Molecular Allergen Profiling of Dual Mite Sensitization in Severe Allergic Rhinitis.

BACKGROUND: Mites are the most prevalent source of indoor allergens. The present study used a component-resolved diagnosis (CRD) approach to investigate the mite-specific IgE sensitization profile for Dermatophagoides pteronyssinus and Blomia tropicalis. We also assessed the performance of a commercially available CRD approach in patients with severe allergic rhinitis. METHODS: We selected 63 consecutive patients with dual sensitization to D pteronyssinus and B tropicalis and persistent severe rhinitis according to the ARIA guidelines. We performed skin prick tests with standardized extracts and determined specific serum IgE to both mites, along with serum specific IgE to Der p 1, Der p 2, Der p 23, Der p 10, and Blo t 5. RESULTS: Fifty-eight and 59 patients had positive sIgE to the whole extracts of D pteronyssinus and B tropicalis, respectively. While 91.67% of patients were sensitized to specific IgE to Der p 1, Der p 2, and/or Der p 23, specific IgE to Blo t 5 (≥0.3 ISU-E) was not detected in most of the serum samples (55%). CONCLUSIONS: Although the combination panel of the commercially available major allergens Der p 1, Der p 2, and Der p 23 identified more than 90% of the D pteronyssinus-allergic patients, Blo t 5 performed somewhat poorly in those sensitized to B tropicalis. Improvements in CRD and further research concerning the prevalence and clinical relevance of serodominant allergens are needed to achieve a genuine molecular diagnosis, as well as patient-centered mite allergy-specific immunotherapy.

European Survey on Adverse Systemic Reactions in Allergen Immunotherapy (EASSI): a real-life clinical assessment.

BACKGROUND: Outside clinical trials, data on systemic reactions (SRs) due to allergen immunotherapy (AIT) are scarce. METHODS: A prospective, longitudinal, web-based survey of 'real-life' respiratory allergen immunotherapy (AIT) clinical practice was conducted in France, Germany and Spain. SRs were recorded and coded according to the Medical Dictionary for Regulatory Activities (MedDRA) and risk factors associated with SRs were identified. RESULTS: A total of 4316 patients (corresponding to 4363 ongoing courses of AIT) were included. A total of 109 SRs were recorded, and 90 patients (2.1%) presented at least one SR. Most of the SRs occurred in subcutaneous allergen immunotherapy (SCIT) (89%, n = 97). The most frequently reported symptoms were urticaria, rhinitis, dyspnoea and cough. Respiratory symptoms appeared before skin symptoms. Most SRs occurred during the up-dosing phase (75.8%) and were mild in severity (71.6%). Intramuscular adrenaline was administered in 17 SRs, but only 65% of these were subsequently classified as anaphylaxis. Independent risk factors for SRs during SCIT were as follows: the use of natural extracts (odds ratio, OR) [95% confidence interval (CI)] = 2.74 [1.61-4.87], P = 0.001), the absence of symptomatic allergy medications (1.707 [1.008-2.892], P = 0.047), asthma diagnosis (1.74 [1.05-2.88], P = 0.03), sensitization to animal dander (1.93 [1.21-3.09], P = 0.006) or pollen (1.16 [1.03-1.30], P = 0.012) and cluster regimens (vs rush) (4.18 [1.21-14.37], P = 0.023). A previous episode of anaphylaxis increased the risk for anaphylaxis in SCIT (OR [95% CI] = 17.35 [1.91-157.28], P = 0.01). CONCLUSION: AIT for respiratory allergy is safe, with a low number of SRs observed in real-life clinical practice. A personalized analysis of risk factors could be used to minimize SRs.

Assessment of a new brand of determinants for skin testing in a large group of patients with suspected beta-lactam allergy.

BACKGROUND: Skin testing with major and minor determinants of benzylpenicillin is recommended standard practice for the evaluation of patients with immediate hypersensitivity reactions to beta-lactams. However, commercial reagents for this purpose were recently dropped from the European market. OBJECTIVE: In the present study, we assessed a new brand of reagents for use in skin testing in patients with suspected penicillin allergy. METHODS: Prick tests and intradermal tests were performed with benzylpenicilloyl polylysine (PPL) and minor determinant mixture (MDM). Penicillin G, amoxicillin, and the culprit beta-lactam were also tested. If skin tests were negative, a single-blind oral challenge test was performed with the culprit active principle or penicillin. If both skin tests and challenge tests were negative, the same procedure was repeated between 2 and 4 weeks later. RESULTS: A total of 636 patients were assessed. The allergy study was positive in 69 patients. Skin tests with PPL were positive in 30 patients (46.8%) and with MDM in 28 (43.7%). Sixteen patients displayed a positive reaction to both PPL and MDM (25%), while 42 patients (65.6%) had a positive reaction to either PPL or MDM alone. Thirty-two patients had positive skin test reactions to penicillin G or another p-lactam antibiotic. Five patients in whom a negative result was obtained in skin tests had a positive reaction to oral challenge. CONCLUSIONS: Our results indicate that a new brand of determinants that is commercially available in Europe is a reliable and useful tool for the diagnosis of beta-lactam allergy. The new reagents are a safe alternative to the previously available brand.

[Evaluation of nasal inflammation]. / Valoración de la inflamación nasal.

In the reaction of immediate hypersensibility to alergene is joined to its specific type IgE antibody, also united to the high affinity receptors for IgE (FccI) of the effecters cells fundamentally mastocites and basophiles. The interbreeding of these molecules Fcc to RI, after the union ofpolyvalent antigenes to IgE, active these cells, producing three biologic responses: excitosis of the preformed content of its granules, synthesization of lipidic mediators and citoquine secretion. The inflammation mediators are in last term, substances responsible of the clinic symptomatology. They can be divided generally in preformed mediators (biogene amines and macromolecules of the granules) and of new synthese mediators (lipidic and citoquine mediators).

Pertinence of Telehealth in a Rush Conversion to Virtual Allergy Practice During the COVID-19 Outbreak

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Trypanosoma cruzi phospho enol pyruvate carboxykinase (ATP-dependent): transition metal ion requirement for activity and sulfhydryl group reactivity.

We studied the transition metal ion requirements for activity and sulfhydryl group reactivity in phospho enol pyruvate carboxykinase (PEP-carboxykinase; ATP:oxaloacetate carboxylase (transphosphorylating), EC 4.1.1.49), a key enzyme in the energy metabolism of the protozan parasite Trypanosoma (Schizotrypanum) cruzi. As for other PEP-carboxykinases this enzyme has a strict requirement of transition metal ions for activity, even in the presence of excess Mg2+ ions for the carboxylation reaction; the order of effectiveness of these ions as enzyme activators was: Co2+ > Mn2+ > Cd2+ > Ni2+ >> Fe2+ > VO2+, while Zn2+ and Ca2+ had no activating effects. When we investigated the effect of the varying type or concentration of the transition metal ions on the kinetic parameters of the enzyme the results suggested that the stimulatory effects of the transition metal center were mostly associated with the activation of the relatively inert CO2 substrate. The inhibitory effects of 3-mercaptopicolinic acid (3MP) on the enzyme were found to depend on the transition metal ion activator: for the Mn(2+)-activated enzyme the inhibition was purely non-competitive (Kii = Kis) towards all substrates, while for the Co(2+)-activated enzyme the inhibitor was much less effective, produced a mixed-type inhibition and affected differentially the interaction of the enzyme with its substrates. The modification of a single, highly reactive, cysteine per enzyme molecule by 5,5'-dithiobis (2-nitro-benzoate) (DTNB) lead ton an almost complete inhibition of Mn(2+)-activated T. cruzi PEP-carboxykinase; however, in contrast with the results of previous studies in vertebrate and yeast enzymes, the substrate ADP slowed the chemical modification and enzyme inactivation but did not prevent it. PEP and HCO3- had no significant effect on the rate or extent of the enzyme inactivation. The kinetics of the enzyme inactivation by DTNB was also dependent on the transition metal activator, being much slower for the Co(2+)-activated enzyme than for its Mn(2+)-activated counterpart. When the bulkier but more hydrophobic reagent N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) was used the enzyme was slowly and incompletely inactivated in the presence of Mn2+ and ADP afforded almost complete protection from inactivation; in the presence of Co2+ the enzyme was completely resistant to inactivation. Taken together, our results indicate that the parasite enzyme has a specific requirement of transition metal ions for activity and that they modulate the reactivity of a single, essential thiol group, different from the hyperreactive cysteines present in vertebrate or yeast enzymes.

Molecular allergen profiling of dual mite sensitization in severe allergic rhinitis

BACKGROUND: Mites are the most prevalent source of indoor allergens. The present study used a component-resolved diagnosis (CRD) approach to investigate the mite-specific IgE sensitization profile for Dermatophagoides pteronyssinus and Blomia tropicalis. We also assessed the performance of a commercially available CRD approach in patients with severe allergic rhinitis. METHODS: We selected 63 consecutive patients with dual sensitization to D pteronyssinus and B tropicalis and persistent severe rhinitis according to the ARIA guidelines. We performed skin prick tests with standardized extracts and determined specific serum IgE to both mites, along with serum specific IgE to Der p 1, Der p 2, Der p 23, Der p 10, and Blo t 5. RESULTS: Fifty-eight and 59 patients had positive sIgE to the whole extracts of D pteronyssinus and B tropicalis, respectively. While 91.67% of patients were sensitized to specific IgE to Der p 1, Der p 2, and/or Der p 23, specific IgE to Blo t 5 (≥0.3 ISU-E) was not detected in most of the serum samples (55%). CONCLUSIONS: Although the combination panel of the commercially available major allergens Der p 1, Der p 2, and Der p 23 identified more than 90% of the D pteronyssinus-allergic patients, Blo t 5 performed somewhat poorly in those sensitized to B tropicalis. Improvements in CRD and further research concerning the prevalence and clinical relevance of serodominant allergens are needed to achieve a genuine molecular diagnosis, as well as patient-centered mite allergy-specific immunotherapy

INTRODUCCIÓN: Los ácaros son los alérgenos de interior más prevalentes. El presente estudio investiga el perfil de sensibilización a Dermatophagoides pteronyssinus y Blomia tropicalis, así como el rendimiento del diagnóstico por componentes (CRD) disponible comercialmente en pacientes con rinitis alérgica grave persistente. MATERIAL Y MÉTODOS: Seleccionamos 63 pacientes con rinitis grave persistente (Guía ARIA) con sensibilización dual a D. pteronyssinus y B. tropicalis. Se realizaron pruebas cutáneas en prick con extractos estandarizados, IgE sérica específica a ambos ácaros además de IgE específica a alérgenos individuales Der p 1, Der p 2, Der p 23, Der p 10 y Blo t 5. RESULTADOS: Cincuenta y ocho y 59 pacientes presentaron IgE específica positiva a extractos crudos de D. pteronyssinus y B. tropicalis, respectivamente. Aunque el 91,67% mostraron sensibilización a Der p 1, Der p 2 y/o Der p 23, Blo t 5 (≥0,3 ISU-E) no fue detectado en la mayoría (55%) de las muestras estudiadas. CONCLUSIONES: Aunque la combinación de alérgenos principales Der p 1, Der p 2 Der p 23, pudo identificar más del 90% de los pacientes sensibilizados a D. pteronyssinus, Blo t 5 presentó un rendimiento diagnóstico muy limitado para aquellos sensibilizados a B. tropicalis. Conocer la prevalencia y relevancia clínica de los alérgenos acarianos serodominantes en cada territorio contribuiría a una mejor identificación de sensibilizaciones genuinas en la era de la medicina de precisión

Success with multidisciplinary team work: experience of a primary immunodeficiency unit

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Experimental design and reporting standards for improving the internal validity of pre-clinical studies in the field of pain: Consensus of the IMI-Europain consortium.

Background and aims Pain is a subjective experience, and as such, pre-clinical models of human pain are highly simplified representations of clinical features. These models are nevertheless critical for the delivery of novel analgesics for human pain, providing pharmacodynamic measurements of activity and, where possible, on-target confirmation of that activity. It has, however, been suggested that at least 50% of all pre-clinical data, independent of discipline, cannot be replicated. Additionally, the paucity of "negative" data in the public domain indicates a publication bias, and significantly impacts the interpretation of failed attempts to replicate published findings. Evidence suggests that systematic biases in experimental design and conduct and insufficiencies in reporting play significant roles in poor reproducibility across pre-clinical studies. It then follows that recommendations on how to improve these factors are warranted. Methods Members of Europain, a pain research consortium funded by the European Innovative Medicines Initiative (IMI), developed internal recommendations on how to improve the reliability of pre-clinical studies between laboratories. This guidance is focused on two aspects: experimental design and conduct, and study reporting. Results Minimum requirements for experimental design and conduct were agreed upon across the dimensions of animal characteristics, sample size calculations, inclusion and exclusion criteria, random allocation to groups, allocation concealment, and blinded assessment of outcome. Building upon the Animals in Research: Reportingin vivo Experiments (ARRIVE) guidelines, reporting standards were developed for pre-clinical studies of pain. These include specific recommendations for reporting on ethical issues, experimental design and conduct, and data analysis and interpretation. Key principles such as sample size calculation, a priori definition of a primary efficacy measure, randomization, allocation concealments, and blinding are discussed. In addition, considerations of how stress and normal rodent physiology impact outcome of analgesic drug studies are considered. Flow diagrams are standard requirements in all clinical trials, and flow diagrams for preclinical trials, which describe number of animals included/excluded, and reasons for exclusion are proposed. Creation of a trial registry for pre-clinical studies focused on drug development in order to estimate possible publication bias is discussed. Conclusions More systematic research is needed to analyze how inadequate internal validity and/or experimental bias may impact reproducibility across pre-clinical pain studies. Addressing the potential threats to internal validity and the sources of experimental biases, as well as increasing the transparency in reporting, are likely to improve preclinical research broadly by ensuring relevant progress is made in advancing the knowledge of chronic pain pathophysiology and identifying novel analgesics. Implications We are now disseminating these Europain processes for discussion in the wider pain research community. Any benefit from these guidelines will be dependent on acceptance and disciplined implementation across pre-clinical laboratories, funding agencies and journal editors, but it is anticipated that these guidelines will be a first step towards improving scientific rigor across the field of pre-clinical pain research.

Screening of horse polyclonal antibodies with a random peptide library displayed on phage: identification of ligands used as antigens in an ELISA test to detect the presence of antibodies to equine arteritis virus.

A random hexapeptide fusion-phage library was screened to isolate phages that bind to antibodies present in horse sera positive for equine arteritis virus (EAV). Analysis of the peptide sequences displayed by isolated phages identified seven groups. 25% of the isolated phages used as antigens in an ELISA test were specifically recognised by a pool of sera which was positive for EAV in virus neutralisation test (VN). Five of these, when used as antigen in ELISA, detected greater than 50% of sera (n = 30) containing antibodies to EAV as detected by VN. When these five phages were pooled together and used as antigen in ELISA, the detection was improved. The sensitivity and specificity of the ELISA were 99 and 71%, respectively, compared with the EAV neutralisation test (n = 200). This study has shown the potential that phage display libraries have for identifying peptide sequences which could be used as antigen in diagnostic ELISAs.

Expression of the nonstructural protein NS1 of equine influenza A virus: detection of anti-NS1 antibody in post infection equine sera.

The nucleotide sequence of the nonstructural protein NS1 of the influenza virus A/equine 2/Suffolk/89 was determined and found to be 97% identical to that of A/equine 2/Miami/63. A similar level of identity was shown for the deduced NS1 amino acid sequence. The NS1 gene was expressed, in its entirety and in part, as fusion proteins with glutathione S-transferase using the pGEX-3X expression vector. Antibodies to NS1 protein were detected in serum samples from ponies experimentally infected with influenza virus, but not in animals vaccinated with whole inactivated virus or in unprimed control animals. The antigenic determinant(s) of NS1 protein appear to be located in the C-terminal half of the protein. The implications of these findings are discussed with reference to the use of NS1 protein as a differential diagnostic marker for influenza virus infection in the presence of high levels of circulating antibody to influenza haemagglutinin generated by recent vaccination.

Utilisation of bacteriophage display libraries to identify peptide sequences recognised by equine herpesvirus type 1 specific equine sera.

Three filamentous phage random peptide display libraries were used in biopanning experiments with purified IgG from the serum of a gnotobiotic foal infected with equine herpesvirus-1 (EHV-1) to enrich for epitopes binding to anti-EHV-1 antibodies. The sequences of the amino acids displayed were aligned with protein sequences of EHV-1, thereby identifying a number of potential antibody binding regions. Presumptive epitopes were identified within the proteins encoded by genes 7 (DNA helicase/primase complex protein), 11 (tegument protein), 16 (glycoprotein C), 41 (integral membrane protein), 70 (glycoprotein G), 71 (envelope glycoprotein gp300), and 74 (glycoprotein E). Two groups of sequences, which aligned with either glycoprotein C (gC) or glycoprotein E (gE), identified type-specific epitopes which could be used to distinguish between sera from horses infected with either EHV-1 or EHV-4 in an ELISA using either the phage displaying the peptide or synthetic peptides as antigen. The gC epitope had been previously identified as an immunogenic region by conventional monoclonal antibody screening whereas the gE antibody binding region had not been previously identified. This demonstrates that screening of phage display peptide libraries with post-infection polyclonal sera is a suitable method for identifying diagnostic antigens for viral infections such as EHV-1.