Evidence of Cross-Reactivity between Different Seed Storage Proteins from Hazelnut (Corylus avellana) and Walnut (Juglans regia) Using Recombinant Allergen Proteins.

Seed storage proteins are extremely stable allergens in nuts, seeds, and legumes and are responsible for the most severe allergic reactions to these foods. The cross-reactivity between seed storage proteins from different sources has not been studied at a molecular level so far. This study aimed to ascertain the cross-reactivity between walnut and hazelnut seed storage proteins using recombinant allergens. Sera from 13 consecutive patients with severe primary walnut and/or hazelnut allergy and hypersensitive to both nuts were studied. IgE specific for rCor a 9, rCor a 14, and rJug r 1 was measured, and inhibition experiments were carried out by measuring IgE reactivity after absorption of patients' sera with freshly prepared walnut extract. All 13 sera showed strong IgE reactivity against walnut 2S albumin, Jug r 1, 12 reacted to hazelnut 2S albumin, Cor a 14, and 8 to the hazelnut legumin, Cor a 9. In inhibition experiments, absorption of sera with whole walnut extract led to the complete disappearance of IgE reactivity to Jug r 1 in 12/13 cases, as expected, but also to the complete disappearance of specific IgE to Cor a 14 in 9/12 sera, and of IgE reactivity to Cor a 9 in 7/8. In the remaining cases a dramatic drop in IgE reactivity was observed. The study shows that patients primarily allergic to either walnut or hazelnut showing a skin or serological reactivity to the other nut also are potentially at risk of severe allergic reactions caused by cross-reactivity between 2S albumins and legumins.

Aedes communis Reactivity Is Associated with Bee Venom Hypersensitivity: An in vitro and in vivo Study.

Mosquito bite is usually followed by a local reaction, but severe or systemic reaction may, in rare cases, occur. Allergic reactions to Aedes communis (Ac) may be underestimated due to the lack of reliable diagnostic tools. In this multicenter study, 205 individuals reporting large local reactions to Ac were enrolled and studied for cutaneous or IgE reactivity to Ac, Blattella germanica, Penaeus monodon, and Dermatophagoides pteronyssinus. Extract and molecular IgE reactivity to bees, wasps, hornets, and yellow jacket venoms were also studied in 119 patients with a clinical history of adverse reaction to Hymenoptera. Immunoblot (IB) analysis and immunoCAP IgE inhibition experiments were carried out in selected sera. Ac sensitization was recorded in 96 (46.8%) patients on SPT. Strict relationship between Ac and D. pteronyssinus, B. germanica, P. monodon, or Apis mellifera reactivity on SPT was observed. Ac IgE recognition was seen in 60/131 (45.8%) patients, 49 (81.6%) of them SPT positive, and 5/14 IB reactors. Ac IgE sensitization was associated with Tabanus spp, A. mellifera, Vespula vulgaris, and Polistes dominula reactivity. A strict relationship between Ac IgE reactivity and Api m 1, Api m 2, Api m 3, Api m 5, and Api m 10 was recorded. IgE reactivity to AC was inhibited in 9/15 cases after serum absorption with the A. mellifera extract. Both SPT and IgE Ac reactivity is observed in about half of patients with a history of large local reactions to mosquito bites. The significant relationship between Ac sensitization and either extract or single bee venom components is suggestive of a "bee-mosquito syndrome" occurrence.

Detection of pan-allergens in commercial pollen extracts for allergen immunotherapy.

BACKGROUND: Up to 50% of patients with pollen allergy are sensitized to at least 1 of the 2 pollen pan-allergens profilin and polcalcin. These allergens could have clinical relevance but the content of profilin and polcalcin in commercial extracts for allergen immunotherapy (AIT) is unknown. OBJECTIVE: To detect these pan-allergens in commercial pollen extracts for AIT from various sources. METHODS: Immunoglobulin E (IgE) reactivity to Phl p 7 and Bet v 2 of sera from 18 adults hypersensitive to profilin and/or polcalcin was investigated by enzyme-linked immunosorbent assay before and after absorption with grass, birch, ragweed, pellitory, and olive pollen extracts for AIT from different producers. Immunoblot inhibition experiments also were carried out using the same allergens. RESULTS: Birch, grass, ragweed, and olive pollen extracts for AIT contained large amounts of profilin, inducing 80% to 90% inhibition in most cases; Parietaria AIT extract appeared to contain little profilin. On immunoblot, grass and birch pollen extracts for sublingual AIT completely absorbed IgE specific for rBet v 2. Interestingly, only grass pollen extracts induced a significant inhibition of IgE binding to rPhl p 7 on enzyme-linked immunosorbent assay and immunoblot. A grass pollen allergoid lost most of its inhibitory potency, suggesting a much weakened affinity for specific IgE. CONCLUSION: With the exception of Parietaria, commercial extracts for AIT of most pollens are rich in profilin and, hence, potentially able to desensitize to this allergen; in contrast, only grass pollen extracts seem rich in polcalcin. These are the pollens to use in case of severe symptoms induced by pollen pan-allergens.

The nature of melon allergy in ragweed-allergic subjects: A study of 1000 patients.

Previous studies suggest cross-reactivity between specific ragweed pollen and melon allergens. This study was designed to clarify the origin of the cross-reactivity between ragweed pollen and the gourd family. One thousand ragweed-allergic subjects were interviewed about the presence of oral allergy syndrome (OAS) induced by melon or watermelon and were divided into reactive to ≤3 seasonal allergen sources or >3 seasonal allergen sources. Patients reporting melon and/or watermelon allergy underwent a skin-prick test (SPT) with fresh melon and, after 2006, also with profilin-enriched date palm pollen extract. Because no IgE reactivity to melon extract was detected in vitro, ELISA was performed using date palm pollen extract, and inhibition experiments were performed using grass pollen, date palm profilin, and bovine serum albumin (BSA) as inhibitors. Six hundred forty-six and 354 subjects reacted to ≤3 seasonal allergens or >3 seasonal allergens, respectively; 4/646 (1%) and 81/354 (23%) reported a history of melon/watermelon-induced OAS (p < 0.0001). Forty-three of 46 (93%) melon reactors scored positive on SPT with the profilin-enriched extract, which was positive in 0/2 (0%) versus 43/44 (98%) reactive to ≤3 or >3 seasonal allergen sources, respectively (p < 0.0001). in vitro, serum from melon-allergic subjects showed a strong IgE reactivity to the profilin-enriched date palm pollen extract, which was abolished by preabsorption with both grass pollen extract and date palm pollen extract, but not by BSA. In ragweed pollen-allergic subjects, melon allergy is most likely associated with cross-sensitization to the plant pan-allergen profilin and not to specific ragweed pollen allergens. This study confirms the association between profilin sensitization and melon allergy.

Respiratory and skin allergy to Galleria mellonella (bee moth).

This case report describes a patient with bee moth-induced rhinoconjunctivitis, asthma and contact urticaria. Immunoblot analysis showed IgE reactivity to two distinct bee moth proteins at 23 and 70 kDa, respectively. ELISA inhibition studies excluded cross-reactivity to the other popular live bait, fly larva.

Respiratory allergy to lipid transfer protein.

BACKGROUND: Due to unclear reasons, allergy to lipid transfer protein (LTP) is frequent in Mediterranean countries but rare in Northern Europe. OBJECTIVE: We report a paradigmatic case of primarily airborne sensitization to LTP that might explain the geographical distribution of this type of food allergy. METHODS: A 21-year-old woman began having severe perennial rhinitis 6 months after she started working in a wholesale fruit storehouse in Southern Italy where large amounts of fruits, including peaches, were handled; symptoms subsided when she left the workplace for >5 days and relapsed as soon as she was back at work. Later on, she developed severe food allergies to peach, hazelnut, peanut, apricot, plum and tomato. The patient underwent a nasal challenge with peach peel extract, and IgE reactivity was assessed by immunoblot analysis. RESULTS: In vivo and in vitro analyses showed sensitivity to LTP. The nasal challenge with peach peel extract (6 microg protein) induced acute, severe respiratory symptoms. On immunoblot with peach peel extract patient's serum reacted uniquely against LTP, as demonstrated by inhibition assays with the recombinant peach protein. CONCLUSION: LTP may induce sensitization via the respiratory tract due to inhalation of air-dispersed food particles, and this may precede the onset of food allergy. If this way of sensitization were effective in the majority of LTP allergic patients (e.g. by exposure to peaches showing intact fuzz in areas where peaches are grown and directly sold on the market) our findings could explain the strange geographical distribution of this type of food allergy.

Detection of some safe plant-derived foods for LTP-allergic patients.

BACKGROUND: Lipid transfer protein (LTP) is a widely cross-reacting plant pan-allergen. Adverse reactions to Rosaceae, tree nuts, peanut, beer, maize, mustard, asparagus, grapes, mulberry, cabbage, dates, orange, fig, kiwi, lupine, fennel, celery, tomato, eggplant, lettuce, chestnut and pineapple have been recorded. OBJECTIVE: To detect vegetable foods to be regarded as safe for LTP-allergic patients. METHODS: Tolerance/intolerance to a large spectrum of vegetable foods other than Rosaceae, tree nuts and peanut was assessed by interview in 49 subjects monosensitized to LTP and in three distinct groups of controls monosensitized to Bet v 1 (n = 24) or Bet v 2 (n = 18), or sensitized to both LTP and birch pollen (n = 16), all with a history of vegetable food allergy. Patients and controls underwent skin prick test (SPT) with a large spectrum of vegetable foods. The absence of IgE reactivity to foods that were negative in both clinical history and SPT was confirmed by immunoblot analysis and their clinical tolerance was finally assessed by open oral challenge (50 g per food). RESULTS: All patients reported tolerance and showed negative SPT to carrot, potato, banana and melon; these foods scored positive in SPT and elicited clinical symptoms in a significant proportion of patients from all three control groups. All patients tolerated these four foods on oral challenge. Immunoblot analysis confirmed the lack of IgE reactivity to these foods by LTP-allergic patients. CONCLUSION: Carrot, potato, banana and melon seem safe for LTP-allergic patients. This finding may be helpful for a better management of allergy to LTP.

Cerebrovascular disease and varicella in children.

Varicella-associated stroke has been reported with increasing frequency in recent years. In many cases, diagnosis is difficult because of the late onset of manifestations after the acute infectious episode. Four cases of cerebrovascular disease after varicella infection were observed. Three children presented hemiparesis and one facial paresis. The neuroradiological findings comprised stenosis/occlusion of middle cerebral artery or nucleo capsular signal alteration. Because, several pathogenetic mechanisms have been proposed as the cause of stroke, the relationship between prothrombotic conditions, antipospholipid antibodies and stroke in these patients is discussed. The difficulty in defining the pathogenesis of the ischemic episode is related to problems in the choice of antithrombotic treatment, which is still not standardized and must be decided on individual basis. In the event of rapid onset of stroke after exanthem high dose antiviral therapy seems to be justified. On the basis of our experience and of literature data on varicella-associated stroke, we recommend that VZV infection be taken into account in every episode of stroke in children.

Cleavage of von Willebrand factor by ADAMTS-13 in vitro: effect of temperature and barium ions on the proteolysis kinetics.

The multimeric size of von Willebrand factor (VWF) is regulated by the specific cleaving metalloprotease, ADAMTS-13. Laboratory assays for ADAMTS-13 are useful for identifying severe protease-deficient activity. ADAMTS-13 activity is currently assayed by prolonged dialysis of plasma at 37 degrees C in low-ionic-strength denaturing buffer. We investigated the effect of temperature and divalent cation supplementation on the kinetics of VWF proteolysis by ADAMTS-13 in vitro. Proteolysis was monitored for 24 h at 37, 22, and 4 degrees C, in the presence or absence of barium ions, by measuring the binding of VWF to collagen. Complete VWF proteolysis was observed at 37 degrees C in the presence of BaCl2, while about 25% VWF still bound to collagen when BaCl2 supplementation was omitted. Proteolysis kinetics at 22 and 4 degrees C was slower but complete, even in the absence of added barium. A subphysiological temperature might influence the proteolysis kinetics by determining minor variations of the ADAMTS-13 structure, or further modification of the VWF substrate. We describe a simple procedure to analyse the kinetics of VWF proteolysis that is suitable for routine diagnostic use. Furthermore, we offer new insight into the biochemistry of ADAMTS-13.

von Willebrand factor multimer composition is modified following oral methionine load in women with thrombosis, but not in healthy women.

Hyperhomocysteinemia is associated with an increased risk of venous and arterial thrombosis, probably by inducing endothelial damage. Von Willebrand factor (VWF) is an endothelial marker protein. It is a plasma multimeric molecule that plays a thrombophilic role. Our purpose was to investigate VWF changes in patients with thrombosis following oral methionine load. We evaluated homocysteine levels and VWF parameters (plasma levels, activity, proteolysis fragments, and multimer composition) before and after methionine load in 42 women with venous or arterial thrombosis and in 36 healthy women. Methionine load induced mild hyperhomocysteinemia in 10 patients and two controls. No changes in VWF levels and activity were observed, but an increased amount of VWF proteolysis fragments was found post-load in patients and controls. VWF multimer composition was unaffected in controls, while a decrease of the largest VWF multimers was found in women with thrombosis. Homocysteine levels inversely correlated with the amount of the largest multimers in hyperhomocysteinemic patients. Large VWF molecules were probably released from endothelial cells following load, and rapidly cleaved by the specific VWF-cleaving protease. VWF proteolysis was enhanced in mild hyperhomocysteinemic patients, thus leading to downregulation of VWF size to smaller multimers.

Isoform identification, recombinant production and characterization of the allergen lipid transfer protein 1 from pear (Pyr c 3).

Non-specific lipid transfer proteins belonging to LTP1 family represent the most important allergens for non pollen-related allergies to Rosaceae fruits in the Mediterranean area. Peach LTP1 (Pru p 3) is a major allergen and is considered the prototypic allergenic LTP. On the contrary, pear allergy without pollinosis seems to be under-reported when compared to other Rosaceae fruits suggesting that the as-yet-uncharacterized pear LTP1 (Pyr c 3) has in vivo a low allergenicity. We report here on the identification of four cDNAs encoding for LTP1 in pear fruits. The two isoforms exhibiting amino acid sequences most similar to those of peach and apple homologues were obtained as recombinant proteins. Such isoforms exhibited CD spectra and lipid binding ability typical of LTP1 family. Moreover, pear LTP1 mRNA was mainly found in the peel, as previously shown for other Rosaceae fruits. By means of IgE ELISA assays a considerable immunoreactivity of these proteins to LTP-sensitive patient sera was detected, even though allergic reactions after ingestion of pear were not reported in the clinical history of the patients. Finally, the abundance of LTP1 in protein extracts from pear peel, in which LTP1 from Rosaceae fruits is mainly confined, was estimated to be much lower as compared to peach peel. Our data suggest that the two isoforms of pear LTP1 characterized in this study possess biochemical features and IgE-binding ability similar to allergenic LTPs. Their low concentrations in pear might be the cause of the low frequency of LTP-mediated pear allergy.

Rice: another potential cause of food allergy in patients sensitized to lipid transfer protein.

Recent studies show that the lipid transfer protein (LTP), the major Rosaceae allergen in patients not sensitized to birch pollen, is a largely cross-reacting allergen. Moreover, it is a potentially hazardous allergen due to its stability upon thermal treatment and pepsin digestion. The present study reports 3 cases of rice-induced anaphylaxis in LTP-allergic patients. In vitro inhibition studies, carried out using LTP purified from both rice and apple as well as whole peach extract, show that LTP was the relevant allergen in these patients and demonstrate the cross-reactivity between rice LTP and peach/apple LTP.

Giant ragweed specific immunotherapy is not effective in a proportion of patients sensitized to short ragweed: analysis of the allergenic differences between short and giant ragweed.

BACKGROUND: Short ragweed and giant ragweed pollen allergens are considered largely cross-reactive, and it is generally believed that 1 species is sufficient for skin testing and immunotherapy. However, in the area north of Milan (a zone widely invaded only by short ragweed), about 50% of patients submitted to injection specific immunotherapy with giant ragweed showed little or no clinical response, but showed an excellent outcome if they were shifted to short ragweed specific immunotherapy. OBJECTIVE: To investigate allergenic differences between short and giant ragweed. METHODS: IgE reactivity to short ragweed of sera from 16 patients allergic to ragweed was assessed by immunoblot before and after absorption with short and giant ragweed. Moreover, 41 ragweed-monosensitive patients underwent skin prick test with both ragweed species. RESULTS: In several cases, preabsorption of sera with giant ragweed extract was unable to inhibit IgE reactivity fully against both a 43-kd allergen and other allergens at different molecular weights in short ragweed. On skin prick test, short ragweed induced larger wheals than giant ragweed in the majority of patients, and 6 of 41 (15%) patients were strongly short ragweed-positive but giant ragweed-negative. The immunoblot with the serum from 1 of these subjects showed a strong IgE reactivity to short ragweed at about 43 kd in the absence of any reactivity to giant ragweed. CONCLUSION: Short and giant ragweed are not allergenically equivalent. Allergenic differences involve both the major allergens Amb a 1-2/Amb t 1-2 and some minor allergens. In patients allergic to ragweed, both diagnosis in vivo and immunotherapy should always be performed by using the ragweed species present in that specific geographic area.