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BACKGROUND: Cow's milk is one of the most common causes of food allergy. In two-thirds of patients, adverse symptoms following milk ingestion are caused by IgE-mediated allergic reactions, whereas for one-third, the mechanisms are unknown. Aim of this study was to investigate whether patients suffering from non-IgE-mediated cow's milk protein intolerance can be distinguished from persons without cow's milk protein intolerance based on serological measurement of IgG and IgA specific for purified cow's milk antigens. METHODS: We determined IgG(1-4) subclass and IgA antibody levels to purified recombinant ?S1-casein, ?S2-casein, ?-casein, ?-casein, ?-lactalbumin, and ?-lactoglobulin in four patient groups by ELISA: Patients with IgE-mediated cow's milk allergy (CMA, n=25), patients with non-IgE-mediated cow's milk protein intolerance (CMPI, n=19), patients with gastrointestinal symptoms not associated with cow's milk ingestion (GI, n=15) and control persons without gastrointestinal problems (C, n=26). Cow's milk-specific IgE levels were determined by ImmunoCAP. RESULTS: Only CMA patients had IgE antibodies to cow's milk. Cow's milk allergic patients mounted the highest IgG(1) and IgG(4) antibody levels to ?S1-casein, ?S2-casein, ?-casein, ?-casein, and ?-lactalbumin. No elevated levels of IgG(4) , IgA, and complement-binding IgG subclasses (IgG(1) , IgG(2) , IgG(3) ) to purified cow's milk allergens were found within the CMPI patients compared to persons without cow's milk protein intolerance (GI and C groups). CONCLUSION: Cow's milk protein intolerant patients cannot be distinguished from persons without cow's milk protein intolerance on the basis of IgG subclass or IgA reactivity to cow's milk allergens.

SummaryBackground Cow’s milk is one of the most common causes of food allergy affectingapproximately 2.5% of infants in the first years of their life. However, only limitedinformation regarding the allergenic activity of individual cow’s milk allergens is available.ObjectiveToanalysethefrequencyofIgEreactivityandtodeterminetheallergenicactivityofindividual cow’s milk allergens.MethodsA nitrocellulose-based microarray, based on purified natural and recombinant cow’smilk allergens was used to determine IgE reactivity profiles using sera from 78 cow’s milk-sensitized individuals of varying ages. The allergenic activity of the individual allergens wastested using patients’ sera for loading rat basophil leukaemia cells (RBL) expressing thea-chain of the human receptor FceRI.Results Using the microarray and the RBL assay, cow’s milk allergens were assessed forfrequency of IgE recognition and allergenic activity. Moreover, the RBL assay alloweddistinguishing individuals without or with mild clinical reactions from those with severesystemic or gastrointestinal symptoms as well as persons who grew out cow’s milk allergyfrom those who did not.Conclusions Component-resolved testing using milk allergen microarrays and RBL assaysseems to provide useful additional diagnostic information and may represent a basis forfuture forms of prophylactic and therapeutic strategies for cow’s milk allergy.Keywords Basophil degranulation, cow’s milk allergy, diagnosis, microarray, recombinantallergenSubmitted 15 March 2010; revised 8 June 2010; accepted 13 July 2010

The present paper is part of a study dealing with various aspects of reproduction of two Costa Rican Clusia species offering resin as a floral reward. It provides data on the floral development and flower (especially stamen and staminode) anatomy of one of the species, Clusia valerioi . In the early stages, both male and female flowers develop in the same manner. The bracts are distinguished by a decussate arrangement from the five sepals and five petals, which emerge in a spiral manner. In the male flowers the apical meristem forms five meristematic mounds (common stamen primordia) that are pentagonally arranged around the apical meristem in epipetalous position. From these mounds, the primordia of the proper stamina emerge in 3–5 whorls. Direction is centrifugal. In the centre, five hemispherical bulges arise which develop into carpel primordia. These, however, cease growth, stay rudimentary and are hidden by the stamens in the mature male flower. The adult stamens consist mainly of a thick angular filament column, while the two anthers situated at the flattened top are very small. One anther is annular and surrounds a second, hemispherical one right in the centre. At the periphery, these two pollen sacs (provided with a distinct wall of customary anatomy) are surrounded by a ring-like protuberance of the filament. The resin canals are situated at the periphery of the filament. Their schizogenous development is documented in cross sections. At anthesis, the resin is released from the ring-like filament protuberance by burst of the single-layered epidermis. In the female flower, the five meristematic mounds produce two whorls of staminode primordia. The development of the staminodes does not essentially differ from that of the fertile stamens, but some staminodes lack the central pollen sac and the other tissues do not develop into pollen grains. An attempt is made to derive the peculiar stamen morphology of Clusia valerioi and similar species from conventional stamens. Three hypotheses are proposed and discussed.

but robust approach towards the identification of markers that enable the prediction of the outcome of oral immunotherapy for cow’s milk allergy. By means of ELISA, the authors determined specific IgE, IgA, IgG subclass levels against cow’s milk extract, natural single cow’s milk allergens and the egg allergen ovalbumin in 40 children with cow’s milk allergy before and after immunotherapy. Seventy-six children with no history of cow’s milk allergy and 56 children who recovered spontaneously from their milk allergy served as a reference. The researchers found differences in antibody levels between groups and concluded that high levels of cow’s milk-specific IgE, IgA and IgG before immunotherapy may be good indicators for the failure of tolerance, similar to constant IgA and IgE levels during immunotherapy. The course of IgE levels during therapy has been described differently in different articles; some studies observed that levels of IgE remained constant whereas others noticed a decrease or increase. A clearer picture is reported for allergen-specific IgG 4 levels: low cow’s milk-specific IgG 4 levels indicate a persistent allergy and also a negative outcome of oral immunotherapy. The role of IgA during tolerance development has been investigated less and needs to be studied in more detail. The importance of Savilahti et al.’s [6] article is the topic of the study, the search for objective markers which can be easily evaluated during the course of an immunotherapy and help to predict the outcome. Allergen-specifCow’s milk allergy strongly affects the quality of life of patients and their families and the only specific and causative treatment besides avoidance is specific immunotherapy (SIT). However, SIT must be performed over a long period, is often accompanied by severe side effects and its success rate is affected by a variety of factors, among them the patient’s compliance. Quite often, patients treated with SIT are not properly selected and monitored during therapy. The monitoring of the effects of SIT, especially the immunological parameters, may provide useful information regarding the continuation or discontinuation of treatment. Diagnostic surrogate markers for the prediction of the outcome of immunotherapy are still missing and badly needed. Several rather cumbersome approaches towards suitable markers have been pursued. Complex gene expression profiles, in the case of venom immunotherapy [1] , and impaired pulmonary function in conjunction with age, in the case of house dust mite immunotherapy, have been used as indicators for a lower success rate [2] . Other markers include the reduction of allergen-specific T cell proliferation, shown in sublingual immunotherapy studies involving birch pollen [3] and house dust mite [4] . Ideally, the use of biomarkers should already be possible during treatment, in order to predict the outcome [5] . The article by Savilahti et al. [6] in International Archives of Allergy and Immunology is a rather conservative Published online: July 10, 2014

The aim of the present study was to develop a surface display ELISA (SD-ELISA) for IgG-serum reaction against bovine casein αS1 (CSN1S1). In a SD-ELISA, the antigen is displayed on the surface of Escherichia coli using the autodisplay technology and whole cells of E. coli are used to coat the microplates for serum testing. After establishing the setup of the SD-ELISA with polyclonal rabbit antiserum against bovine CSN1S1, the SD-ELISA was validated with 20 human sera, of which 10 sera were proven to have an IgG-mediated reaction against bovine CSN1S1 and 10 sera were shown to be negative for this reaction. Receiver operating characteristics (ROC) analysis revealed sensitivity of 100% and a specificity of 100% at a cut-off value of 0.133. Furthermore, human serum of 48 patients with known reactivity against human CSN1S1 (31 positive and 17 negative) was examined by the newly developed SD-ELISA to exclude cross-reactivity. Twenty human sera showed an IgG-mediated reaction against bovine CSN1S1. Eleven of these sera were positive for the reactivity against human CSN1S1, and nine were negative. In conclusion it was demonstrated that the performance of SD-ELISA is comparable to established ELISA without loss in sensitivity or specificity. Based on the advantages of this method – in particular no need for time-consuming and expensive antigen production and purification – the SD-ELISA is a potent alternative to convenient methods for identification and especially high-throughput screening of new antigens in the field of food allergies.

Milk is one of the first components introduced into human diet. It also represents one of the first allergen sources, which induces IgE-mediated allergies in childhood ranging from gastrointestinal, skin, and respiratory manifestations to severe life-threatening manifestations, such as anaphylaxis. Here we isolated a cDNA coding for a major cow’s milk allergen, αS1-casein, from a bovine mammary gland cDNA library with allergic patients’ IgE Abs. Recombinant αS1-casein was expressed in Escherichia coli, purified, and characterized by circular dichroism as a folded protein. IgE epitopes of αS1-casein were determined with recombinant fragments and synthetic peptides spanning the αS1-casein sequence using microarrayed components and sera from 66 cow’s milk-sensitized patients. The allergenic activity of rαS1-casein and the αS1-casein-derived peptides was determined using rat basophil leukemia cells transfected with human FcεRI, which had been loaded with the patients’ serum IgE. Our results demonstrate that rαS1-casein as well as αS1-casein-derived peptides exhibit IgE reactivity, but mainly the intact rαS1-casein induced strong basophil degranulation. These results suggest that primarily intact αS1-casein or larger IgE-reactive portions thereof are responsible for IgE-mediated symptoms of food allergy. Recombinant αS1-casein as well as αS1-casein-derived peptides may be used in clinical studies to further explore pathomechanisms of food allergy as well as for the development of new diagnostic and therapeutic strategies for milk allergy.

BACKGROUND: alpha-Lactalbumin (alpha-La) is a major cow's milk (CM) allergen responsible for allergic reactions in infants. OBJECTIVE: We performed molecular, structural, and immunologic characterization of alpha-La. METHODS: Recombinant alpha-lactalbumin (ralpha-La) was expressed in Escherichia coli, purified to homogeneity, and characterized by means of mass spectrometry and circular dichroism, and its allergenic activity was studied by using microarray technology, as well as in a basophil histamine release assay. IgE epitope mapping was performed with synthetic peptides. RESULTS: According to circular dichroism analysis, ralpha-La represented a folded protein with a high thermal stability and refolding capacity. ralpha-La reacted with IgE antibodies from 57.6% of patients with CM allergy (n = 66) and induced the strongest basophil degranulation with sera from patients with CM allergy who had exhibited gastrointestinal symptoms or severe systemic reactions on CM exposure. ralpha-La contained sequential and conformational IgE epitopes. Superposition of IgE-reactive peptides onto the 3-dimensional structure of alpha-La revealed a close vicinity of the N- and C-terminal peptides within a surface-exposed patch. CONCLUSIONS: ralpha-La can be used for the diagnosis of patients with severe allergic reactions to CM and serves as a paradigmatic tool for the development of therapeutic strategies for CM allergy.

Allergy diagnosis based on purified allergen molecules provides detailed information regarding the individual sensitization profile of allergic patients, allows monitoring of the development of allergic disease and of the effect of therapies on the immune response to individual allergen molecules. Allergen microarrays contain a large variety of allergen molecules and thus allow the simultaneous detection of allergic patients' antibody reactivity profiles towards each of the allergen molecules with only minute amounts of serum. In this article we summarize recent progress in the field of allergen microarray technology and introduce the MeDALL allergen-chip which has been developed for the specific and sensitive monitoring of IgE and IgG reactivity profiles towards more than 170 allergen molecules in sera collected in European birth cohorts. MeDALL is a European research program in which allergen microarray technology is used for the monitoring of the development of allergic disease in childhood, to draw a geographic map of the recognition of clinically relevant allergens in different populations and to establish reactivity profiles which are associated with and predict certain disease manifestations. We describe technical advances of the MeDALL allergen-chip regarding specificity, sensitivity and its ability to deliver test results which are close to in vivo reactivity. In addition, the usefulness and numerous advantages of allergen microarrays for allergy research, refined allergy diagnosis, monitoring of disease, of the effects of therapies, for improving the prescription of specific immunotherapy and for prevention are discussed. This work was supported by the European Commission’s Seventh Framework Programme under grant agreement No. 261357 and in part by project F4605 of the Austrian Science Fund (FWF) and the Christian Doppler Research Association, Austria