergens by direct ELISA and Immunoblot analysis. Cross-reactivity between mugwort pollen and fennel was checked by cross-inhibition experiments. Fennel and mugwort allergens selected on the basis of IgE reactivity and inhibition tests were excised from SDS-PAGE gels and microsequenced. The amino acid sequences obtained were used to screen the NCBI database using the protein BLAST software. RESULTS: On ELISA inhibition experiments, serum absorption with fennel extract completely inhibited the IgE response to mugwort. On immmunoblot analysis periodate treatment caused the disappearance of all bands of IgE reactivity except one at about 60 kDa. The 60 kDa bands from both mugwort and fennel PAGE-SDS gels revealed the presence of distinct proteins. The N-terminal amino acid sequencing gave the same major amino acid sequence corresponding to an Api g 5-like allergen. The MS/MS spectra were analyzed and a provided evidence of a fennel-specific protein with sequence similarity to phosphoglyceromutase from Apium graveolens. CONCLUSION: A 60 kDa allergen, highly homologous to Api g 5, was recognized in fennel by patient's IgE. Inhibition experiments showed a high degree of cross-reactivity between this fennel allergen and the homologous mugwort pollen allergen. This allergen might be responsible for the mugwort-celery-spice syndrome.

Italian study on buckwheat allergy: prevalence and clinical features of buckwheat-sensitized patients in Italy.

Buckwheat allergy is considered a rare food allergy outside of Asia. In Europe, buckwheat has been described mainly as a hidden allergen. Data on the prevalence of buckwheat hypersensitivity in non-Asian countries is very poor. The aim of this multicenter study was to evaluate the prevalence of buckwheat sensitization and its association with other sensitizations among patients referred to allergy clinics in different geographic areas of Italy. All patients referred to 18 Italian allergy clinics from February through April 2011 were included in the study and evaluated for sensitization to buckwheat and other allergens depending on their clinical history. A total of 1,954 patients were included in the study and 61.3 percent of them were atopic. Mean prevalence of buckwheat sensitization was 3.6 percent with significant difference between Northern (4.5 percent), Central (2.2 percent) and Southern (2.8 percent) regions. This is, to our knowledge, the largest epidemiological survey on buckwheat allergy reported outside of Asia. Buckwheat is an emerging allergen in Italy, being more frequently associated to sensitization in Northern regions.