Sensitive detection of major food allergens in breast milk: first gateway for allergenic contact during breastfeeding.

Food allergy is recognized as a major public health issue, especially in early childhood. It has been hypothesized that early sensitization to food allergens maybe due to their ingestion as components dissolved in the milk during the breastfeeding, explaining reaction to a food, which has never been taken before. Thus, the aim of this work has been to detect the presence of the food allergens in breast milk by microarray technology. We produced a homemade microarray with antibodies produced against major food allergens. The antibody microarray was incubated with breast milk from 14 women collected from Fundación Jiménez Díaz Hospital. In this way, we demonstrated the presence of major foods allergens in breast milk. The analysis of allergens presented in breast milk could be a useful tool in allergy prevention and could provide us a key data on the role of this feeding in tolerance induction or sensitization in children.

Molecular characterization of contact urticaria in patients with melon allergy.

BACKGROUND: The relevance of contact allergy to plant-related food has recently emerged. Oral allergy syndrome is one of the most characteristic symptoms of fruit allergy, although it also causes systemic reactions. Plant-food allergy is increasing at the same time as pollen allergy, and fruit-induced allergic contact urticaria could be rising as well. OBJECTIVES: The present study was carried out in order to investigate whether one particular primary melon-peel allergen is responsible for contact urticaria. METHODS: Fourteen patients presenting with contact urticaria after touching melon peel were evaluated. A melon-peel extract was prepared and analysed by immunoblotting using the patients' sera. Molecular characterization of IgE-binding bands was performed using mass spectrometry. Melon-peel lipid transfer protein (LTP) was purified. Inhibition studies and contact challenge with the protein were performed to confirm IgE reactivity to the purified allergen. RESULTS: An IgE-binding band of ~8-9 kDa was observed in an immunoblotting assay with all the patients' sera and was identified as an LTP. The melon-peel LTP was purified in two chromatography steps. Inhibition studies confirmed LTP as a major allergen in patients with melon-peel contact urticaria. Contact challenge with melon-peel LTP was performed in five patients, all of whom had positive results, exhibiting itchy erythema and hives in the area of contact. CONCLUSIONS: This study confirmed our previous findings that melon-peel LTP is a major allergen and is responsible for contact allergy. This knowledge may be used to improve both diagnosis and treatment of patients allergic to melon.

Spanish human proteome project: dissection of chromosome 16.

The Chromosome 16 Consortium forms part of the Human Proteome Project that aims to develop an entire map of the proteins encoded by the human genome following a chromosome-centric strategy (C-HPP) to make progress in the understanding of human biology in health and disease (B/D-HPP). A Spanish consortium of 16 laboratories was organized into five working groups: Protein/Antibody microarrays, protein expression and Peptide Standard, S/MRM, Protein Sequencing, Bioinformatics and Clinical healthcare, and Biobanking. The project is conceived on a multicenter configuration, assuming the standards and integration procedures already available in ProteoRed-ISCIII, which is encompassed within HUPO initiatives. The products of the 870 protein coding genes in chromosome 16 were analyzed in Jurkat T lymphocyte cells, MCF-7 epithelial cells, and the CCD18 fibroblast cell line as it is theoretically expected that most chromosome 16 protein coding genes are expressed in at least one of these. The transcriptome and proteome of these cell lines was studied using gene expression microarray and shotgun proteomics approaches, indicating an ample coverage of chromosome 16. With regard to the B/D section, the main research areas have been adopted and a biobanking initiative has been designed to optimize methods for sample collection, management, and storage under normalized conditions and to define QC standards. The general strategy of the Chr-16 HPP and the current state of the different initiatives are discussed.

Two-photon cooperative absorption in colliding cold Na atoms.

Two-photon cooperative absorption is common in solid-state physics. In a sample of trapped cold atoms, this effect may open up new possibilities for the study of nonlinear effects. The experiment described herein starts with two colliding Na atoms in the S hyperfine ground state. The pair absorb two photons, resulting in both a P(1/2) and a P(3/2) atom. This excitation is observed by ionization using an external light source. A simple model that considers only dipole-dipole interactions between the atoms allows us to understand the basic features observed in the experimental results. Both the pair of generated atoms and the photons originating from their decay are correlated and may have interesting applications that remain to be explored.

An odorant-binding protein as a new allergen from Siberian hamster (Phodopus sungorus).

A case of anaphylaxis following a bite from a Siberian hamster (SH; Phodopus sungorus) is described. Skin prick tests with hair, urine and salivary gland extracts from SH were positive, while the tests were negative for hair extracts from other rodents. IgE immunoblotting with the patient serum revealed 3 IgE-binding bands of about 18, 21 and 23 kDa. When the patient's serum was preincubated with rabbit, mouse and gerbil hair extracts, no inhibition of the 3 SH IgE-binding bands was demonstrated. Proteins extracted from the 3 bands were analyzed by N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry, and peptides were sequenced. IgE-binding bands were identified as being an odorant-binding protein belonging to the lipocalin family. Analysis of the 3 IgE-binding bands found in the hair, urine and salivary glands of SH showed a new allergenic protein lacking cross-reactivity with allergens from other rodents. The 3 bands likely correspond to isoforms of a single allergen.

Identification of sole parvalbumin as a major allergen: study of cross-reactivity between parvalbumins in a Spanish fish-allergic population.

BACKGROUND: Fish allergy is becoming an important health problem in Spain, a country with the third highest level of fish consumption after Japan and Portugal. The most common fish allergens are parvalbumins. In our area, the most widely consumed fish species are lean, such as whiff (Lepidorhombus whiffiagonis) and sole (Solea solea). Adverse reactions to fish are usually related to these species, a fact that is largely unknown to allergists in other countries. OBJECTIVE: The aim of this study was to identify and purify the major allergen implicated in allergic response to sole and evaluate the IgE cross-reactivity of purified parvalbumins from whiff and sole, which are phylogenetically close, and more distant species (i.e. cod and salmon). METHODS: Eighteen Spanish fish-allergic patients with a positive history of type I allergy to fish were recruited from the clinic. Total protein extracts and purified parvalbumins from whiff and sole were tested for their IgE-binding properties by combining two-dimensional Western blotting and mass spectrometry. The extent of cross-reactivity between these parvalbumins along with cod and salmon parvalbumins was investigated by IgE ELISA inhibition assay. RESULTS: An IgE-binding spot of approximately 14 kDa was identified as parvalbumin and confirmed as a major allergen in sole extract, which is recognized by almost 70% of the patients. Whiff parvalbumin was recognized by 83.4% of the patients. High cross-reactivity was determined for all purified parvalbumins by IgE inhibition assay. CONCLUSIONS AND CLINICAL RELEVANCE: Sole and whiff parvalbumin were confirmed as major allergens. The parvalbumins of sole, whiff, cod and salmon were highly cross-reactive, thus suggesting a high amino acid sequence identity between them.

Phosphorylation reduces the allergenicity of cow casein in children with selective allergy to goat and sheep milk.

This study aimed to characterize the role of phosphorylation of caseins in selective allergy to goat milk (GM) and sheep milk (SM) in patients with good tolerance to cow milk (CM). We performed skin prick tests with milk and caseins from CM, GM, and SM and immunoblotting and specific immunoglobulin (Ig) E determinations with milk and casein from cow and GM and SM. Sensitization to milk and caseins from goat and sheep was demonstrated in all 3 patients by skin tests, determination of specific IgE, or both. Immunoblotting confirmed that GM/SM proteins but not CM proteins were involved in the allergic symptoms. IgE reacted with several protein bands from the caseins and milk extracts of both sheep and goat. Phosphorylation was involved in the different allergenicity of CM caseins. We report the implication of phosphorylation in the allergenicity of caseins involved in selective allergy to GM and SM.

[Cardiovascular risk study in patients with renin-angiotensin system blockade by means of the proteone of circulating extracellular vesicles]. / Estudio del riesgo cardiovascular en pacientes con bloqueo del sistema renina-angiotensina a través del proteoma de las vesículas extracelulares circulantes.

INTRODUCTION: Extracellular vesicles (EVs) are released to the bloodstream by certain cell types due to transport, activation and cell death processes. Blood count of EVs from platelet and endothelial origin has been proved to be a cardiovascular risk biomarker. Thus, EVs proteome might reflect the underlying cellular processes in hypertensive patients with albuminuria. MATERIAL AND METHODS: Protein content of circulating EVs was analyzed by liquid chromatography coupled to mass spectrometry. EVs were isolated by an ultracentrifugation protocol optimized in order to avoid contamination by blood plasma proteins. Purity of the isolated fraction was verified by electronic and confocal microscopy, and by flow cytometry. RESULTS: We hereby show a method to isolate circulating EVs from hypertensive patients with/without albuminuria with high yield and purity. Besides, we provide a reference proteome of the EVs of these patients, composed of 2,463 proteins, and prove that the proteins carried by these vesicles are associated with crucial processes involved in the inherent cardiovascular risk. CONCLUSION: The proteome of circulating EVs is an interesting source of indicators in the evaluation of cardiovascular risk in hypertensive patients with renin-angiotensin system blockage.

The covalent interaction of C3 with IgG immune complexes.

Antigens (Ags) are converted into immune complexes (antigen-antibody complexes, IC) as soon as they encounter their specific antibodies (Abs). In fluids containing complement, the process of IC formation and fixation of complement components occur simultaneously. Hence, the formation of Ag-Ab-complement complexes is the normal way of eliminating Ags from a host. C3b-C3b-IgG covalent complexes are immediately formed on interaction of serum C3 with IgG-IC. These C3b-C3b dimers constitute the core for the assembly of C3/C5-convertase on the IC, which are subsequently converted into iC3b-iC3b-IgG by the complement regulators. These complexes are detected on SDS-PAGE by two bands of molecular composition, C3alpha65-C3alpha43 (band A) and C3alpha65-heavy chain of the Ab (band B), which correspond to C3b-C3b and C3b-IgG covalent interaction respectively, and that identify opsonized IC (C3b-IC). C3b can attach to Fab and Fc regions of the Ab molecule with similar efficiency. The presence of multiple C3b binding regions on IgG is considered an advantageous characteristic that facilitates the elimination of Ags in the form of C3b(n)-IC. Ab molecules on the IC recognize the Ag, and also serve as a very good acceptor for C3b binding. In this way, Ags, even if they have no acceptor sites for C3b, can be efficiently processed and removed. When C3 is activated in serum by IC or other activators, secondary C3b-IgG covalent complexes are generated, with bystander monomeric circulating IgG, and thus constitute, physiological products of complement activation. These complexes gain importance when IgG concentration is extremely high as in cases of infusion of intravenous IgG (IVIG) in several pathologies. The covalent attachment of activated complement C3 (C3b, iC3b, C3 d,g) to Ags or IC links innate and adaptative immunity by targeting Ags to different cells of the immune system (follicular dendritic cells, phagocytes, B cells). Hence C3b marks Ags definitively, from the earliest contact with the innate immune system until their complete elimination from the host.

Proteolytic activity of the different fragments of factor B on the third component of complement (C3). Involvement of the N-terminal domain of Bb in magnesium binding.

Kinetic experiments measuring the proteolytic activity of Bb and 33Kd fragment (the C-terminal domain of factor B) on C3 were performed in several conditions, in order to assess the role of factor B domains in the catalytic activity and magnesium binding. The experiments were carried out in fluid phase with 125I-C3 or C3(H2O) as substrates and in the presence of nonradioactive C3b as cofactor. The results indicate: (a) The C-terminal domain, 33Kd, possesses proteolytic activity on C3, which is Mg2(+)-independent, whereas proteolysis by Bb is enhanced in 5 mM Mg2+. (b) C3b behaves as cofactor of 33Kd proteolytic activity on C3 and factor H is able to inhibit this activity. (d) Kinetics of C3 proteolysis by 33Kd shows a lag phase which is also displayed by Bb in the absence but not in the presence of Mg2+. Taken together these data are consistent with the involvement of the N-terminal domain of Bb in Mg2+ binding, which results in an enhancement of the proteolytic activity on C3 of the adjacent C-terminal domain. A C3 convertase model accounting for these results is presented.

The interaction of 1-anilino-8-naphthalene sulphonate with human C1q.

C1q has 12 binding sites for 1-anilino-8-naphthalene sulphonate (ANS), two per peripheral subunit. This number increases to 18 upon weak-acid-induced conformational transition in the globular heads. One ANS binding site is present in each C gamma 2 domain of human IgG. ANS is bound by C1q with a higher affinity (Ka = 2.07 X 10(6) M-1) than by the Fc fragment (Ka = 9.07 X 10(4) M-1) of human IgGl. Hence the inhibitory capacity of C1q binding to IgG immune complexes of ANS probably reflects its preferential binding to the globular heads of C1q. The characteristics of ANS-C1q binding may in part explain the hydrophobic component of the C1q-IgG interaction. It is suggested that an ionic-hydrophobic two-step process is involved in the contact between C1q and IgG.

Separation of active and inactive forms of the third component of human complement, C3, by fast protein liquid chromatography (FPLC).

C3(H2O), an inactive form of C3 present to a variable extent in most C3 preparations, has been isolated in 40 min from previously purified C3 using FPLC ion exchange chromatography on a Mono Q column. As many as six peaks were obtained from some C3 preparations, corresponding to different molecular forms of the protein. One of these peaks consisted of a molecular form of C3 with intact alpha and beta chains, a free sulfhydryl group but no hemolytic activity and was identified as C3(H2O). C3(H2O) eluted as a homogeneous peak well resolved from native C3, C3b, high molecular weight aggregates and small degradation fragments. The same C3(H2O) peak was generated from native C3 by repeated freeze-thaw cycles or NH2OH treatment. C3(H2O) alpha chain appeared as a doublet about 2 kDa heavier than native C3 alpha chain in low cross-linked gels. Two forms of C3b could be separated on the Mono S column, both able to form the C3 convertase. The present report describes a very fast method to resolve and isolate to homogeneity C3(H2O) and native C3 from C3 preparations. Both molecular forms of C3 are very suitable for studies of the initial and amplification C3 convertases of the alternative pathway of complement.

Molecular cloning and characterization of an IgE-reactive protein from Anisakis simplex: Ani s 1.

Ingestion of the parasitic nematode Anisakis simplex in undercooked fish can cause severe allergic reactions in some individuals. Using pooled human sera from sensitized patients we have probed an expression library for A. simplex antigens. One positive clone was found to encode a full length 21 kDa protein with strong homology to nematode troponins. The recombinant protein was expressed as a GST-fusion protein and found by immunoblot analysis to react with sera from 20% of allergic patients. The presence of functional EF-hand Ca(2+) binding motifs was demonstrated by gel-shift analysis.

Surface spiral waves in a filamentary vortex

Spiral surface waves emitted from a vertical vortex are studied experimentally. It is found that, similar to what occurs in the scattered wave of electrons for the Aharonov-Bohm effect, the number of arms of these waves is linked to circulation flux and therefore to the number of dislocations in the wave front.