Molecular characterization of allergens in raw and processed kiwifruit.

BACKGROUND: The prevalence of allergy to kiwifruit is increasing in Europe since the last two decades. Different proteins have been identified as kiwifruit allergens; even though with geographic differences, Act d 1, a cysteine protease protein of 30 kDa, and Act d 2, a thaumatin-like protein of 24 kDa, are normally considered the most important. The aim of this study was (i) to identify at molecular level the sensitization pattern in a group of well-characterized patients allergic to kiwifruit and (ii) to assess the role of technological treatments on kiwifruit allergenic potential. METHODS: The differences in the pattern of antigenicity between fresh and processed kiwifruit were evaluated by both immunoelectrophoretic techniques and clinical tests. RESULTS: In the group of patients included in this study, three proteins were identified as major allergens in fresh kiwifruit, as the specific sensitization was present in ≥50% of the subjects. These proteins corresponded to actinidin (Act d 1), pectin methyl aldolase (Act d 6), and thaumatin-like protein (Act d 2). Kiwellin (Act d 5) and proteins of Bet v 1 family (Act d 8/act d 11) were also recognized as minor allergens. Immunoreactivity was totally eliminated by industrial treatments used for the production of kiwifruit strained derivative. CONCLUSIONS: In this group of allergic children, the technological treatments used in the production of kiwifruit strained product reduced drastically the allergenic potential of kiwifruit.

Biochemical and immunochemical evidences supporting the inclusion of quinoa (Chenopodium quinoa Willd.) as a gluten-free ingredient.

To date, the only acceptable therapeutic approach for celiac disease (CD) is a strict elimination from the diet of gluten-containing foods, but this diet does not always guarantee an adequate nutritional intake. Pseudocereals are receiving considerable attention as interesting alternatives for the formulation of gluten-free products, and quinoa grains arise as nutritive substitutes of conventional cereals. The aim of this study was the characterization of different quinoa samples corresponding to 11 quinoa varieties, using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) and immunoblotting techniques to assess their suitability for celiac subjects. Some of these varieties were grown in Italy to assess if the reproduction in a new habitat can guarantee the retention of the "safe" protein pattern. None of the quinoa varieties studied presented protein bands with electrophoretic mobility comparable with those of wheat gliadins, the toxic protein for celiac subjects. All the quinoa samples showed a low binding affinity for both specific anti-gliadin antibodies and IgAs from celiac subjects, confirming that quinoa can be considered as a safe ingredient for celiac patients. However, reliable varieties should be previously selected since the immuno cross-reactivity with anti-gliadin antibodies can vary significantly.

Characterization of the sensitization profile to lupin in peanut-allergic children and assessment of cross-reactivity risk.

BACKGROUND: Case reports of allergy to lupin, due to primary sensitization or cross-reactions with other legumes, are increasing as a consequence of the augmented use of lupin flour in bakery, pasta formulations and other food items. The main allergens that have been associated with the sensitization to lupin are α- and ß-conglutins and, to a lesser extent, γ- and δ-conglutin, but no conclusive data are available so far. The aim of this study was to characterize the sensitization pattern to lupin in a group of 12 Italian children allergic to peanut and identify the specific lupin proteins involved in the cross-reactivity with peanut. METHODS: The immunochemical cross-reactivity among peanut and lupin was evaluated by both in vitro immunoblotting and in vivo fresh food skin prick test (FFSPT). RESULTS: The results showed that ß-conglutin was recognized by cutaneous IgEs from 7/12 peanut-allergic children in FFSPT and serum IgEs from 5/12 in immunoblotting, while 4/12 and 8/12 patients tested positive to γ-conglutin in FFSPT and immunoblotting, respectively. No significant immunoreactive responses were observed to α- and δ-conglutins under non-reducing conditions, but they were bound in FFSPT by the sera of 5/12 and 3/12 patients, respectively. CONCLUSION: In this group of allergic children, ß-conglutin has been identified as the major lupin allergen involved both in vitro and in vivo cross-reactivity with peanut proteins. The role of γ-conglutin in the cross-reactivity between lupin and peanut proteins was also relevant and clear, despite the observed unspecificity of the immunoblotting responses.

Children monosensitized to pine nuts have similar patterns of sensitization.

BACKGROUND: Several cases of pine nut allergies and anaphylaxis have been reported in the literature, but only few pine nut allergens have been characterized. The aim of this research is to identify through immunoelectrophoretic techniques the major pine nut allergens in a group of children monosensitized to pine nuts. METHODS: We studied five children with pine nut allergies and no other sensitization to food except to pine nuts, confirmed by in vivo (prick test, prick-to-prick) and in vitro tests (specific IgE determinations [CAP-FEIA]). The protein profile of pine nuts was analyzed by Sodium Dodecyl sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Immunoblotting was performed after incubation of membranes with the sera from the children included in the present study. RESULTS: Immunoblotting (SDS-PAGE) demonstrated five similar bands between 6 and 47 kDa in all the subjects studied. CONCLUSION: These bands should be considered the potential allergens for pine nut allergic children.

Absence of allergenic residues in experimental and commercial wines fined with caseinates.

The possible presence of allergenic residues in wines treated with one of the potassium caseinates used as fining agents has been investigated. Samples of experimental (16) and commercial (63) wines that had been fined with caseinates with or without bentonites were studied. The leading physicochemical characteristics of each wine sample were determined to assess their possible role in promoting or hindering the elimination of allergenic residues. The study used a specifically developed ELISA test as well as an immunoblotting technique in which membranes were incubated with specific anti-caseinate antibody, both tests having high sensitivity for allergenic residues. No detectable allergenic residues were found in any of the samples by either immunochemical test, irrespective of the physicochemical characteristics of the wine, the type and dosage of refining agent and the enological process used.

Molecular insight into IgE-mediated reactions to sesame (Sesamum indicum L.) seed proteins.

BACKGROUND: Food allergy is becoming a major public health concern in recent times. Several sesame seed allergenic proteins have been identified. However, sensitization toward these proteins does not follow a common and unique pattern of clinical reactivity, as shown by the differential geographic recognition of single proteins. OBJECTIVE: To evaluate the sensitization profiles of 18 Italian individuals who experienced clinical symptoms after sesame seed consumption, including 4 anaphylactic reactions. METHODS: Using an in vitro approach, we adopted a 2-dimensional electrophoretic technique combined with immunoblotting analyses by using sera from 18 Italian sesame-allergic patients. RESULTS: We showed the prevalent and almost exclusive reactivity of the sesame 11S globulin. We shed light on the active role of the basic subunit of this globulin family. The limited accessibility of this polypeptide chain, unless the interchain disulphide bonds are cleaved, may be one of the reasons for its structural/functional stability and, thus, great potential for induction of IgE reactivity. CONCLUSIONS: These results confirmed previous findings on the reactivity of the basic subunit of 11S globulin in various legume species. Moreover, this experimental approach proved to be useful for the noninvasive screening of specific reactivities in sensitized patients.

IgE-mediated cross-reactivity among leguminous seed proteins in peanut allergic children.

The immunological cross-reactivity among major protein- and oil-crops, including lupin, lentil, pea, peanut, kidney bean and soybean, has been studied by a combination of in vitro and in vivo experimental approaches: SDS-PAGE separations of legume protein extracts and immuno-blot revelations with 12 peanut-sensitive subjects' sera, Immuno-CAP and Skin Prick tests on the same subjects. The immuno-blotting data showed a wide range of IgE-binding responses both displayed by one subject towards different plant extracts and among subjects. Differences were both quantitative and qualitative. The prevalent responses of most subjects' sera were seen with peanut polypeptides, as expected, as well as with various polypeptides of the other legumes, the most recurrent of which were the basic subunits of the 11S globulins. The distribution of in vivo responses generally paralleled those obtained by in vitro approaches with strong responses elicited by peanut, lentil and pea protein extracts, especially by most sensitive subjects, thus providing a consistent overall set of results. In this work, the comparison of various approaches has allowed us to get an overall broad picture of the immunological cross-reactivities among proteins of widely used different seed species and to hypothesize the role of most conserved specific polypeptides.

A method for the analysis of milk and egg allergens for the atopy patch test.

The patch test with food antigens (atopy patch test, APT) has been reported as a more specific method than prick or RAST for the early detection of cow's milk and/or egg sensitizations in children. Standardization of APT extracts is a major issue on the road towards full clinical exploitation of this assay. Here, we used sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to characterize sensitivity and specificity of commercial preparations of APT for milk and egg allergies, which are expected to improve the reliability of this test, when compared with fresh food allergen sources. We found that: (i) SDS-PAGE is an appropriate technique for quality control of APT and (ii) commercial milk and egg APT are equivalent to fresh food preparations in terms of allergen content. Clinical trials aimed at characterizing sensitivity and specificity of APT in the diagnosis of food allergy in children will benefit from this technique.

Molecular aspects of milk allergens and their role in clinical events.

Milk allergy is the most frequent food allergy in childhood. Even though cases of newly developed milk allergy in adulthood are known, this allergy is less frequent in adults since it is normally outgrown by children during the first years of life. One of the reasons why allergy to cow's milk shows its highest prevalence in children is its early introduction into the diets of babies when breast feeding is not possible. The major allergens are caseins and beta-lactoglobulin, but allergies to other minor proteins (immunoglobulins, bovine serum albumin) have also been reported. Milk allergenicity can be reduced by various treatments (mainly hydrolysis), meaning that formulas based on cow's milk can often be safely fed to children allergic to milk proteins. Cross-reactivity has been described between different mammalian milks and between milk and meat or animal dander. Cross-contamination can result from inadequate cleaning of industrial equipment and constitutes a hidden danger for allergic subjects who unknowingly ingest milk proteins.

Evaluation of the residual antigenicity of dairy whey hydrolysates obtained by combination of enzymatic hydrolysis and high-pressure treatment.

Dairy whey was hydrolyzed for 15 min with five food-grade enzymes (Alcalase, Neutrase, Corolase 7089, Corolase PN-L, and Papain) at atmospheric pressure (0.1 MPa) and in combination with high pressure (HP) at 100, 200, and 300 MPa, applied prior to or during enzymatic digestion. The peptide profile of the hydrolysates obtained was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and their residual antigenicity was assessed by immuno-blotting with anti-beta-lactoglobulin monoclonal antibodies and the sera from pediatric patients allergic to cow's milk proteins. Moreover, to evaluate the presence of residual trace amounts of casein in bovine whey hydrolysates, immunoblotting with anti-cow's milk protein polyclonal antibodies was performed. SDS-PAGE analysis showed that HP treatment increased hydrolysis by the proteases assayed, especially when it was applied during the enzymatic digestion. Positive reactions at the band corresponding to beta-lactoglobulin were detected for Corolase PN-L and Corolase 7089 hydrolysates, except for those obtained under 300 MPa by the last protease. However, the immunochemical reaction was lower in the hydrolysis products obtained under HP than in those obtained at atmospheric pressure and after the HP treatment. On the contrary, no residual immunochemical reactivity associated with beta-lactoglobulin was observed in the hydrolysates obtained by Alcalase and Neutrase under HP, and none was observed in any of the hydrolysis products obtained by Papain. The presence of traces of casein was not significant. These results suggest that HP combined with selected food-grade proteases is a treatment that can remove the antigenicity of whey protein hydrolysates for their use as ingredients of hypoallergenic infant formulae.

Two-dimensional electrophoresis and western-blotting analyses with anti Ara h 3 basic subunit IgG evidence the cross-reacting polypeptides of Arachis hypogaea, Glycine max, and Lupinus albus seed proteomes.

The allergenicity of seed storage proteins, the major components of edible legume seeds, may cause serious reactions in both children and adult population. Updated methodologies for evaluation of the activity of these proteins are needed. In this paper we used two-dimensional (2D) electrophoretic techniques to investigate the immuno-cross-reactivities of anti Ara h 3 basic subunit IgG to the seed proteomes of three legume species, namely, peanut, soybean, and lupin. The seed proteins, extracted with two different procedures, were separated by 2D electrophoresis, and the electrophoretic maps were analyzed by Western blot. In peanut proteome the antibodies strongly reacted with the 23 kDa polypeptides, corresponding to Ara h 3 basic isoforms, the antigen they were raised to, and three unidentified acidic polypeptides near 45 kDa. Remarkable cross-reactivities with lupin and soybean Ara h 3 homologous polypeptides and nonrelated proteins, namely, lupin conglutin gamma and soybean Bg7S, were detected. Therefore, these proteins may be regarded as new putative allergens. The present findings show the potentiality of 2D electrophoresis in the identification of food allergens and open the way to the traceability of the new cross-reacting proteins in the food chain.

One- and two-dimensional electrophoretic identification of IgE-binding polypeptides of Lupinus albus and other legume seeds.

The prevalence of food allergies in the world population requires integrated approaches to identify new potential allergens, especially those of plant origin. The aim of this work was the allergen in vitro analysis of Lupinus albus seed proteome, a promising food protein source, and the assessment of IgE cross-reactivities with other more diffused legume species. A combination of one- and two-dimensional gel electrophoresis and immunoblotting analyses with specific IgGs for band identification and lupin-sensitized patients' circulating IgEs for allergenicity studies has been used. Two lupin proteins, namely, conglutin gamma and 11S globulin basic subunits, strongly reacted with all patients' sera. Also, cross-reactivities with the homologous polypeptides of other legume species were observed. Otherwise, no reaction at all was detected with a 2S-type lupin protein. This global electrophoretic approach has allowed the identification of a new potential lupin allergen and confirmed the cross-reactivity among the legume 11S globulin basic subunits.

Immunochemical investigation of allergenic residues in experimental and commercially-available wines fined with egg white proteins.

Proteinaceous egg whites are widely used as a fining agent during the production of red wines. Residues of egg white in the final wine could present a risk for individuals allergic to eggs. This study investigated the presence of allergenic residues in both red and white wines fined with egg whites. Experimental and commercially available wines fined with egg whites, with or without subsequent bentonite fining, were studied. Unfined wines were used as negative controls. The physicochemical characteristics of each wine were determined to assess their possible role in enhancing or hindering the elimination of allergenic residues from wine. The amount of egg white protein residues was investigated both by a specifically developed/validated ELISA test and by immunoblotting. Both immunochemical tests used the same anti-total egg white protein antibody and were highly sensitive to the allergen. No egg white protein was detected in the wines studied in either immunochemical test, irrespective of the physicochemical characteristics of the wine, the type and dosage of the fining agent and the oenological process used. The risk of adverse reactions in egg-allergic individuals should therefore be considered negligible, but the exemption from labelling should be allowed only when the absence of residues is confirmed by analytical controls.

Biochemical and immunochemical characterization of different varieties of amaranth (Amaranthus L. ssp.) as a safe ingredient for gluten-free products.

Celiac disease is a food intolerance triggered by the ingestion of gluten-containing cereals; the only therapy is a strict gluten-free diet for life. In recent years, amaranth flour has received considerable attention as an interesting source for the formulation of gluten-free products due to its high nutritional value and low content of prolamins, the toxic proteins for celiacs. The aim of this study was to characterize 40 amaranth varieties using both SDS-PAGE/immunoblotting and ELISA to assess their possible tolerance by celiac subjects. All of the amaranth samples studied showed similar binding affinities for both specific anti-gliadin antibodies and human IgAs. In most amaranth grains, the content of gluten-like proteins measured by ELISA was <20 ppm. The molecular characterization of amaranth proteins suggests that amaranth is safe for celiacs to consume. It is recommended that the most suitable amaranth varieties are those having the lowest content of proteins cross-reacting with anti-gliadin antibodies.

Meat allergy.

PURPOSE OF REVIEW: This review summarizes the scientific evidence on meat allergy, an unusual disorder, whose prevalence in some European countries (such as Italy) may be increasing. RECENT FINDINGS: Data reported in this review underline some interesting points: in meats rarely consumed, such as kangaroo, whale and seal, the main allergens are only partially correlated to those detected in beef or other usually consumed meats; cross-reactivity and cross-contamination are critical aspects, which should be seriously considered by allergologists. SUMMARY: Meat allergy is normally outgrown during the first years of life, so that it is rare in adults. Beef among mammals and chicken among birds are most frequently involved. The major allergens are serum albumins and immunoglobulins, but there are a few reports of allergies to muscle proteins (actin, myosin and tropomyosin). As meat allergenicity can be reduced by various treatments (heat, homogenization and freeze-drying), the consumption of meat derivatives by children allergic to meat proteins is often permitted. Cross-reactivity has been described between different meats, between meat and milk or eggs and between meat and animal dander. There are some reports of cross-contamination associated with the inadequate cleaning of industrial or butchers' equipment. All these aspects may have serious implications for clinical practice.

Analytical aspects of diterpene alkaloid poisoning with monkshood.

A sensitive and specific method for aconitine extraction from biological samples was developed. Aconitine, the main toxic alkaloid from plants belonging to Aconitum species (family Ranunculaceae), was determined in plant material by an external standard method, and by a standard addition calibration method in biological fluids. Described here is one fatal case and five intoxications of accidental aconitine poisoning following the ingestion of aconite mistaken for an edible grass, Aruncus dioicus (Walt.) Fernald, "mountain asparagus", and Cicerbita alpina (L.) Wallroth. The aconitine content in urine was in the range 2.94 microg/mL (dead patient)-0.20 microg/mL (surviving patients), which was almost two to four times higher than that in plasma.

Analysis of food supplements containing iodine: a survey of Italian market.

Iodine is an essential nutrient for humans as a critical constituent of thyroid hormones; both iodine deficiency and excess have adverse consequences for the thyroid gland. The aims of this research were to compare claimed concentrations of iodine with measured ones in various iodine-supplemented products, estimate the amount of iodine ingested by Italian consumers who use these products, and compare the calculated intakes to the Recommended Daily Allowance and tolerable Upper Level. A convenience sample of 43 food supplements was analyzed for iodine concentration. Analytical values resembled those declared in the label in fewer than half of the examples; in four cases, the maximal daily dose was higher than the tolerable upper level for iodine of 600 microg/day. Labeling of iodine-rich food supplements appears to be unreliable and caution should be exercised in the consumption of food supplements rich in iodine as there is a risk of exceeding the established safe upper level of daily intake.

Identification of the basic subunit of Ara h 3 as the major allergen in a group of children allergic to peanuts.

BACKGROUND: Several proteins have been identified as peanut allergens; among them, Ara h 1 (7S globulin) and Ara h 2 (2S globulin) are usually considered the major allergens. OBJECTIVE: To identify the major allergens in a group of children selected for their specific pattern of immunoreactivity. METHODS: We identified the dominant allergen by using (1) amino acid sequencing of the bands that show the strongest IgE immunoreactivity in 1-dimensional electrophoresis and immunoblotting and (2) specific animal IgGs raised against the dominant immunoreactive band to pinpoint the allergen(s) in peanut proteins separated by 2-dimensional electrophoresis and immunoblotting. To confirm these data, we further examined the peanut proteome using serum samples from the children with the unusual immunoreactivity. RESULTS: We found a group of children with marked peanut allergy who are specifically sensitized to the basic subunit of Ara h 3 (11S globulin family). CONCLUSION: That the dominant immunoreactivity in these patients is in a basic subunit of Ara h 3 was unexpected, because previous studies had indicated that Ara h 3 was only a minor peanut allergen and that the identified allergenic epitopes occurred mainly in the acidic Ara h 3 subunit.

Cross-reactivity between mammalian proteins.

BACKGROUND: Cross-reactivity between food allergens occurs when they share part of their amino acid sequence, or when their three-dimensional molecular structure causes them to have a similar capacity to bind specific antibodies. OBJECTIVES: To review data from our laboratory on cross-reactivity between mammalian proteins (milk and meat allergens). METHODS: Studies used immunoelectrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis/polyacrylamide gel electrophoresis and immunoblotting), and animal monoclonal antibodies. RESULTS: The findings suggest that animal monoclonal antibodies specific for cow's milk proteins are able to recognize the major part of milk proteins from mammals bred in Mediterranean countries (sheep, goat, and buffalo); weak cross-reactivity was observed with milk proteins from mares and donkeys. None of the antibodies used in our studies reacted with proteins from an exotic mammalian species: the camel. Similar cross-reactions were found with human circulating immunoglobulin E from children allergic to milk. With regard to beef allergy, monoclonal antibodies specific for bovine serum albumin cross-reacted only with ovine serum albumin, whereas the number of sera from allergic children able to recognize other mammalian serum albumins depended directly on the closeness of phylogenetic relationship between animal species and inversely on the percent identity with human serum albumin in the main epitopic sequence. CONCLUSION: An area of heterogeneity between animal and human species in a critical amino acid sequence (epitope) of an allergen can determine the degree of immunogenic activity.

Tolerance of heat-treated kiwi by children with kiwifruit allergy.

Kiwifruit allergy is increasing among children but whether heating affects clinical tolerance to kiwifruit is unknown. To assess tolerance to heated kiwifruit in children allergic to fresh kiwifruit. In this prospective trial, 20 children (median age 9.4 yr) with a history of immediate allergic reactions to fresh kiwifruit underwent double-blind placebo-controlled food challenges with steam-cooked (100 degrees C for 5') and industrially homogenised kiwifruit. Skin prick tests with a commercial kiwifruit allergen, raw kiwifruit and double-blind placebo-controlled food challenge with 25 g of fresh kiwifruit were used to confirm the history. Specific kiwifruit IgE to native and homogenized fruit were identified by immunoblotting. Fresh kiwifruit induced positive skin prick wheals in all children (confirmed during challenge in 19 patients). Commercial skin prick test elicited a positive response in five children, steam-cooked kiwifruit in five, and the homogenised kiwifruit preparation in none. UniCAP determinations were positive for kiwifruit in three patients. All children's sera showed specific IgE at immunoblotting with raw kiwifruit and one with the homogenised preparation (major allergens identified: Act c 1 and Act c 2). There was no clinical reactivity following challenge with homogenised kiwifruit but one child reacted to cooked kiwifruit challenge. Industrial heat treatment and homogenisation can make kiwifruit safe for children who are allergic to this increasingly popular fruit. This has dietary implications for children who are allergic to several fruit and vegetable proteins.