Hymenoptera venom allergy in outdoor workers: Occupational exposure, clinical features and effects of allergen immunotherapy.

OBJECTIVES: To describe (i) the clinical characteristics of workers, exposed to hymenoptera stings, with an ascertained diagnosis of Hymenoptera Venom Allergy (HVA), (ii) the specific role of occupational exposure, (iii) the effect of Venom Immunotherapy (VIT) in reducing the severity of allergic episodes in workers exposed to repeated stings of hymenoptera, and (iv) the management of the occupational consequences caused by allergic reactions due to hymenoptera stings. METHODS: Between 2000 and 2013 an observational study, including patients referred to the regional reference hospital of Liguria, Italy, with an ascertained diagnosis of HVA and treated with VIT, was performed. A structured questionnaire was administered to all patients to investigate the occupational features of allergic reactions. These were graded according to standard systems in patients at the first episode, and after re-stings, during VIT. RESULTS: One-hundred and 8four out of the 202 patients referred had a complete data set. In 32 (17.4%) patients, the allergic reaction occurred during work activities performed outdoor. Of these, 31.2% previously stung by hymenoptera at work, and receiving VIT, were re-stung during occupational activity. The grades of reaction developed under VIT treatment resulted clinically less severe than of those occurred at the first sting (p-value = 0.031). CONCLUSION: Our findings confirmed the clinical relevance of HVA, and described its occupational features in outdoor workers with sensitization, stressing the importance of an early identification and proper management of the professional categories recognized at high risk of hymenoptera stings. The Occupational Physician should be supported by other specialists to recommend appropriate diagnostic procedures and the prescription of VIT, which resulted an effective treatment for the prevention of episodes of severe reactions in workers with a proven HVA.

It is time for top-down venomics

The protein composition of animal venoms is usually determined by peptide-centric proteomics approaches (bottom-up proteomics). However, this technique cannot, in most cases, distinguish among toxin proteoforms, herein called toxiforms, because of the protein inference problem. Top-down proteomics (TDP) analyzes intact proteins without digestion and provides high quality data to identify and characterize toxiforms. Denaturing top-down proteomics is the most disseminated subarea of TDP, which performs qualitative and quantitative analyzes of proteoforms up to ~30 kDa in high-throughput and automated fashion. On the other hand, native top-down proteomics provides access to information on large proteins (> 50 kDA) and protein interactions preserving non-covalent bonds and physiological complex stoichiometry. The use of native and denaturing top-down venomics introduced novel and useful techniques to toxinology, allowing an unprecedented characterization of venom proteins and protein complexes at the toxiform level. The collected data contribute to a deep understanding of venom natural history, open new possibilities to study the toxin evolution, and help in the development of better biotherapeutics.(AU)

Key issues in hymenoptera venom allergy: an update

In this review, the Hymenoptera Allergy Committee of the SEAIC analyzes the most recent scientific literature addressing problems related to the diagnosis of hymenoptera allergy and to management of venom immunotherapy. Molecular diagnosis and molecular risk profiles are the key areas addressed. The appearance of new species of hymenoptera that are potentially allergenic in Spain and the associated diagnostic and therapeutic problems are also described. Finally, we analyze the issue of mast cell activation syndrome closely related to hymenoptera allergy, which has become a new diagnostic challenge for allergists given its high prevalence in patients with venom anaphylaxis (AU)

En esta revisión el Comité de Alergia a Himenópteros de la SEAIC ha analizado la literatura científica más reciente sobre los principales problemas diagnósticos de la alergia a himenópteros, así como sobre las dificultades que pueden surgir durante la inmunoterapia con venenos. Se revisan especialmente las novedades relacionadas con el diagnóstico molecular y los perfiles moleculares de riesgo. También se describe la alergia a himenópteros poco habituales y los problemas diagnósticos y terapéuticos que esta conlleva. Por último, se tratan los síndromes de activación mastocitaria clonal, íntimamente relacionados con la alergia a himenópteros, que se han convertido en un nuevo reto diagnóstico para el alergólogo (AU)

Insect venom immunotherapy: analysis of the safety and tolerance of 3 buildup protocols frequently used in Spain

Introduction: Hymenoptera venom immunotherapy (VIT) is an effective treatment but not one devoid of risk, as both local and systemic adverse reactions may occur, especially in the initial phases. We compared the tolerance to 3 VIT buildup protocols and analyzed risk factors associated with adverse reactions during this phase. Materials and Methods: We enrolled 165 patients divided into 3 groups based on the buildup protocol used (3, 4, and 9 weeks). The severity of systemic reactions was evaluated according to the World Allergy Organization model. Results were analyzed using exploratory descriptive statistics, and variables were compared using analysis of variance. Results: Adverse reactions were recorded in 53 patients (32%) (43 local and 10 systemic). Local reactions were immediate in 27 patients (63%) and delayed in 16 (37%). The severity of the local reaction was slight/moderate in 15 patients and severe in 13. Systemic reactions were grade 1-2. No significant association was found between the treatment modality and the onset of local or systemic adverse reactions or the type of local reaction. We only found a statistically significant association between severity of the local reaction and female gender. As for the risk factors associated with systemic reactions during the buildup phase, we found no significant differences in values depending on the protocol used or the insect responsible. Conclusions: The buildup protocols compared proved to be safe and did not differ significantly from one another. In the population studied, patients undergoing the 9-week schedule presented no systemic reactions. Therefore, this protocol can be considered the safest approach (AU)

Introducción: La inmunoterapia con veneno de himenópteros (ITV) es un tratamiento eficaz, pero no está desprovisto de riesgo ya que pueden ocurrir reacciones adversas locales o sistémicas, especialmente en las etapas iniciales del tratamiento. Comparamos la tolerancia de tres protocolos de inicio de ITV y analizamos los factores de riesgo asociados con las reacciones adversas que se produjeron en esta fase. Métodos: Se incluyeron 165 pacientes divididos en tres grupos según el protocolo de iniciación utilizado (3, 4 o 9 semanas). Evaluamos la gravedad de las reacciones sistémicas de acuerdo con el modelo de la Organización Mundial de Alergia. Analizamos los resultados mediante estadística descriptiva exploratoria y comparamos variables mediante el análisis de la varianza. Resultados: Cincuenta y tres pacientes (32%) experimentaron algún tipo de reacción adversa; 43 eran locales y 10 sistémicas. Las reacciones locales fueron inmediatas en 27 pacientes (63%) y tardías en 16 (37%). La gravedad de la reacción local fue leve o moderada en 15 pacientes y grave en 13. Las reacciones sistémicas fueron de grado 1 o 2. No encontramos asociación significativa entre la modalidad de tratamiento y la aparición de reacciones adversas locales o sistémicas o el tipo de reacción local. Solo encontramos una asociación estadísticamente significativa de la gravedad de la reacción local con el sexo femenino. En cuanto a los factores de riesgo asociados con las reacciones sistémicas en la fase de inicio, no se encontraron diferencias significativas en estos valores en función del protocolo utilizado o el insecto responsable. Conclusiones: Los protocolos de inicio comparados demostraron ser seguros y no difirieron significativamente entre sí. En la población estudiada, el protocolo de 9-semanas no produjo reacciones sistémicas, por lo que se puede considerar el protocolo más seguro (AU)

Cross-sectional validation of a quality of life questionnaire in spanish for patients allergic to hymenoptera venom

Introducción: En 2002 se desarrolló el primer cuestionario de calidad de vida para alérgicos a veneno de Vespula (VQLQ). El objetivo de este trabajo es desarrollar y validar una versión en español de este cuestionario. Métodos y Pacientes: Partiendo del VQLQ en inglés se realizó la traducción al español y adaptación cultural del cuestionario con una posterior retraducción consensuada con los autores de la versión original. Así se desarrolló el cuestionario de calidad de vida en español para pacientes alérgicos a veneno de himenópteros (HRQLHA). La validación transversal se realizó mediante un estudio multicéntrico, con diseño test-retest en el que se incluyeron pacientes mayores de 14 años con reacción sistémica por alergia a veneno de Apis, Vespula o Polistes . Se analizó la fiabilidad test-retest, la validez externa y la consistencia interna del cuestionario. Resultados: Se incluyeron 116 pacientes alérgicos a venenos de Apis, Vespula o Polistes . La puntuación media del HRQLHA fue 3.48. El estudio de fiabilidad test-retest mostró un coeficiente de correlación intraclase de 0.85. El HRQLHA mostró una correlación positiva (0.5) con el cuestionario de referencia externa, confirmando su validez. En el análisis de la consistencia interna se encontró una Alpha de Cronbach de 0.95 Conclusiones: El análisis estadístico muestra una elevada fiabilidad test-retest y consistencia interna del HRQLHA, además de su validez externa. En la validación del HRQLHA se han incluido por primera vez pacientes alérgicos a veneno de Polistes, muy prevalente en nuestra área, demostrando la utilidad del cuestionario también en estos pacientes (AU)

Introduction and Objectives: The first quality of life questionnaire for Vespula-allergic patients (Vespid Allergy Quality of Life Questionnaire [VQLQ]) was developed in 2002. Our objective was to perform the cross-sectional validation of the Spanish version of this questionnaire. Patients and Methods: Using the original English-language version of the VQLQ as a starting point, the Spanish translation and cultural adaptation were performed and the first Spanish version was backtranslated into English and discussed with the authors of the original version. The result was the Spanish Health-related Quality of Life Questionnaire for Hymenoptera Venom–Allergic Patients (HRQLHA). Cross-sectional validation was carried out in a multicenter study using a test-retest design. Patients over the age of 14 diagnosed with systemic allergic reaction to venom from Apis, Vespula, or Polistes species were included. The test-retest reliability, external validity, and internal consistency of the questionnaire were measured. Results: The sample comprised 116 patients. The mean HRQLHA score was 3.48. Test-retest reliability showed an intraclass correlation coefficient of 0.85. HRQLHA showed a positive correlation (0.5) with an external questionnaire, thus demonstrating its external validity. Analysis of the internal consistency of the HRQLHA yielded a Cronbach a of 0.95, which can be considered to be excellent. Conclusions: Statistical analysis revealed the high test-retest reliability, internal consistency, and external validity of the HRQLHA.The HRQLHA is the first validated questionnaire to include patients allergic to Polistes species, which is common in our area. Our findings show that the questionnaire is also valid for these patients (AU)

Antigenic cross-reactivity and immunogenicity of Bothrops venomsfrom snakes of the Amazon region

Snakebites are still a critical public health problem in developing countries or isolated areas. In Brazil, the North Region has a high distribution coefficient worsened by the significant number of eventually unreported cases, due to difficulties in access to healthservices, to the natural geographic barriers and the vast territory. In the Rio Negro area, the species Bothrops atrox, Bothrops brazili, Lachesis muta muta and Bothriopsis taeniata arethought to be the major species responsible for snakebites. The aim of this study was to qualitatively and quantitatively determine the antigenic cross-reactivity and expression oftoxins and the immunogenicity of Bothrops venom species of the Amazon and to evaluate the general efficacy of the therapeutic sera. The in vivo assays demonstrated that the defibrinating activity of B. taeniata venom was absent but that the lethal and hemorrhagic properties were more intense than in the B. atrox venom. The results evidence venom variability among the two B. atrox populations from two distinct Amazonian regions, which may reveal a subjacent speciation process. The results point to new aspects that mayguide the improvement of anti-Bothropic therapeutic serum.

Production and characterization of monoclonal antibodies against aflatoxin g(1).

Using semisolid methylcellulose HAT medium selection and non-competitive and competitive indirect enzyme-linked immunosorbent assay, three hybridomas that secreted antibodies for aflatoxin G(1) were selected after fusion of mouse SP2/0 myeloma cells with spleen cells isolated from BALB/c mice that had been immunized with the conjugate of aflatoxin G(1) (AFG(1)) and bovine serum albumin. The aflatoxin-modified protein used to immunize mice was produced chemically by activating aflatoxin G(1) to a 9,10-epoxide derivative, which then covalently bound to the protein. All of these antibodies were found to be specific to AFG(1), and reacted strongly with aflatoxin G(2). These antibodies were designated 1C8, 1C10, and DE7, and had affinities for AFG1 of 5.1 x 10(7), 2.7 x 10(8), and 1.1 x 10(8) liters mol(-1), respectively.

Hiperinmunización de ovinos contra veneno de Bothrops asper del estado Zulia Venezuela: estudio preliminar / Hyperimmunization of ovines against Bothrops asper venom from the Zulia state Venezuela: preliminary study

Un grupo de cuatro ovinos sanos se inmunizó con veneno de Bothrops asper, para estudiar el desarrollo de la respuesta inmune, inducida por la aplicación de un esquema de hiperinmunización. Se tomaron muestras de sangre cada siete días en nueve oportunidades, con el suero obtenido se realizaron pruebas de neutralización en ratones. Se determinó la DE 50, la cual se expresó en µg de veneno neutralizado por ml de suero. Simultáneamente se observaron las condiciones generales de los ovinos durante el esquema de hiperinmunización, no presentándose alteraciones generales ni locales como consecuencia de la inoculación del veneno. En los resultados se observaron variaciones individuales en la magnitud de la elevación del título de anticuerpos y rasgos comunes en el comportamiento de las curvas desarrolladas. El título promedio óptimo (705,5 µg/ml), fue obtenido el día 21 posterior a la primera inoculación. Para ese momento, el ovino con título más alto fue de 840µg/ml y el más bajo, de 593 µg/ml. El promedio más bajo (308 µg/ml) fue observado el día 56. Se recomienda que los animales que serán utilizados en la producción comercial de antiveneno sean evaluados individualmente y seleccionados con base a una respuesta inmune satisfactoria.

A group of four (4) healthy male sheep were immunized with Bothrops asper venom, in order to study the development of the immune response induced by the application of a hiperimmunization protocol. Blood samples were taken from the sheep every seven days in nine different opportunities. With the resultant serum, neutralization tests were done in mice. ED50 was calculated and expressed in µg of neutralized venom per ml of serum, at the same time, general health conditions of sheep were observed during the hiperimmunization protocol. No general or local alterations of health were present as consequence of venom inoculations. Individual variations in the magnitude of the increase of antibody titles were observed in the obtained results. None the less common features in the antibody title curves were also recorded. The mean optimal average (705,5 µg/ml) was obtained on day 21 after the first challenge. At that time, the individual sheep with the highest value was 840 µg/ml and the lowest was 593 µg/ml; the lowest average of all samples (308 µg/ml ) was observed on day 56. It is recommend that animals to be used in the commercial venom antiserum production need to be individually screened and selected based on a satisfactory immune response.

Hypersensitivity to Vespula and Polistes: can we tell the primary sensitization from the clinical history? / Hipersensibilidad a vespula y polistes: ¿se puede determinar la sensibilización predominante a partir de la historia clínica?

Objective: To study the relationship between theprimary sensitization to wasp venoms and the geographicaland seasonal circumstances of the anaphylaxis-induced sting.Methods: We performed a retrospective review of115 patients (age 10-80) who suffered a systemicreaction to a wasp sting. Season and type of locality(urban or rural) at the moment of the sting wererecorded. Serum specific IgE levels to venoms fromVespula and Polistes were measured, and a primarysensitization was determined to whichever genus ofwasp for which the highest class of specific IgE wasobserved. The primary sensitization in relation to thetype of locality and the season was assessed usingthe chi-square test.Results: Most reactions occurred in urban areas(67.8 %), and in the summer season (63.4 %). Mostpatients were sensitized to Vespula venom (94.8 %).Primary sensitization was to Vespula in 56.5 %, toPolistes in 10.4 %, and undetermined in 33 %. Thedistribution of geographical areas did not show significantdifferences in relation to primary sensitization(p > 0.05). Most patients with primary sensitization toVespula suffered the anaphylaxis-induced sting afterthe spring season, with a statistically significant result(p < 0.05).Conclusion: In our population, the probability ofVespula sting is higher than Polistes sting when thereaction occurs after spring. This finding can help usto identify the responsible vespid when the diagnostictests do not provide an accurate result

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Studies on the affinity chromatography purification of anti-patulin polyclonal antibodies by enzyme linked immunosorbent assay and electrophoresis.

Patulin is a mycotoxin produced by fungal species that frequently grow on fruit and vegetables. It presents risks, particularly for children consuming compotes and fruit juices. Thus, it is important to have methods such as immunoassays to screen a large number of samples. In the relevant literature, previous studies on the production of antibodies against patulin derivatives described qualitative tests for a patulin derivative or showed slight responses. The present study reinvestigated the production of polyclonal antibodies against patulin and their purification since crude antiserum could react non-specifically in immunoassays. Patulin-hemiglutarate was synthesized and conjugated to bovine serum albumin as the immunogen for the immunization of five New Zealand white rabbits. The immunoglobulin G (IgG) fraction was isolated twice by affinity chromatography using Sepharose-LS gel and recombinant G-protein. Classic affinity chromatography using Sepharose-LS gel was unable to eliminate serum albumin from the IgG fraction and the use of recombinant G-protein was efficient to isolate the purified IgG. Titres and specificity were determined by indirect competitive enzyme-linked immunosorbent assay. Patulin-hemiglutarate-ovalbumin gave complete displacement, while patulin displaced 30% of bound antibodies. Thus, a fraction of the antibodies are specific for free patulin. The non-specific binding increased with patulin concentrations. The electrophilic properties of patulin might also induce intermolecular cross-links in vitro that hinder the possibility of responses displacement when free patulin is used.

The equine antitoxins supply system for biological poisons in Japan.

Recently, the equine antitoxin supply in Japan has sharply decreased; then it is apparent that a stable supply produced solely by private industry cannot be relied upon. The Ministry of Health, Labor and Welfare (MHLW), therefore, purchases vaccines and equine antitoxins from manufacturers who could not otherwise independently provide an adequate antitoxin supply to hospitals. This supply system is called the 'Kokuyu vaccine system.' Under this system, MHLW purchases, stores and distributes vaccines and antitoxins to hospitals. This system has worked efficiently and effectively so far and may be a good model for establishing a stable antitoxin supply system in other countries.

Toward antibody-catalyzed hydrolysis of organophosphorus poisons.

We report here our preliminary results on the use of catalytic antibodies as an approach to neutralizing organophosphorus chemical weapons. A first-generation hapten, methyl-alpha-hydroxyphosphinate Ha, was designed to mimic the approach of an incoming water molecule for the hydrolysis of exceedingly toxic methylphosphonothioate VX (1a). A moderate protective activity was first observed on polyclonal antibodies raised against Ha. The results were further confirmed by using a mAb PAR 15 raised against phenyl-alpha-hydroxyphosphinate Hb, which catalyzes the hydrolysis of PhX (1b), a less toxic phenylphosphonothioate analog of VX with a rate constant of 0.36 M(-1) x min(-1) at pH 7.4 and 25 degrees C, which corresponds to a catalytic proficiency of 14,400 M(-1) toward the rate constant for the uncatalyzed hydrolysis of 1b. This is a demonstration on the organophosphorus poisons themselves that mAbs can catalytically hydrolyze nerve agents, and a significant step toward the production of therapeutically active abzymes to treat poisoning by warfare agents.

Experimental studies on genetically determined susceptibility to mercury-induced autoimmune response.

Individual differences in susceptibility may be caused by genetical as well as external variables. In mice as well as rats, autoimmune response can be induced by mercury or gold. The present data demonstrates that the autoimmune response depends qualitatively on the H-2 haplotype. If a strain of mice does not have the susceptible haplotype, no autoimmunity will be induced. However, even among the responding strains significant differences were observed. A clear correlation exists between renal mercury deposition and the autoimmune response in susceptible strains of mice. There was a threshold for induction of the autoimmune response in genetically susceptible mice. When exposure was interrupted, the mercury deposition decreased as did the antinucleolar antibody titre. Thresholds exist within susceptible strains below which no autoimmune response is induced.