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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) α2β1 integrin-binding motif in the N-terminal domain of donkey sequence only. Because integrin α2β1 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 andRGDmotifs (named pDGE-RGD) showed the greatest activity, and its mechanism of antiviral action was characterized; pDGE-RGD binds to integrin α2β1 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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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 α-amylase inhibitor, 30-kDa endochitinases A and -B, 19 kDa zein-β precursor, and 26kDa zein-α 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. [ABSTRACT FROM AUTHOR]

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 · kg · day) 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.

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.

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 (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.

Soybean containing products are widely consumed, thus reliable methods for detection of soy in foods are needed in order to make appropriate risk assessment studies to adequately protect soy allergic patients. Six methods were compared using eight food products with a declared content of soy: a direct sandwich ELISA based on polyclonal rabbit antibody (ab) to raw soy flakes, a commercial and an in-house competitive ELISA both based on ab to denatured, 'renatured' soy protein, an enzyme-allergosorbent test (EAST) inhibition based on two sera from soy allergic patients, histamine release (HR) using basophils passively sensitized with patient serum and a PCR method detecting soy DNA. Eight food products were selected as model foods to test the performance of the methods. There was an overall good agreement between the methods in terms of ranks of soy content but not the quantity. The sandwich ELISA aimed at native soy proteins had the lowest detection limit of 0.05 ppm, but only identified soy in 5/8 products, and generally in lower amounts compared to other methods. The competitive ELISA had a higher detection limit of 21 ppm, but seemed more successful in detecting processed soy. Only HR, EAST inhibition and PCR detected soy in all eight products. In spite of a general good correlation in terms of ranks of soy content, more than a single method may be necessary to confirm the presence of soy in foods.

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.

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.

Background: In a previous study a 9-kd lipid-transfer protein (LTP) was identified as the major allergen of raw maize in a population of 22 anaphylactic patients. However, the stability of this protein in cooked maize is unknown., Objective: We investigated the allergenicity of 5 maize hybrids and its modification after different thermal treatments by using sera from anaphylactic patients and patients with positive double-blind, placebo-controlled food challenges., Methods: Five maize hybrids were extracted by using different methods, obtaining the water-soluble, zein, total zein, glutelin, and total protein fractions. The IgE-binding capacity of the different extracts, both raw and after thermal treatment, was investigated by means of SDS-PAGE immunoblotting. A 9-kd heat-stable allergen was purified by means of HPLC and sequenced. Changes in its secondary structure during and after heating from 25 degrees C to 100 degrees C were monitored by means of circular dichroism., Results: All raw maize hybrids showed similar protein and IgE-binding profiles. The SDS-PAGE of all the heat-treated hybrids demonstrated a decreased number of stained bands in respect to the raw samples. The IgE immunoblotting demonstrated that the major allergen of the water-soluble, total zein, total protein, and glutelin fractions was a 9-kd protein identified by means of amino acid sequence as an LTP and a sub-tilisin-chymotrypsin inhibitor (in total zein fraction). The IgE-binding capacity of this 9-kd protein remained unchanged after thermal treatments, even though circular dichroism demonstrated an altered secondary structure., Conclusions: Maize LTP maintains its IgE-binding capacity after heat treatment, thus being the most eligible candidate for a causative role in severe anaphylactic reactions to both raw and cooked maize.