A Novel Donkey Milk-derived Human Milk Fortifier in Feeding Preterm Infants: A Randomized Controlled Trial.

OBJECTIVES: The purpose of the present randomized controlled clinical trial was to compare the use of donkey milk-derived fortifier (DF) with commercial bovine milk-derived fortifier (BF) in very preterm or very-low-birth-weight newborns, in terms of feeding tolerance. METHODS: This trial included 156 newborns born at <32 weeks of gestational age and/or with a birth weight ≤1500 g. Newborns were randomized 1:1 to receive enteral feeding with either a BF-arm, or a new, DF-arm for 21 days. The fortification protocol was the same for both study arms, and the 2 diets were designed to be isoproteic and isocaloric. Feeding tolerance was assessed by a standardized protocol. RESULTS: The risk of feeding intolerance tended to be lower in DF-arm than in BF-arm, with a relative risk reduction of 0.63 (95% confidence interval: -0.29, +0.90). The mean number of episodes per newborn of feeding intolerance and feeding interruptions (any duration) were consistently lower in the DF-arm than in the BF-arm. Episodes of bilious gastric residuals and vomiting were significantly lower in the DF-arm. Time needed to reach full enteral feeding (150 mL ·â€Škg ·â€Šday) and daily weight increase between the first day of exclusive enteral feeding (ie, without administering intravenous fluids) and discharge were similar in the BF- and DF-arms. CONCLUSIONS: These results suggest that DF improve feeding tolerance when compared with standard bovine-derived fortifiers, with a similar auxological outcome.

Nutritional adequacy of a novel human milk fortifier from donkey milk in feeding preterm infants: study protocol of a randomized controlled clinical trial.

BACKGROUND: Fortification of human milk is a standard practice for feeding very low birth weight infants. However, preterm infants often still experience suboptimal growth and feeding intolerance. New fortification strategies and different commercially available fortifiers have been developed. Commercially available fortifiers are constituted by a blend of ingredients from different sources, including plant oils and bovine milk proteins, thus presenting remarkable differences in the quality of macronutrients with respect to human milk. Based on the consideration that donkey milk has been suggested as a valid alternative for children allergic to cow's milk proteins, due to its biochemical similarity to human milk, we hypothesized that donkey milk could be a suitable ingredient for developing an innovative human milk fortifier. The aim of the study is to evaluate feeding tolerance, growth and clinical short and long-term outcomes in a population of preterm infants fed with a novel multi-component fortifier and a protein concentrate derived from donkey milk, in comparison to an analogous population fed with traditional fortifier and protein supplement containing bovine milk proteins. METHODS: The study has been designed as a randomized, controlled, single-blind clinical trial. Infants born <1500 g and <32 weeks of gestational age were randomized to receive for 21 days either a combination of control bovine milk-based multicomponent fortifier and protein supplement, or a combination of a novel multicomponent fortifier and protein supplement derived from donkey milk. The fortification protocol followed is the same for the two groups, and the two diets were designed to be isoproteic and isocaloric. Weight, length and head circumference are measured; feeding tolerance is assessed by a standardized protocol. The occurrence of sepsis, necrotizing enterocolitis and adverse effects are monitored. DISCUSSION: This is the first clinical study investigating the use of a human milk fortifier derived from donkey milk for the nutrition of preterm infants. If donkey milk derived products will be shown to improve the feeding tolerance or either of the clinical, metabolic, neurological or auxological outcomes of preterm infants, it would be an absolute innovation in the field of feeding practices for preterm infants. TRIAL REGISTRATION: ISRCTN - ISRCTN70022881 .

Identification of Equine Lactadherin-derived Peptides That Inhibit Rotavirus Infection via Integrin Receptor Competition.

Human rotavirus is the leading cause of severe gastroenteritis in infants and children under the age of 5 years in both developed and developing countries. Human lactadherin, a milk fat globule membrane glycoprotein, inhibits human rotavirus infection in vitro, whereas bovine lactadherin is not active. Moreover, it protects breastfed infants against symptomatic rotavirus infections. To explore the potential antiviral activity of lactadherin sourced by equines, we undertook a proteomic analysis of milk fat globule membrane proteins from donkey milk and elucidated its amino acid sequence. Alignment of the human, bovine, and donkey lactadherin sequences revealed the presence of an Asp-Gly-Glu (DGE) α2ß1 integrin-binding motif in the N-terminal domain of donkey sequence only. Because integrin α2ß1 plays a critical role during early steps of rotavirus host cell adhesion, we tested a minilibrary of donkey lactadherin-derived peptides containing DGE sequence for anti-rotavirus activity. A 20-amino acid peptide containing both DGE and RGD motifs (named pDGE-RGD) showed the greatest activity, and its mechanism of antiviral action was characterized; pDGE-RGD binds to integrin α2ß1 by means of the DGE motif and inhibits rotavirus attachment to the cell surface. These findings suggest the potential anti-rotavirus activity of equine lactadherin and support the feasibility of developing an anti-rotavirus peptide that acts by hindering virus-receptor binding.

Rice-induced anaphylaxis: IgE-mediated allergy against a 56-kDa glycoprotein.

BACKGROUND: Although rice (Oryza sativa) is one of the most common cereals produced and consumed around the world, there have been only a few reports on immediate hypersensitivity reactions after ingestion of rice. Few clinical studies on rice allergy in Asia have been reported concerning rhinitis, asthma and atopic dermatitis. In this case study, we identify allergens presumably responsible for anaphylaxis after ingestion of rice in a German patient. METHODS: Prick-to-prick tests, determination of specific IgE and the basophil activation test (BAT) were performed to confirm IgE-mediated allergy. IgE reactivity was further analyzed by immunoblotting of protein extracts from cooked commercial rice products. Rice allergens were purified, subjected to N-terminal sequencing and characterized by IgE binding and IgE inhibition assays using additional sera from 8 subjects with sensitization to rice and/or a history of hypersensitivity symptoms after rice ingestion. RESULTS: Prick-to-prick tests were positive to raw and cooked rice (basmati rice and long-grain rice) and preparations of different rice extracts. Specific IgE against rice (f9) was 1.87 kU(A)/l. The BAT showed specific IgE-mediated activation of basophils after stimulation with rice extracts. Four IgE-reactive rice proteins with an apparent molecular weight of 49, 52, 56 and 98 kDa were identified. Interestingly, only binding to the 56-kDa glycoprotein was at least partially independent from cross-reactive carbohydrate determinants (CCD), whereas IgE binding to the other rice proteins was completely inhibited by pre-incubation with the CCD MUXF derived from bromelain. CONCLUSIONS: Yet unidentified high-molecular-weight allergens from rice seeds, predominantly a 56-kDa glycoprotein, seem to be responsible for anaphylaxis after consumption of rice in a German patient.

Maize food allergy: lipid-transfer proteins, endochitinases, and alpha-zein precursor are relevant maize allergens in double-blind placebo-controlled maize-challenge-positive patients.

Italian patients with maize anaphylaxis have been shown to have IgE toward two major maize allergens: an alpha-amylase inhibitor and a 9-kDa LTP. A complete study on maize food allergens in patients with positive maize double-blind, placebo-controlled food challenge (DBPCFC) is lacking. The objective was to utilize the three maize protein fractions to identify and characterize the most relevant IgE-binding proteins recognized by the sera of Italian and Swiss patients with either a positive maize-DBPCFC or a history of maize-induced anaphylaxis. Osborne's protein fractions of maize were extracted to obtain water-soluble, total zein, and total protein fractions. Protein IgE-binding capacity was investigated by SDS-PAGE immunoblotting using the sera from DBPCFC-positive patients and from patients with maize-induced anaphylaxis. Purified maize LTP was used to inhibit the IgE immunoblotting of the three protein fractions. IgE immunoblotting demonstrated that the 9-kDa LTP was recognized by all the Italian patients and by none of the Swiss patients. Other allergens were: 14-kDa alpha-amylase inhibitor, 30-kDa endochitinases A and -B, 19 kDa zein-beta precursor, and 26 kDa zein-alpha precursor; a newly described allergen, the globulin-2 precursor, identified in the total protein fraction. It is noteworthy that maize LTP and endochitinase were cross-reactive with grape LTP and one grape endochitinase. LTP was found to be the only major allergen in Italian patients with either positive maize challenge or a history of maize-induced anaphylaxis. We have identified other maize allergens in subjects with maize food allergy, as grape cross-reactive endochitinase, however, the clinical significance of these proteins needs to be investigated in larger groups of patients with allergy to these food items.

Milk fat globule membrane components--a proteomic approach.

The milk fat globule membrane (MFGM) is the membrane surrounding lipid droplets during their secretion in the alveolar lumen of the lactating mammary gland. MFGM proteins represent only 1-4% of total milk protein content; nevertheless, the MFGM consists of a complex system of integral and peripheral proteins, enzymes, and lipids. Despite their low classical nutritional value, MFGM proteins have been reported to play an important role in various cellular processes and defense mechanisms in the newborn. Using a proteomic approach, such as high-resolution, two-dimensional electrophoresis followed by direct protein identification by mass spectrometry, it has been possible to comprehensively characterize the subcellular organization of MFGM. This chapter covers the description of MFGM proteomics from the first studies about 10 years ago through the most recent papers. Most of the investigations deal with MFGMs from human and cow milk.

Wheat IgE-mediated food allergy in European patients: alpha-amylase inhibitors, lipid transfer proteins and low-molecular-weight glutenins. Allergenic molecules recognized by double-blind, placebo-controlled food challenge.

BACKGROUND: Three main problems hamper the identification of wheat food allergens: (1) lack of a standardized procedure for extracting all of the wheat protein fractions; (2) absence of double-blind, placebo-controlled food challenge studies that compare the allergenic profile of Osborne's three protein fractions in subjects with real wheat allergy, and (3) lack of data on the differences in IgE-binding capacity between raw and cooked wheat. METHODS: Sera of 16 wheat-challenge-positive patients and 6 patients with wheat anaphylaxis, recruited from Italy, Denmark and Switzerland, were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis/immunoblotting of the three Osborne's protein fractions (albumin/globulin, gliadins and glutenins) of raw and cooked wheat. Thermal sensitivity of wheat lipid transfer protein (LTP) was investigated by spectroscopic approaches. IgE cross-reactivity between wheat and grass pollen was studied by blot inhibition. RESULTS: The most important wheat allergens were the alpha-amylase/trypsin inhibitor subunits, which were present in all three protein fractions of raw and cooked wheat. Other important allergens were a 9-kDa LTP in the albumin/globulin fraction and several low-molecular-weight (LMW) glutenin subunits in the gluten fraction. All these allergens showed heat resistance and lack of cross-reactivity to grass pollen allergens. LTP was a major allergen only in Italian patients. CONCLUSIONS: The alpha-amylase inhibitor was confirmed to be the most important wheat allergen in food allergy and to play a role in wheat-dependent exercise-induced anaphylaxis, too. Other important allergens were LTP and the LMW glutenin subunits.

IgE-mediated food allergy diagnosis: Current status and new perspectives.

In June 2005, the work of the EU Integrated Project EuroPrevall was started. EuroPrevall is the largest research project on food allergy ever performed in Europe. Major aims of the project are to generate for the first time reliable data on the prevalence of food allergies across Europe and on the natural course of food allergy development in infants. Improvement of in vitro diagnosis of food allergies is another important aim of the project. The present review summarizes current knowledge about the clinical presentation of food allergy and critically reviews available diagnostic tools at the beginning of the project period. A major problem in diagnosis is a relatively poor 'clinical specificity', i. e. both positive skin tests and in vitro tests for specific IgE are frequent in sensitized subjects without food allergy symptoms. So far, no in vitro test reliably predicts clinical food allergy. EuroPrevall aims at improving the predictive value of such tests by proceeding from diagnosis based on allergen extracts to purified allergen molecules, taking into account the affinity of the IgE-allergen interaction, and evaluating the potential of biological in vitro tests such as histamine release tests or basophil activation tests including assays performed with permanently growing cell lines.

Germin-like protein Cit s 1 and profilin Cit s 2 are major allergens in orange (Citrus sinensis) fruits.

Oranges are clinically relevant allergenic foods. To date, orange allergens have not been characterized in detail. The study is aimed at analyzing the sensitization profile in orange-sensitized subjects with and without clinical allergy, and to identify orange allergens. Fifty-six sensitized subjects with self-reported reactions to orange were grouped into reactors (anaphylaxis or multiple episodes of immediate reactions and/or positive challenge tests) and non-reactors (negative open food challenge tests). Allergens were characterized by IgE immunoblotting, N-terminal sequencing, IgE-inhibition assays, and mediator release assays were performed to determine the allergenic potency of orange profilin. Of 56 subjects, 23 were classified as orange allergic showing mainly an oral allergy syndrome. Of 23 subjects classified as orange allergic, 22 were sensitized to profilin, Cit s 2. In patients with mono-sensitization to profilin in vitro histamine releases up to 75% from basophils were induced using orange extract and purified plant profilins. Of the allergic patients 78% were sensitized to germin-like protein, Cit s 1. Both allergens showed retained IgE reactivity in heat-processed orange juice. Interestingly, subjects with and without clinical allergy showed a comparable sensitization profile. Profilin and germin-like proteins are major orange allergens. The potential clinical relevance of orange profilin was indicated by its strong capacity to release histamine from basophils. However, a predominant sensitization to both allergens in subjects without symptoms also indicates a high frequency of clinically insignificant sensitization.

Hazelnut (Corylus avellana) vicilin Cor a 11: molecular characterization of a glycoprotein and its allergenic activity.

In Europe, hazelnuts (Corylus avellana) are a frequent cause of food allergies. Several important hazelnut allergens have been previously identified and characterized. Specific N-glycans are known to induce strong IgE responses of uncertain clinical relevance, but so far the allergenic potential of glycoproteins from hazelnut has not been investigated. The aim of the study was the molecular characterization of the glycosylated vicilin Cor a 11 from hazelnut and the analysis of its allergenic activity. Although MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS showed that one of two potential glycosylation sites of Cor a 11 was glycosylated, CD spectroscopy indicated that recombinant and natural Cor a 11 share similar secondary structures. Thus to analyse the impact of the glycan residues of Cor a 11 on IgE binding, the allergenic activity of natural glycosylated Cor a 11 and recombinant Cor a 11 was compared. In addition, the IgE sensitization pattern to recombinant Cor a 11, Cor a 1, Cor a 2 and Cor a 8 of 65 hazelnut allergic patients was determined in vitro. The prevalence of IgE reactivity to hazelnut vicilin Cor a 11 was below 50%. Basophil histamine-release assays were used to determine the allergenic activity of both natural and recombinant Cor a 11 in comparison with Cor a 1, a birch (Betula verrucosa) pollen-related major hazelnut allergen. Both forms of Cor a 11 induced mediator release from basophils to a similar extent, indicating that the hazelnut allergic patients had cross-linking IgE antibodies binding to the protein backbone and not to carbohydrate structures. In comparison to Cor a 1, a 10000-fold higher concentration of Cor a 11 was required to induce similar basophil mediator release. In conclusion, the hazelnut vicilin Cor a 11 is a minor allergen both in regard to prevalence and allergenic potency, whereas its glycan does not contribute to its allergenic activity.

Cloning and characterization of two catechol 1,2-dioxygenase genes from Acinetobacter radioresistens S13.

Two novel catechol 1,2-dioxygenase (C 1,2-O) genes have been isolated from an Acinetobacter radioresistens strain that grows on phenol or benzoate as sole carbon and energy source. Designated as catA(A) and catA(B), they encode proteins composed of 314 and 306 amino acids, whose deduced sequences indicate that they have approximately 53% identity, whereas their NH2-terminal and COOH-terminal regions have no sequences in common. This may explain their different thermal and pH stability. Polyclonal antibodies raised against an amino-terminal CatA(A) peptide or the whole CatA(B) protein were used to establish their inducible and differential expression patterns upon bacterial growth in phenol or benzoate. The CatA(A) protein (IsoA) was induced by both phenol and benzoate though with different kinetics, whereas the catA(B) product (IsoB) was constitutively produced at low levels that increased only during growth in the presence of benzoate.

The proteomic approach to analysis of human milk fat globule membrane.

Lactating mammary epithelial cells do not adhere to the classical rule of lipid secretion since lipids are secreted enveloped by the milk fat globule membrane (MFGM) via a budding process. Historically, biochemical studies of MFGM proteins focused on the analysis of single molecules. Today, thanks to proteomic technologies, it is now possible to comprehensively analyse the protein component of MFGM. Questions concerning: (1) protein organization within the MFGM structure; (2) correlation to the intracellular secretion pathway; and (3) display of multiple specialized functions, can now be addressed. Proteomics allows for rapid identification of the total MFGM proteins thereby providing a global functional screen of the lactating cell. In addition to their nutritional value, human MFGM proteins have important protective functions against bacteria, viruses and protozoa. Identifying the complex nature of protein-protein interactions would further enhance our present knowledge of MFGM function. A speculative molecular architecture of the human MFGM is proposed.

Cow's milk allergens identification by two-dimensional immunoblotting and mass spectrometry.

Cow's milk allergy (CMA) has become a common disease in early childhood, its prevalence ranging from 1.6% to 2.8% among children younger than 2 years of age. The role of different cow's milk protein (CMP) in the pathogenesis of CMA is still controversial. Even if the proteins most frequently and most intensively recognized by immunoglobulin E (IgE) seem to be the most abundant in milk (caseins and beta-lactoglobulin), with an although great variability all milk proteins appear to be potential allergens, even those that are present in trace amounts (i.e., lactoferrin, IgG, and BSA). In this work proteomics techniques have been applied for CMP allergens analysis. Allergens have been identified by immunoblotting following resolution of CMP components by two-dimensional electrophoresis. Sera from 20 milk-allergic subjects, as proven by oral provocation test, CAP-RAST and skin prick test, have been used for cow's milk major allergen identification. Cow's milk proteins and their isoforms were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. In our group of patients, the prevalence of CMP allergens, i.e., the total number of subjects sensitized to CMP divided by the total number of the subjects enrolled in the study, was: 55% alpha(s1)-casein, 90% alpha(s2)-casein, 15% beta-casein, 50% kappa-casein, 45% beta-lactoglobulin, 45% BSA, 95% IgG-heavy chain, 50% lactoferrin, and 0% alpha-lactalbumin.

Effect of two pasteurization methods on the protein content of human milk.

The Holder method is the recommended pasteurization method for human milk banks, as it ensures the microbiological safety of human milk (HM). The loss of some biologically active milk components, due to the heat treatment, is a main limit to the diffusion of donor HM. High-temperature short-time (HTST) pasteurization may be an alternative to maintain the nutritional and immunological quality of HM. The aim of the present study was to compare the impact of Holder and HTST pasteurization on the HM protein profile. The protein patterns of HTST-treated milk and raw milk were similar. The Holder method modified bile salt-stimulated lipase, lactoferrin and components of the immune system. The HTST method preserved the integrity of bile salt-stimulated lipase, lactoferrin and, to some extent, of IgAs. Holder pasteurization decreased the amount of bile salt-stimulated lipase and inactivated the remaining molecules, while the HTST method did not alter its activity. Pasteurization increased the bioavailable lysine quantity. HTST pasteurization seems to better retain the protein profile and some of the key active components of donor HM.

Generation of a comprehensive panel of crustacean allergens from the North Sea Shrimp Crangon crangon.

BACKGROUND: Published data on crustacean allergens are incomplete. The identification of tropomyosin (TM), arginine kinase (AK), sarcoplasmic Ca-binding protein (SCP) and myosin light chain (MLC) as shrimp allergens are all important contributions but additional allergens are required for the development of a complete set of reagents for component resolved diagnosis and the exploration of novel vaccination strategies. METHODS: The North Sea shrimp (Crangon crangon), which is frequently consumed in Europe, served as a model organism in this study. TM and AK were directly cloned from mRNA based on sequence homology and produced as recombinant proteins. Additional IgE-reactive proteins were isolated by preparative SDS-PAGE and identified by mass spectrometry and corresponding cDNAs were cloned and expressed in E. coli. The relevance of the 6 cloned crustacean allergens was confirmed with sera of 31 shrimp-allergic subjects, 12 of which had a positive double-blind, placebo-controlled food challenge (DBPCFC) to shrimp and 19 a convincing history of food allergy to shrimp, including 5 cases of anaphylaxis. Quantitative IgE measurements were performed by ImmunoCAP. RESULTS: Six recombinant crustacean proteins: TM, AK, SCP, a novel MLC, troponin C (TnC), and triosephosphate isomerase (TIM) bound IgE in ImmunoCAP analysis. Specific IgE to at least one of these single shrimp allergens was detected in 90% of the study population, thus the in vitro diagnostic sensitivity was comparable to that of shrimp extract (97%). In 75% of the subjects, the combined technical sensitivity was similar to or greater with single shrimp allergens than with natural shrimp extract. CONCLUSIONS: We identified six IgE-binding proteins from C. crangon, three of which have not before been described as allergens in crustaceans. This extensive panel of shrimp allergens forms a valuable asset for future efforts towards the identification of clinically relevant biomarkers and as a basis to approach patient-tailored immunotherapeutic strategies.

Green bean (Phaseolus vulgaris): a new source of IgE-binding lipid transfer protein.

Green beans belong to the Fabaceae family, which includes widely consumed species, such as beans, peanuts, and soybeans. In the literature, few cases have described allergic reactions upon the exposure to green bean boiling steam or ingestion. Here, we describe five patients reporting documented adverse reactions upon the ingestion of cooked green beans, and we characterize the responsible allergen. Fresh and cooked green beans were tested by a prick + prick technique. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and IgE immunoblotting were performed with boiled vegetable extract, and the N-terminal sequence of the immunoreactive protein was obtained by analyzing the excised band in a protein sequencer. Immunoblotting inhibition of cooked green bean with in-house-purified peach lipid transfer protein (LTP) Pru p 3 was performed. An interesting green bean protein was chromatographically purified, tested with a pool serum, and inhibited with Pru p 3. Moreover, its molecular mass was determined by mass spectrometry. Prick + prick tests with raw and cooked green beans were positive for all of the patients. IgE immunoblotting showed that all of the patients reacted toward a unique IgE-binding protein at about 9 kDa. The obtained N-terminal sequence revealed the following amino acids: Ala-Ile-Ser-X-Gly-Qln-Val-Thr-Ser-Ser-Leu-Ala, corresponding to an LTP. A complete inhibition of the IgE binding to this protein, in both raw and purified extract, was obtained by purified peach Pru p 3, confirming previous IgE immunoblotting results.

Detailed proteomic analysis on DM: insight into its hypoallergenicity.

Successful therapy in cow milk (CM) protein allergy rests upon completely eliminating CM proteins from the child's diet: it is thus necessary to provide a replacement food. Donkey milk (DM) has recently aroused scientific and clinical interest, above all among paediatric allergologists. A deeper knowledge of proteins in DM is necessary to evaluate the immunological and physiological properties of this natural substitute for cow's milk. The paper offers a detailed comparative analysis among the protein fractions of DM, CM and human milk, following an extensive proteomic study of the casein and whey proteins of DM performed by narrow pH range 2-DE. The detailed protein composition and structural features reported in this study provide insight into the molecular reasons for the hypoallergenicity of DM. Whole DM might constitute a valid substitute of CM in feeding children with CM protein allergy and it might also constitute the basis for formulas suitable for allergic subjects in the first year of life.

Donkey's milk detailed lipid composition.

Donkey's milk (DM) has recently aroused scientific interest, above all among paediatric allergologists. A deeper knowledge of both proteins and fats in donkey's milk is necessary to evaluate the immunological, physiological and nutritional properties. By using the most refined techniques for fatty acids analysis, the paper offers a detailed comparative analysis of the lipid fractions of DM as well as of human and cow milk, also indicating the distribution of fatty-acid moieties among sn-1/3 and sn-2 positions of the glycerol backbone. In DM the position of fatty acids on glycerol backbone, above all of long chain saturated fatty acids, is very similar to that of human milk: this fact, in conjunction with the relatively high contents of medium-chain triglycerides, makes the lipids in DM, through quantitatively reduced, highly bioavailable. The high PUFA n-3 content of donkey's milk, and especially its low n-6/n-3 ratio, acquires particular interest in subjects affected by cow's milk protein allergy. Whole DM might also constitute the basis for formulas suitable for subjects in the first year of life.

Tomato allergy: detection of IgE-binding lipid transfer proteins in tomato derivatives and in fresh tomato peel, pulp, and seeds.

There is an increasing consumption of tomatoes worldwide: fresh in salads, cooked in household sauces, or industrially processed. Although many tomato allergens have been identified, there is no information in the literature on the allergenic components found in commercial tomato products. The primary aim of the study was to evaluate the allergenic profile of commercial tomato products by skin prick tests (SPTs) and IgE/immunoblotting in tomato-allergic subjects. The secondary end point was the study of the IgE-binding profile of tomato peel, pulp, and seeds. Forty tomato-allergic patients, reporting oral allergy syndrome (OAS) at different grades of severity for fresh and, in some cases, also for cooked tomato, were selected on the basis of positive tomato allergy history or open food challenge (OFC). They were evaluated by SPTs with different experimental tomato extracts. SDS-PAGE/immunoblotting was performed to detect tomato allergens, which were then identified by Edman degradation. Twenty-three patients (57.5%) presented first-grade OAS at the OFC, whereas 17 (42.5%) reported severe symptoms. Ten of these 17 patients (25%) reported allergic reactions to cooked tomatoes; in immunoblotting tests, their sera reacted only to lipid transfer protein (LTP). In commercial products, LTP was the only detectable allergen. In contrast to other LTP-containing fruits, in tomato, an IgE-binding LTP was identified not only in the peel but also in the pulp and seeds. This study demonstrates that, in fresh tomato, different LTP isoforms are present and allergenic. Industrial tomato derivatives still contain LTP, thus presenting a problem for LTP-allergic patients.

Analysis of major proteins and fat fractions associated with mare's milk fat globules.

Although several studies have aimed to identify mare's milk proteins, only the major whey proteins and some caseins have yet been characterized. Incomplete sequencing of the equine genome and the difficulty of recovering highly hydrophobic proteins mean that little is known to date about the proteins associated with milk fat globules, which have been shown to play an important role in newborns' defense mechanisms. The fat fraction, in particular the distribution of unsaturated fatty acids, has been more extensively studied, but complex lipids are only partially elucidated. This study reports a 2-DE approach combined with a powerful method for de novo protein sequencing, and quali-quantitative data on complex lipid composition determined by high performance TLC (HPTLC) and GC. The presence in mare's milk of long-chain highly unsaturated fatty acids, and the evidence of close similarity between equine and human milk fat globule membrane proteins, support the use of mare's milk for human nutrition.