Anaphylaxis to Vespa velutina nigrithorax: Pattern of Sensitization for an Emerging Problem in Western Countries.

OBJECTIVE: To define the sensitization pattern of patients with anaphylaxis to Vespa velutina nigrithorax (VVN). METHODS: We studied 100 consecutive Spanish patients with anaphylaxis to Hymenoptera venom and systematically determined specific IgE (sIgE) to whole venoms (Vespula species, Polistes dominula, Apis mellifera, Vespa crabro, and Dolichovespula maculata) and their molecular components (rApi m 1, rApi m 5, rApi m 10, rVes v 1, rVes v 5, rPol d 5, and cross-reactive carbohydrates). Specific IgE to VVN venom and its antigen 5 (nVesp v 5) were measured in a subsample. RESULTS: Seventy-seven patients had anaphylaxis to VVN. Of these, only 16 (20.8%) reported previous VVN stings, but were stung by other Hymenoptera. Positive sIgE (>0.35 kUA/L) to each of the whole venoms was detected in >70% of patients (Vespula species in 100%). The components showing >50% positivity were rApi m 5 (51.4%), rPol d 5 (80.0%), and rVes v 5 (98.7%). This pattern was similar to that of Vespula species anaphylaxis (n=11) but different from that of A mellifera anaphylaxis (n=10). Specific IgE to nVesp v 5 was positive in all patients (n=15) with VVN anaphylaxis and was correlated with sIgE to both rVes v 5 (R=0.931) and rPol d 5 (R=0.887). CONCLUSIONS: VVN has become the commonest cause of Hymenoptera anaphylaxis in our area. Most cases report no previous VVN stings. Their sensitization pattern is similar to that of patients with anaphylaxis to other Vespidae. Specific IgE to antigen-5 from VVN, Vespula species, and P dominula are strongly correlated in patients with VVN anaphylaxis.

Anaphylaxis to Vespa velutina nigrithorax: Pattern of Sensitization for an Emerging Problem in Western Countries

Objective: To define the sensitization pattern of patients with anaphylaxis to Vespa velutina nigrithorax (VVN). Methods: We studied 100 consecutive Spanish patients with anaphylaxis to Hymenoptera venom and systematically determined specific IgE (sIgE) to whole venoms (Vespula species, Polistes dominula, Apis mellifera, Vespa crabro, and Dolichovespula maculata) and their molecular components (rApi m 1, rApi m 5, rApi m 10, rVes v 1, rVes v 5, rPol d 5, and cross-reactive carbohydrates). Specific IgE to VVN venom and its antigen 5 (nVesp v 5) were measured in a subsample. Results: Seventy-seven patients had anaphylaxis to VVN. Of these, only 16 (20.8%) reported previous VVN stings, but were stung by other Hymenoptera. Positive sIgE (>0.35 kUA/L) to each of the whole venoms was detected in >70% of patients (Vespula species in 100%). The components showing >50% positivity were rApi m 5 (51.4%), rPol d 5 (80.0%), and rVes v 5 (98.7%). This pattern was similar to that of Vespula species anaphylaxis (n=11) but different from that of A mellifera anaphylaxis (n=10). Specific IgE to nVesp v 5 was positive in all patients (n=15) with VVN anaphylaxis and was correlated with sIgE to both rVes v 5 (R=0.931) and rPol d 5 (R=0.887). Conclusions: VVN has become the commonest cause of Hymenoptera anaphylaxis in our area. Most cases report no previous VVN stings. Their sensitization pattern is similar to that of patients with anaphylaxis to other Vespidae. Specific IgE to antigen-5 from VVN, Vespula species, and P dominula are strongly correlated in patients with VVN anaphylaxis (AU)

Component-resolved diagnosis of vespid venom-allergic individuals: phospholipases and antigen 5s are necessary to identify Vespula or Polistes sensitization.

BACKGROUND: Cross-reactivity between hymenoptera species varies according to the different allergenic components of the venom. The true source of sensitization must therefore be established to ensure the efficacy of venom immunotherapy. OBJECTIVE: In the Mediterranean region, Polistes dominulus and Vespula spp. are clinically relevant cohabitating wasps. A panel of major vespid venom allergens was used to investigate whether serum-specific IgE (sIgE) could be used to distinguish sensitization to either vespid. METHODS: Fifty-nine individuals with allergic reactions to vespid stings and positive ImmunoCAP and/or intradermal tests to vespid venoms were studied. sIgE against recombinant and natural venom components from each wasp species was determined using the ADVIA Centaur(®) system. RESULTS: sIgE against recombinant antigen 5s sensitization to be detected in 52% of the patients tested (13/25). The sensitivity increased to 80% (20/25), when using natural antigen 5s, and to 100% with the complete panel of purified natural components, because the sIgE was positive to either the antigen 5s (Pol d 5/Ves v 5) or to the phospholipases (Pol d 1/Ves v 1) of the two vespids, or to both components at the same time. In 69% of cases, it was possible to define the most probable sensitizing insect, and in the rest, possible double sensitization could not be excluded. Vespula hyaluronidase was shown to have no additional value as regards the specificity of the assay. CONCLUSIONS: The major allergens of P. dominulus' and Vespula vulgaris' venom, namely phoshpholipases and antigen 5s, are required to discriminate the probable sensitizing species in vespid-allergic patients.

Assessment of airborne soy-hull allergen (Gly m 1) in the Port of Ancona, Italy.

BACKGROUND: Epidemic asthma outbreaks are potentially a very high-risk medical situation in seaport towns where large volumes of soybean are loaded and unloaded Airborne allergen assessment plays a pivotal role in evaluating the resulting environmental pollution. OBJECTIVE: The aim of this study was to measure the airborne Gly m 1 allergen level in the seaport of Ancona in order assess the soybean-specific allergenic risk for the city. METHODS: Allergen and PM10 were evaluated at progressive distances from the port area. Allergen analysis was performed by monoclonal antibody-based immunoassay on the sampled filters. Daily meteorological data were obtained from the local meteorological station. For estimating the assimilative capacity of the atmosphere, an approach based on dispersive ventilation coefficient was tried. RESULTS: The allergen concentrations detected were low (range = 0.4-171 ng/m3). A decreasing gradient of the airborne allergen from the unloading area (22.1 +/- 41.2 ng/m3) to the control area (0.6 +/- 0.7 ng/m3) was detected. The concentration of the airborne Gly m 1 was not coupled with the presence of the soy-carrying ships in the port. A statistically significant relationship between airborne allergen, PM10 and local meteorological parameters quantifies the association with the atmospheric condition. CONCLUSION: Airborne Gly m 1 is part of the atmospheric dust of Ancona. The low level of this allergen seems consistent with the absence of asthma epidemic outbreak.

Profilin sensitization detected in the office by skin prick test: a study of prevalence and clinical relevance of profilin as a plant food allergen.

BACKGROUND: Profilin, a pan-allergen present in all eukaryotic cells, is one of the main causes of cross-sensitization between pollen and plant-derived foods, but its clinical relevance as a food allergen is still debated. OBJECTIVE: To investigate the prevalence of profilin sensitization in a pollen-allergic population and its clinical relevance as a food allergen. METHODS: Two hundred consecutive patients with pollen allergy underwent skin prick tests (SPT) with purified natural date palm profilin (Pho d 2; 50 microg/mL; Alk Abello, Madrid, Spain). Those reporting adverse reactions to foods (confirmed by SPT with either commercial food extracts or fresh foods) underwent SPT with an apple extract containing uniquely Mal d 1 (2 microg/mL; ALK-Abello), and with a commercial peach extract containing uniquely lipid transfer protein (LTP 30 microg/mL; ALK-Abello). RESULTS: Sixty patients (30%) showed skin reactivity to date palm profilin, Pho d 2. All were sensitized to grass pollen, and most of them reacted to birch, mugwort, ragweed and plantain pollen as well. SPT with pellitory and cypress scored negative in a high proportion of profilin reactors [26/60 (43%) and 33/60 (55%), respectively]. More than one half (34/60 [57%]) of profilin reactors had food allergy; 21 of these were monosensitized to profilin, 11 were sensitized to both profilin and Bet v 1 homologous protein, one to both profilin and LTP, and one to all the three allergens. The large majority of profilin-allergic patients reported oral allergy syndrome as the only food-induced symptom and were able to tolerate the offending foods if they were cooked or otherwise processed. Twenty-eight of 34 reported reactivity to two or more plant-derived foods. Rosaceae, tree nuts, melon and watermelon, tomato, pineapple, citrus fruits and banana were the more frequently offending foods. CONCLUSION: Profilin should be considered a clinically relevant food allergen. Allergy to melon, watermelon, tomato, banana, pineapple and orange may be considered as a marker of profilin hypersensitivity. This study underlines the clinical importance of being able to diagnose hypersensitivity to single food allergenic proteins by SPT, particularly when the relevant food allergen sources contain several allergens that show different chemical/physical features and, hence, completely different risk profiles.

The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Accidente cerebrovascular: análisis de la casuística en el servicio de medicina de un hospital de baja complejidad / Cerebrovascular accident: case analysis at the medicine unit of an hospital of low complexity

El accidente cerebrovascular (ACV) constituye uno de los problemas de salud pública más relevantes en la actualidad, debido a su alta incidencia, a la mortalidad e invalidez que ocasiona, a los altos costos que genera y a la carencia de estrategias terapéuticas para un adecuado manejo. Se analizó el total de pacientes egresados del servicio de medicina del Hospital Santo Tomás de Limache durante el año 2003, con el objeto de identificar las características de ellos, su evolución intrahospitalaria y plantear mejores estrategias de manejo futuras. Se encontró una distribución equitativa entre ambos sexos, con un promedio de edad de 72 años. Entre las comorbilidades destacó la alta frecuencia de hipertensión arterial 86,96 por ciento, fueron menos frecuentes la fibrilación articular 21,74 por ciento y la diabetes mellitus 17,39 por ciento. Casi un tercio de los pacientes presentaban antecedentes de ACV previos 27,91 por ciento. Las complicaciones más frecuentes encontradas durante la hospitalización fueron la infección del tracto urinario, la insufiencia renal aguda y la neumonía

Resultados del tratamiento quirúrgico de las fracturas de acetábulo / Acetabulum fracture: results of surgical treatment

Objetivo: Evaluación de los resultados del tratamiento con reducción abierta y fijación interna de las fracturas desplazadas de acetábulo, intervenidas quirúrgicamente entre enero de 1989 y diciembre de 1998. Material y Método: Estudio retrospectivo de 57 pacientes con una edad promedio de 34, 6 años y un tiempo medio de seguimiento de 66,3 meses. El mecanismo lesional correspondió en 45 casos a accidentes de tráfico (78,9 por ciento) y en 12 casos a aplastamientos (21,1 por ciento). De acuerdo con la clasificación de Judet y Letournel hubo 24 fracturas simples (42,1 por ciento) y 33 fracturas complejas (57,9 por ciento). La fijación interna se realizó con placa y tornillo en 35 casos (61,4 por ciento) y con tornillos en 22 casos (38,6 por ciento). Resultados: La reducción postoperatoria fue anatómica en 27 casos (47,3 por ciento), satisfactoria en 16 casos (28,1 por ciento) y deficiente en 14 casos (24,6 por ciento). Los resultados funcionales fueron excelentes en 22 casos (38,6 por ciento), buenos en 18 casos (31,6 por ciento), regulares en 6 casos (10,5 por ciento) y malos en 11 casos (19,3 por ciento). Las complicaciones precoces fueron redesplazamiento de la fractura en 3 casos (5,3 por ciento), hematoma postoperatoria en 2 casos (3,5 por ciento), parálisis del nervio ciático en 2 casos (3,5 por ciento), trombosis venosa profunda en un caso (1,8 por ciento) infección profunda en un caso (1,8 por ciento) e infección superficial en un caso (1,8 por ciento). Las complicaciones tardías fueron osificación heterotópica en 7 casos (12,3 por ciento), artrosis postraumática en 6 casos (10,5 por ciento) y necrosis avascular de la cabeza femoral en 4 casos (7,0 por ciento). Conclusión: La reducción abierta y fijación interna es un método eficaz para el tratamiento de las fracturas desplazadas de acetábulo, pues permite reconstruir la articulación coxofemoral, consiguiendo buenos resultados clínicos en el largo plazo.

Solution structure of allergenic 2 S albumins.

The NMR solution structures at different levels of refinement of three different 2 S albumin seed proteins, the recombinant pronapin precursor from Brassica napus, the recombinant RicC3 from Ricinus communis and the methionine-rich protein from sunflower ( Helianthus annuus ), are described. The resulting common structure consists of a bundle of five alpha-helices, folded in a right-handed superhelix. The structure is very similar to that of other plant proteins: the hydrophobic protein from soybean, non-specific lipid transfer proteins and amylase/trypsin inhibitors. Analogies and differences in the structures of these families, as well as their possible relationship to allergenicity, are discussed.

Allergenic diversity of the olive pollen.

A great number of allergenic proteins have been detected in olive pollen extracts. To date, nine allergens have been isolated and characterized, which have been called Ole e 1 to Ole e 9. The most prevalent olive allergen is Ole e 1, which affects more than 70% of patients hypersensitive to olive pollen, but others, such as Ole e 2, Ole e 8, and Ole e 9, have been demonstrated to be major allergens, and Ole e 6 or Ole e 7 reach high values of clinical incidence. Many of these allergens, such as Ole e 2 (profilin) and Ole e 3 (polcalcin), are involved in cross-reactivities, which agrees with their adscription to panallergenic families. Among the many olive allergens of high molecular mass, only Ole e 9 (46 kDa) has been characterized. The allergen is a polymorphic and glycosylated beta-1,3-glucanase, which belongs to a pathogenesis-related (PR-2) protein family. In addition to the polypeptide epitopes, Ole e 1 also exhibits IgE-binding determinants in the carbohydrate, which are recognized by more than 60% of the sera from patients sensitive to the whole allergen, although the level of such glycan-specific IgE seems not to be clinically relevant in the overall content of the sera. Recent advances in the elucidation of the structure of the Ole e 1-oligosaccharide component allows us to explain the antigenicity of the molecule. Finally, the recombinant production of several allergens from olive pollen in both bacterial and eukaryotic cells has allowed us to resolve problems derived from the polymorphism and scarcity of the natural forms of these allergens. The biological equivalence between the natural and recombinant forms lets us initiate studies on the design of mixtures for clinical purposes, in which hypoallergenic derivatives of these allergens could play a definitive role.

Major venom allergen of yellow jackets, Ves v 5: structural characterization of a pathogenesis-related protein superfamily.

Ves v 5 is one of three major allergens found in yellow-jacket venom: phospholipase A(1) (Ves v 1), hyaluronidase (Ves v 2), and antigen 5 (Ves v 5). Ves v 5 is related by high amino acid sequence identity to pathogenesis-related proteins including proteins from mammals, reptiles, insects, fungi, and plants. The crystal structure of Ves v 5 has been solved and refined to a resolution of 1.9 A. The majority of residues conserved between the pathogenesis-related proteins can be rationalized in terms of hydrogen bonding patterns and hydrophobic interactions defining an alpha-beta-alpha sandwich core structure. A small number of consensus residues are solvent exposed (including two adjacent histidines) and located in an elongated cavity that forms a putative active site. The site has no structural resemblance to previously characterized enzymes. Homologous antigen 5's from a large number of different yellow jackets, hornets, and paper wasps are known and patients show varying extents of cross-reactivity to the related antigen 5's. The structure of Ves v 5 allows a detailed analysis of the epitopes that may participate in antigenic cross-reactivity, findings that are useful for the development of a vaccine for treatment of insect allergy.

The spectrum of olive pollen allergens.

Olive pollen is one of the most important causes of seasonal respiratory allergy in Mediterranean countries, where this tree is intensely cultivated. Among the high number of protein allergens detected in this pollen, 8 - Ole e 1 to Ole e 8 - have been isolated and characterized. Ole e 1 is the most frequent sensitizing agent, affecting more than 70% of the patients suffering of olive pollinosis, although others, such as Ole e 4 and Ole e 7, have also been shown to be major allergens. In this context, the prevalence of many olive pollen allergens seems to be dependent on the geographical area where the sensitized patients live. Some of the olive allergens have been revealed as members of known protein families: profilin (Ole e 2), Ca(2+)-binding proteins (Ole e 3 and Ole e 8), superoxide dismutase (Ole e 5) and lipid transfer protein (Ole e 7). No biological function has been demonstrated for Ole e 1, whereas Ole e 4 and Ole e 6 are new proteins without homology to known sequences from databases. cDNAs encoding for Ole e 1, Ole e 3 and Ole e 8 have been overproduced in heterologous systems. The recombinant products were correctly folded and exhibited the functional activities of the natural allergens. In addition to the Oleaceae family, other species, such as Gramineae or Betulaceae, contain pollen allergens structurally or immunologically related to those of the olive tree. This fact allows to detect and evaluate antigenic cross-reactivities involving olive allergens. The aim of this research is the development of new diagnostic tools for olive pollinosis and new approaches to improve the classical immunotherapy.

Recombinant allergens with reduced allergenicity but retaining immunogenicity of the natural allergens: hybrids of yellow jacket and paper wasp venom allergen antigen 5s.

The homologous venom allergen Ag 5s from the yellow jacket (Vespula vulgaris) and paper wasp (Polistes annularis) have 59% sequence identity of their respective 204 and 205 amino acid residues, and they have low degrees of antigenic cross-reactivity in insect allergic patients and in animal models. Hybrids containing different segments of these two vespid Ag 5s were expressed in yeast. Circular dichroism spectroscopy suggests the hybrids to have the secondary structure of natural Ag 5. Inhibition ELISA with human and murine Abs suggests the hybrids to have the discontinuous B cell epitopes of the natural Ag 5 but with an altered epitope density. The hybrids were immunogenic in mice for B and T cell responses to both Ag 5s. The N-terminal region of Ag 5 was found to contain its dominant B cell epitope(s). Hybrids containing 10-49 residues of yellow jacket Ag 5 showed 100- to 3000-fold reduction in allergenicity when tested by histamine release assay with basophils of yellow jacket-sensitive patients. Our findings suggest that hybrids represent a useful approach to map the discontinuous B cell epitope-containing regions of proteins. They also suggest that Ag 5 hybrids may be useful immunotherapeutic reagents in man.

Assignment of the disulfide bonds of Ole e 1, a major allergen of olive tree pollen involved in fertilization.

The most prevalent allergen from olive tree pollen, Ole e 1, consists of a single polymorphic polypeptide chain of 145 amino acids which includes six cysteine residues at positions 19, 22, 43, 78, 90 and 131. By using an homogeneous form of the allergen expressed in Pichia pastoris, the array of the disulfide bridges has been elucidated. Specific proteolysis with thermolysin and reverse-phase HPLC separation of the peptides allowed the determination of the disulfide bond between Cys43 and Cys78. Another thermolytic product, which contained three peptides linked by the remaining four cysteines, was digested with Glu-specific staphylococcal V8 protease and the products isolated by reverse-phase HPLC. Amino acid compositions and Edman degradation of the peptide products indicated the presence of the disulfide bonds at Cys19-Cys90 and Cys22-Cys131. These data can help in the analysis of the three-dimensional structure of the protein as well as in studies of its allergenic determinants.

Expressions of recombinant venom allergen, antigen 5 of yellowjacket (Vespula vulgaris) and paper wasp (Polistes annularis), in bacteria or yeast.

Antigen 5 is a major allergen of vespid venom. It has partial sequence identity with proteins from diverse sources. The biologic function of Ag 5 and its related proteins is not known. We are interested in the expression of Ag 5 with the native conformation of the natural protein since its B cell epitopes are mainly of the discontinuous type. When expressed in bacteria, recombinant Ag 5 formed an insoluble intracellular product, and it did not translocate from cytoplasm to periplasm by the addition of a pelB leader sequence to the cloned protein. When expressed in yeast Pichia pastoris, Ag 5 was secreted because the cloned protein contained a yeast alpha signal leader sequence. Recombinant Ag 5 from yeast was shown to have the native structure of the natural protein and the recombinant Ag 5 from bacteria did not. This was shown by comparison of their solubility, electrophoretic behavior, disulfide bond content, CD spectrum, and binding of IgE antibodies from allergic patients and IgG antibodies from mice immunized with natural Ag 5 or recombinant Ag 5s from yeast or bacteria. These studies were made with Ag 5s from yellowjacket (Vespula vulgaris) and paper wasp (Polistes annularis).

IgE-binding and histamine-release capabilities of the main carbohydrate component isolated from the major allergen of olive tree pollen, Ole e 1.

BACKGROUND: Pollen from olive trees (Olea europaea ) is a cause of pollinosis and an aggravating of asthma in Mediterranean regions. Recently, Ole e 1, the major allergen from olive tree pollen, has been isolated and its amino acid sequence has been elucidated. It is a glycoprotein whose carbohydrate moiety is involved in an IgE-binding epitope responsible for cross-reactivity among plant glycoproteins. However, the allergenicity of the free carbohydrate side chains remains to be clarified. OBJECTIVE: The purpose of this study was to isolate the main carbohydrate component of Ole e 1 allergen and analyze its IgE-binding and histamine-release capabilities. METHODS: Deglycosylation treatment of Ole e 1 with PNGase F and gel exclusion chromatography were used to isolate the main sugar component of the allergen. Sera of patients who are allergic to olive pollen and sera sensitive to Ole e 1 have been used in dot blotting assays of IgE binding to the isolated carbohydrate. Heparinized whole blood obtained from patients sensitive to Ole e 1 were stimulated by the free carbohydrate; the resulting histamine release was measured. RESULTS: The main sugar component of Ole e 1 has been isolated. Free carbohydrate was able to bind IgE from sera of patients allergic to olive pollen; the sera of 65% of these patients contained anticarbohydrate reacting IgE, and 100% of those patients were sensitive to Ole e 1. The free carbohydrate promoted in vitro histamine release from basophils of sensitized patients. CONCLUSION: The carbohydrate moieties of allergenic glycoproteins can constitute significant determinants on the binding to IgE of the sera from patients who are hypersensitive and can be responsible for inducing histamine release from blood cells.

Expression of yellow jacket and wasp venom Ag5 allergens in bacteria and in yeast.

Antigen 5 (Ag5), of unknown biological function, is one of the major venom allergens of vespids and fire ants. We have compared the expression of Ag5 in bacteria and in yeast. Recombinant Ag5 from bacteria formed an insoluble intracellular product, which was not properly folded, but that produced in Pichia pastoris was secreted to the extracellular medium. Immunochemical characterizations showed the secreted Ag5 to have the native structure of the natural protein. This is of interest since the B cell epitopes of Ag5 are mainly of the discontinuous type. These studies were made with Ag5s from yellow jacket (Vespula vulgaris) and paper wasp (Polistes annularis), and with hybrid Ag5 molecules that contained partial sequences of these two species. In vitro allergenicity studies with sera from yellow jacket-sensitive patients showed that some of these hybrid molecules had a greatly reduced allergenicity but retained the immunogenicity of the natural allergen. This could be of importance for immunotherapy of this type of allergy.