Detection of a novel 20 kDa shrimp allergen showing cross-reactivity to house dust mites.

BACKGROUND: Allergy to crustacean shellfish is one of the most common IgE-mediated food allergies, and tropomyosin has been identified as the major allergen. However, not all subjects affected by this allergy are IgE-positive to tropomyosin. AIMS: To evaluate whether sera of patients with shrimp allergy but negative for tropomyosin react to other allergen(s); and to evaluate the role such allergen(s) may play in cross-reactivity between crustaceans and house dust mites (HDMs). METHODS: Three different pools of sera-one from subjects with shellfish allergy and HDMs positivity, but negative for recombinant and native tropomyosin (rPen a 1 and nPen m 1) (Pool 2); a second from subjects with tropomyosin and HDMs positivity (Pool 1); and the last from subjects allergic only to HDMs (Pool 3) were submitted to immunoblotting. Subsequently, a 20 kDa protein- enriched fraction of shrimp extract was used at two different concentrations (10 and 100 microg/mL) to pre-absorb the Pool 2 serum and to evaluate, by ELISA assay, the level of inhibition on shrimp and HDMs-coated wells, respectively. RESULTS: The Pool 2 serum showed IgE reactivity against a 20 kDa component. Its pre-absorption with an enriched fraction of 20 kDa protein caused an inhibition of 56% in IgE binding to shrimp extract at a concentration of 100 microg/mL, and of 14% and 35% to HDMs extract at concentrations of 10 and 100 microg/mL, respectively, as measured by ELISA assay. CONCLUSIONS: The 20 kDa component seems to be a new crustacean allergen and it could play a role in cross-reactivity with HDMs.

Clinical and immunological correlates of pre-co-seasonal sublingual immunotherapy with birch monomeric allergoid in patients with allergic rhinoconjunctivitis.

Sublingual immunotherapy is safe and efficacious in the treatment of patients with allergic rhinitis. The clinical and biological efficacy of modified allergens (allergoids) has not been fully clarified. We investigated in birch allergic patients the effect of a pre-co-seasonal sublingual immunotherapy regimen with a modified allergen extract on clinical parameters and on T cell proliferation and regulatory cytokine production (IL-10, TGF-beta). We found that during the birch pollen season symptoms and drug usage scores were 30 and 40 percent improved, respectively, in treated versus control subjects (p<0.0001 for both comparisons) whereas well days were 23.5 (33 percent) versus 16.9 (23 percent) (p=0.0024), respectively. Bet v 1 allergen specific proliferation decreased (p = 0.0010), whereas IL-10 transcription increased (p=0.0010) in treated, but not in control patients. Moreover, TGF-beta transcription was increased, although not significantly (p=0.066), following immunotherapy. Thus, sublingual immunotherapy with modified allergen in birch-allergic subjects was safe, clinically efficacious and associated with the reduction of allergen-specific proliferation and with the increased production of the IL-10 regulatory cytokine.

Detection of a novel allergen in raw tomato.

The study reports a case of "pure" tomato allergy in an adult female. The responsible allergen was partially characterized by immunoblot analysis, pepsin digestion, and heating. It had a molecular weight of about 9 kDa and was heat-labile and pepsin-resistant, thus confirming the clinical history. Unfortunately, due to the failure of both 2-dimensional electrophoresis analysis and N-terminal sequencing experiments, it was not possible to characterize the protein further. Based on a comparison with currently known tomato allergens, this seems to be a novel allergen protein.

Date palm pollen allergoid: characterization of its chemical-physical and immunological properties.

BACKGROUND: Date palm (DP) pollen can cause allergic symptoms in people living in different countries. Specific immunotherapy with allergenic extracts by subcutaneous route is effective to cure allergic people. However, the risk of side effects has led to explore safer therapeutic modalities. The aim of our work was to evaluate IgE cross-reactivity between DP and autochthonous palm (European fan palm, EFP) pollen extracts, to chemically modify DP extract with potassium cyanate in order to obtain an allergoid, and to characterize it. METHODS: By radioallergosorbent test inhibition, immunoblotting (IB) and skin prick test, in vitro and in vivo allergenic activities of native and modified DP extracts were compared. By SDS-PAGE and IB, we compared the protein profile and IgE-binding capacity of both native and modified DP, as well as of EFP extracts. By IB inhibition, IgE cross-reactivity of native DP and EFP extracts was evaluated. By ELISA, the capacity of modified DP-induced IgG to react with native DP extract was determined. RESULTS: Radioallergosorbent test inhibition, IB and skin prick test results demonstrated that modified DP was significantly less allergenic than native DP extract. The SDS-PAGE profile showed that potassium cyanate treatment of DP extract did not alter the molecular weight of its components. In addition, no difference was observed between native DP and EFP extracts. Subsequent IB inhibition data evidenced the existence of a strong IgE cross-reactivity between native DP and EFP extracts. ELISA results indicated that the administration of modified DP in mice was able to induce specific IgG also recognizing native DP extract. CONCLUSIONS: Modified DP extract (allergoid) seems to be a good candidate for immunotherapy of patients affected by specific allergy.

Diagnostic value of three different latex extracts.

The diagnosis of latex allergy is made on clinical history, but a confirmatory skin prick test (SPT) or a serological assay based on a commercial latex extract is always recommendable. Different raw materials can be used in the preparation of commercial latex extracts. Such extracts can consequently show both different qualitative profiles and a different diagnostic potential. Therefore, the selection of a proper latex extract is essential for in vitro and in vivo diagnosis of latex allergy. In the present study three different latex extracts, prepared from different raw materials (ammoniated -AL-, serum -SL-, or rubber particles -RPE- latex), are compared by in vitro techniques using sera from twenty patients with latex allergy. SDS-PAGE technique was used to compare the antigenic profile of the three latex extracts. Subsequently, their allergenic profiles were evaluated by immunoblotting technique using the individual sera from the twenty latex allergic patients. The diagnostic potential of the three latex extracts was also evaluated using direct Radio-Allergo-Sorbent Test (RAST) as well as skin prick tests (SPTs). In order to establish the more appropriate latex extract in a perspective of in vivo diagnosis of latex sensitization, the same latex extracts were subsequently compared by an in vivo SPT involving ten of the above subjects. The SDS-PAGE profiles of the three latex extracts examined were quite different. SL extract showed numerous bands comprised between 10-100 kDa. RPE extract was characterized by two intense bands at 14 and 20 kDa while AL extract showed the poorer antigenic composition. Analogously, immunoblotting analysis evidenced a different profile in relation to both different patients and extracts. For only two out of the twenty sera, direct RAST results showed a same positive class in relation to the different latex extracts used. SPT with SL extract showed, in respect to the other extracts (AL, RPE), a significantly higher wheal. This study showed that SL extract is able to express the best in vitro and in vivo diagnostic potential. Thus, its use should be preferred for the diagnosis of patients affected by latex allergy.

Peach fuzz contains large amounts of lipid transfer protein: is this the cause of the high prevalence of sensitization to LTP in Mediterranean countries?

BACKGROUND: Allergy to lipid transfer protein (LTP) is quite common in the Mediterranean countries but virtually absent in Northern Europe. The reasons for this latitude-dependent distribution are unclear. One hypothesis is that peach, the primary sensitizer to LTP, may lose in part its allergenicity as a consequence of treatments (handling, brushing, washing, and packaging) preceding marketing in Northern European. Peach surface fuzz might represent a potential vehicle of LTP. OBJECTIVE: To detect LTP in peach fuzz, and compare IgE reactivity to peach fuzz and peel of sera from LTP-allergic patients. METHODS: IgE reactivity to peach peel and peach fuzz extract was measured by ELISA using sera from 2 LTP-allergic PATIENTS. Purified peach LTP was used in inhibition studies. RESULTS: Both sera strongly reacted both to peach peel and fuzz but reactivity to fuzz was stronger than to peel. Pre-absorption of one serum with peach LTP caused an 87% reduction of IgE reactivity to peach fuzz extract. CONCLUSION: Peach fuzz contains large amounts of LTP and might be a potential vehicle of this allergen causing sensitization in genetically predisposed subjects. Fuzz loss during pre-marketing handling of peaches might be at the basis of the geographic differences that characterize allergy to LTP.

PT with heat-processed apple peel extract to detect LTP hypersensitivity.

BACKGROUND: Allergen-resolved diagnosis of food allergy may be essential in the clinical practice, particularly in patients allergic to foods that may contain both labile and stable allergens. However, presently available diagnostic tests are not useful in this sense. OBJECTIVE: To assess the clinical usefulness of SPT with a heat-processed apple peel extract as an easily available means to detect hypersensitivity to lipid transfer protein (LTP), an extremely stable and potentially hazardous apple allergen. METHODS: Raw and heat-processed (100 degrees C x 10 min) apple peel extract (100 microg/ml) were used to carry out SPT in 23 patients with apple-allergy, 15 of which considered as probably sensitized to labile allergens (Maid 1, Mal d 4) and 8 to stable allergens (Mal d 3, LTP), respectively on the basis of the presence/absence of IgE reactivity to birch pollen. IgE reactivity to the heat-processed apple peel extract was further analyzed by immunoblot. RESULTS: Altogether SPT with raw apple extract scored positive in 20/23 (87%) patients, including 12/15 patients considered as probably sensitized to labile allergens and 8/8 patients considered as probably sensitized to LTP. In contrast, the heat-processed apple extract induced a wheal-and-flare reaction only in the 8 (100%) presumptive LTP reactors. Immunoblot analysis showed IgE reactivity to a 10 kDa protein (LTP) in heat-processed apple extract. CONCLUSION: The heat-resistance of stable apple allergens like LTP can be usefully exploited to prepare extracts for allergen-resolved diagnosis in-vivo.

Why do lipid transfer protein-hypersensitive patients tolerate bean (and other legumes)?

BACKGROUND: Patients allergic to nonspecific lipid transfer protein (LTP) frequently score positive on SPT with legumes but virtually never report adverse reactions eating these foods. OBJECTIVE: This study investigated the IgE reactivity to legumes of LTP-allergic patients and aimed to establish whether legumes can be considered safe in LTP-allergic patients. METHODS: Skin reactivity as well as clinical allergy to bean and pea were evaluated in a large cohort of LTP-hypersensitive patients. Sera from 12 patients showing clinical allergy to a number of botanically unrelated plant-derived foods and high levels of IgE to peach LTP were employed in in vitro studies (ELISA, ELISA inhibition, SDS-PAGE/immunoblot) aiming to investigate IgE reactivity to bean. RESULTS: Preabsorption of patients' sera with boiled bean extract did not cause any loss of IgE reactivity to peach LTP (whereas boiled apple totally abolished it). Immunoblot analysis did not show any IgE reactivity to bean proteins at about 10 kDa, and the SDS profile of bean showed little or no staining at 10 kDa. CONCLUSION: Sera from LTP-allergic patients do not seem to show any IgE reactivity to 10-kDa proteins in bean. Whether this is caused by epitopic differences between Rosaceae and bean LTPs or by the fact that LTP is not expressed in bean remains to be established. This study explains why virtually all LTP-allergic patients tolerate legumes and suggests that these foods should be considered safe for patients sensitized to this protein.

Cross-reactivity between IgE-binding proteins from Anisakis simplex and Dermatophagoides pteronyssinus.

An association was found between Anisakis simplex (As) and Dermatophagoides pteronyssinus (Dp) sensitization. One recent study shows a cross-reactivity between As and Dp and tropomyosin (tr)is suspected as being one of the proteins responsible of this cross-reaction. The aim of our study was: 1) to confirm the cross-reactivity between Dp and As; 2) to determine the importance of tr in this cross reaction. SDS-PAGE analysis of Dp and As (metabolic and somatic) extracts was carried out. Then an IgE immunoblotting test using serum from a patient who had specific IgE only to Dp and As and immunoblotting inhibition experiments using Dp extract and tr as inhibitors were performed. We found that patients serum reacted: 1) against larval As antigens with a molecular weight (mw) of 25 kilodalton (kD) and a mw > than 100 kD, 2) against various metabolic As antigens with a mw > than 100 kD, a mw ranging approximately from 35 to 50 kD, and a mw around 20 kD, and 3) against Dp proteins with mw between 35 and 55 kD. Preincubation of patient's serum with Dp extract caused the disappearance of reactivity against antigens with a mw > than 100 kD in both larval and metabolic As extracts and against proteins with mw ranging approximately from 35 to 50 kD in the metabolic As extract. Preincubation of patients serum with As extract caused the disappearance of reactivity against antigens with mw between 35 and 55 kD in the Dp extract. Pre-incubation of patients serum with tr did not induce any change in the immunoblotting profile. The results show that 1) cross-reactive components between Dp and As are some proteins with a mw ranging approximately from 35 to 50 kD and with a mw > than 100 kD, and 2) tr is not involved in cross-reactivity between As and Dp.

Airborne allergy to sunflower seed.

BACKGROUND: There is increasing evidence that bird fanciers may develop airborne allergies to unusual allergens. OBJECTIVE: To detect the allergen source in a bird fancier with a history of asthma associated with bird cage cleaning activities and with contact with a Brazil parrot. METHODS: SPT with a large series of both airborne and food allergens were carried out. IgE reactivity to allergens causing wheal and flare reactions was confirmed by in-vitro investigations including ELISA/ELISA inhibition and immunoblot analysis. RESULTS: Strong skin reactivity to sunflower seed was observed. Immunoblot analysis showed IgE reactivity to low m.w. proteins, most probably 2S albumin, and ELISA inhibition studies showed the absence of cross-reactivity to mustard. CONCLUSION: Sunflower seed dust may sensitize patients via the respiratory tract. Differently from previously reported cases of sunflower seed allergy, no cross-reactivity to 2S albumin from botanically unrelated seeds was found.

Bee moth (Galleria mellonella) allergic reactions are caused by several thermolabile antigens.

BACKGROUND: Exposure and contact with bee moth (Galleria mellonella) larvae (Gm) can cause an allergic reaction both in anglers and breeders. We described the case of an amateur fisherman who experienced an allergic reaction using Gm but not using heat-treated Gm (h-Gm) (mummies). The aim of this study was to demonstrate by immunoblotting and radioallergosorbent test (RAST)-inhibition experiments the loss of allergenic epitopes in h-Gm extracts. METHODS: Galleria mellonella larvae and h-Gm were homogenized and extracted at 10% (w/v) in 0.5 M phosphate-buffered saline, pH 7.4 containing 0.5% NaN(3) for 16 h at 4 degrees C. Gm and h-Gm extracts were electrophoresed in a 10% polyacrylamide precast Nupage Bis-Tris gel at 180 mA for 1 h and the resolved proteins stained with 0.1% Coomassie brilliant blue and the molecular weight calculated. For the immunoblotting detection of allergenic components the resolved extracts were transferred onto a nitrocellulose membrane and incubated with the patient's serum. Bound specific-IgE was detected by peroxidase-conjugated anti-human IgE. RAST inhibition experiments were performed according to the Ceska method. RESULTS: The protein profile of Gm and h-Gm extracts resulted markedly different in number, intensity and the position of bands, indicating that heat-treatment modifies the chemical-physical characteristics of the protein contents. The Gm extract showed a strong-coloured band at 73 kDa and more than 20 components ranging from 12 to 133 kDa; h-Gm showed two main band at 77 and 38 kDa and about 15 faint bands between 20 and 133 kDa apparently without any correspondence to the bands present in the Gm extract. Immunoblotting with the patient's serum demonstrated several bands of reactivity with the Gm extract ranging from 20 to 100 kDa and no recognizable bands, but only a diffuse smear with h-Gm. When used in a RAST inhibition experiment the h-Gm extract demonstrated an inability to compete with the Gm one for the binding to patient's IgE serum. CONCLUSIONS: The h-Gm seems to lose the allergenic epitopes and has two advantages for anglers: to avoid new possible sensitizations as well as allergic symptoms in sensitized people, without interfering with their skills and satisfaction in their fishing performance.

Allergenic relevance of Cupressus arizonica pollen extract and biological characterization of the allergoid.

BACKGROUND: Cupressaceae (cypress) pollens can cause pollinosis in winter. However, the lack of specific commercial extracts combined with the early pollination period of cypress trees make a precise diagnosis difficult. The need for a reliable and effective cypress extract for diagnostic and therapeutic purposes is increasingly felt. METHODS: Mixed or single Cupressus arizonica, lusitanica and sempervirens pollen extracts precipitated with ammonium sulfate (PPT) were compared by direct RAST, RAST inhibition and SDS-PAGE techniques. The major allergen of C. arizonica (Cup a 1), purified by anion exchange chromatography, was checked by immunoblotting experiments before chemical modification, in parallel with a C. arizonica extract, with potassium cyanate (KCNO) to obtain a monomeric allergoid. The allergoid extract was characterized for its biological, chemico-physical and immunological features by RAST inhibition, SDS-PAGE and ELISA assays. RESULTS: Direct RAST, RAST inhibition, and SDS-PAGE data indicated that the PPT C. arizonica pollen extract showed the most allergenic potential, and it can be considered representative of the Cupressus spp. Immunoblotting data confirmed Cup a 1 as a major allergen. RAST inhibition and ELISA showed that modified PPT C. arizonica extract had less IgE reactivity than the native, non-modified extract, while preserving the immunogenic capacity typical for an allergoid. Finally, the SDS-PAGE profile of Cup a 1 allergoid was similar to native Cup a 1 allergen, suggesting the modified C. arizonica extract shows the characteristics of a monomeric allergoid. CONCLUSIONS: The PPT C. arizonica pollen extract shows good in vitro diagnostic potential and its chemically modified form offers the features of a monomeric allergoid. It might therefore lend itself to the development of a product to be administered by the sublingual or oromucosal route for immunotherapy of individuals with cypress pollinosis.

Immunological cross-reactivity between lipid transfer proteins from botanically unrelated plant-derived foods: a clinical study.

BACKGROUND: Lipid transfer proteins (LTP) are highly conserved and widely distributed throughout the plant kingdom. Recent studies demonstrated immunological cross-reactivity between LTP from many botanically unrelated fruits and vegetables and concluded that LTP are pan-allergens. This study aimed to evaluate the clinical relevance of such cross-reactivity in a group of subjects monosensitized to LTP. METHODS: Twenty LTP-hypersensitive patients were selected from a population of about 600 subjects with history of Rosaceae allergy by means of: 1) negative skin prick test (SPT) with a commercial birch pollen extract; 2) positive SPT with a commercial plum extract, rich in LTP but virtually lacking both Bet v 1-like proteins and profilin; 3) in-vitro IgE reactivity to the 9-10 kDa fraction of peach peel or immunoblot with peach peel showing a single band at 10 kDa; and 4) total inhibition of reactivity to whole peach extract (containing Bet v 1-related allergen, profilin, and LTP) by purified peach LTP on enzyme-linked immunoassay (ELISA). Allergy to foods other than Rosaceae was ascertained by careful interview and analysis of medical recordings. SPT with a large series of plant-derived foods were carried out as well. The cross reactivity between LTPs from botanically unrelated plant-derived foods was assessed by ELISA inhibition tests using walnut and peanut extracts as substrate, and peach LTP as inhibitor. RESULTS: All patients reported allergic reactions after the ingestion of at least one from a large number of vegetable foods other than Rosaceae, and in several cases clinical reactions were very severe (anaphylaxis, asthma, urticaria/angioedema). Nuts and peanuts were the most frequently reported causes of allergic reactions (80% and 40% of patients, respectively). All patients showed positive SPT to several non-Rosaceae food extracts. SPT with nuts, peanut, legumes, celery, rice, and corn were positive in the majority of patients. In ELISA inhibition studies, absorption of sera with peach LTP caused complete inhibition of IgE reactivity to walnut and peanut in all cases. CONCLUSION: LTP is a clinically relevant pan-allergen. Most Rosaceae-allergic, LTP-hypersensitive patients experience adverse reactions after ingestion of botanically unrelated plant-derived foods as well. In view of the high prevalence and severity of the allergic reactions induced, hazelnut, walnut, and peanut should be regarded as potentially hazardous for these patients.