Allergens in allergy diagnosis: a glimpse at emerging new concepts and methodologies

Allergic diseases are important concern of public health. A reliable diagnosis is of utmost importance for the management of allergic patients both when immunotherapy is planned and when the treatment is essentially based on the avoidance of the allergy source. However, the available diagnostic systems sometimes fail to detect specific IgE antibodies thus impairing the correct diagnosis. The traditional test systems are generally based on the use of protein extracts derived from the allergenic sources whose composition is very variable and cannot be standardized. The development of a new methodology combining the so-called allergenic molecule-based diagnosis with the multiplex microarray technology and allowing the analysis of multiple purified allergens in a single test represents an important improvement in allergy diagnosis. In addition, the biochemical and immunological characterisation of individual allergens has provided new insights into the understanding of allergen-IgE recognition that could be exploited for further improvements of allergy diagnostic tests

Allergen Micro-Bead Array for IgE Detection: A Feasibility Study Using Allergenic Molecules Tested on a Flexible Multiplex Flow Cytometric Immunoassay

Background: Allergies represent the most prevalent non infective diseases worldwide. Approaching IgE-mediated sensitizations improved much by adopting allergenic molecules instead of extracts, and by using the micro-technology for multiplex testing. Objective and Methods: To provide a proof-of-concept that a flow cytometric bead array is a feasible mean for the detection of specific IgE reactivity to allergenic molecules in a multiplex-like way. A flow cytometry Allergenic Moleculebased micro-bead Array system (ABA) was set by coupling allergenic molecules with commercially available micro-beads. Allergen specific polyclonal and monoclonal antibodies, as well as samples from 167 allergic patients, characterized by means of the ISAC microarray system, were used as means to show the feasibility of the ABA. Three hundred and thirty-six sera were tested for 1 or more of the 16 selected allergens, for a total number of 1,519 tests on each of the two systems. Results: Successful coupling was initially verified by detecting the binding of rabbit polyclonal IgG, mouse monoclonal, and pooled human IgE toward three allergens, namely nDer s 1, nPen m 1, and nPru p 3. The ABA assay showed to detect IgE t

Anti-TNFα Drugs and Interleukin Inhibitors: Epidemiological and Pharmacovigilance Investigation in COVID-19 Positive Patients

Unlabelled: Cytokine patterns and immune activation in patients with Coronavirus 2019 (COVID-19) seem to resemble the case of rheumatoid arthritis (RA), psoriasis and inflammatory bowel disease (IBD). Biological drugs, such as anti-tumor necrosis factor α (TNFα) and interleukin (IL) inhibitors, appear to be protective against adverse outcomes of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). However, these treatments are associated with an increased risk of secondary infections. The aim of the study was to examine the association between the use of immunomodulatory drugs and the risk of SARS-CoV-2-associated positivity, hospitalization and death compared to other commonly prescribed treatment regimens among patients with immune-mediated inflammatory diseases. Methods: All patients with RA, Psoriasis and IBD were included in this observational analysis and treated with anti-TNFα, IL-inhibitors, Methotrexate (MTX) and Sulfasalazine drugs during the year 2020-2021. The population consisted of 932 patients and demographic, clinical and pharmacological data were analyzed. Results: Although no significant differences were observed between patients treated with biological and synthetic drugs in terms of hospitalization and death, the multivariate logistic model showed that the type of drug influences the possibility of COVID-19 positivity. Conclusions: The results of this analysis support the use of biological drugs and justify further research investigating the association of these biological therapies with COVID-19 outcomes

Plant-Made Bet v 1 for Molecular Diagnosis

Allergic disease diagnosis is currently experiencing a breakthrough due to the use of allergenic molecules in serum-based assays rather than allergen extracts in skin tests. The former methodology is considered a very innovative technology compared with the latter, since it is characterized by flexibility and adaptability to the patient's clinical history and to microtechnology, allowing multiplex analysis. Molecular-based analysis requires pure allergens to detect IgE sensitization, and a major goal, to maintain the diagnosis cost-effective, is to limit their production costs. In addition, for the production of recombinant eukaryotic proteins similar to natural ones, plant-based protein production is preferred to bacterial-based systems due to its ability to perform most of the post-translational modifications of eukaryotic molecules. In this framework, Plant Molecular Farming (PMF) may be useful, being a production platform able to produce complex recombinant proteins in short time-frames at low cost. As a proof of concept, PMF has been exploited for the production of Bet v 1a, a major allergen associated with birch (Betula verrucosa) pollen allergy. Bet v 1a has been produced using two different transient expression systems in Nicotiana benthamiana plants, purified and used in a new generation multiplex allergy diagnosis system, the patient-Friendly Allergen nano-BEad Array (FABER). Plant-made Bet v 1a is immunoreactive, binding IgE and inhibiting IgE-binding to the Escherichia coli expressed allergen currently available in the FABER test, thus suggesting an overall similar though non-overlapping immune activity compared with the E. coli expressed form

Biological occupational allergy: Protein microarray for the study of laboratory animal allergy (LAA)

Background: Laboratory Animal Allergy (LAA) has been considered a risk for the workers since 1989 by the NIOSH. About one third of the Laboratory Animal Workers (LAWs) can manifest symptoms to LAA as asthma, rhinitis, conjunctivitis and cutaneous reactions. The prevalence of LAA-induced clinical symptoms has been estimated with a great variability (4–44%) also due to the different methodologies applied. Objective: Evaluate the prevalence of IgE positivity to mouse and rat allergens in LAWs and assess which factors are predisposing to sensitization among subjects exposed to laboratory animals in the workplace. Methods: One hundred LAWs were invited to fill out a questionnaire regarding current allergic symptoms, atopic history, home environment, previous and current occupational history. IgE reactivity versus specific allergens was evaluated with ImmunoCAP ISAC. Results: Out of one hundred LAWs, 18% had a serum susceptibility to mouse and/or rat allergens and 42% reported to have occupational allergy symptoms. Combining the results acquired by ImmunoCAP ISAC and questionnaire, 17% of LAWs have been defined as LAWs-LAA positive since they present a positive IgE response and allergy symptoms, 1% LAWs-LAA sensitized, 25% LAWs-LAA symptomatic and 57% LAWs-LAA negative. Presence of previous allergy symptoms in work and life environment were significantly related to LAWs-LAA positive/sensitized. Conclusions: The study aimed to define the immunological profile of LAWs using the proteomic array as an innovative approach in the study of environmental and occupational exposure to allergens. We suggested a definition of LAWs-LAA considering serum IgE response and presence of allergy symptoms. The proposed approach has the advantage to provide a standard methodology for evaluating the specific IgE responsiveness to animal allergens in specific workplace also considering the immunological profile of workers referred to exposure in life and occupational environment

Medicina 2019, 55, 504 / Similar Allergenicity to Different Artemisia Species Is a Consequence of Highly Cross-Reactive Art v 1-Like Molecules

Background and objectives: Pollens of weeds are relevant elicitors of type I allergies. While many Artemisia species occur worldwide, allergy research so far has only focused on Artemisia vulgaris. We aimed to characterize other prevalent Artemisia species regarding their allergen profiles. Materials and Methods: Aqueous extracts of pollen from seven Artemisia species were characterized by gel electrophoresis and ELISA using sera from mugwort pollen-allergic patients (n = 11). The cDNA sequences of defensinproline-linked proteins (DPLPs) were obtained, and purified proteins were tested in a competition ELISA, in rat basophil mediator release assays, and for activation of Jurkat T cells transduced with an Art v 1-specific TCR. IgE cross-reactivity to other allergens was evaluated using ImmunoCAP and ISAC. Results: The protein patterns of Artemisia spp. pollen extracts were similar in gel electrophoresis, with a major band at 24 kDa corresponding to DPLPs, like the previously identified Art v 1. Natural Art v 1 potently inhibited IgE binding to immobilized pollen extracts. Six novel Art v 1 homologs with high sequence identity and equivalent IgE reactivity were identified and termed Art ab 1, Art an 1, Art c 1, Art f 1, Art l 1, and Art t 1. All proteins triggered mediator release and cross-reacted at the T cell level. The Artemisia extracts contained additional IgE cross-reactive molecules from the nonspecific lipid transfer protein, pectate lyase, profilin, and polcalcin family. Conclusions: Our findings demonstrate that DPLPs in various Artemisia species have high allergenic potential. Therefore, related Artemisia species need to be considered to be allergen elicitors, especially due to the consideration of potential geographic expansion due to climatic changes.(VLID)440247

Tolerability of a fully maturated cheese in cow's milk allergic children: biochemical, immunochemical, and clinical aspects.

BACKGROUND: From patients' reports and our preliminary observations, a fully maturated cheese (Parmigiano-Reggiano; PR) seems to be well tolerated by a subset of cow's milk (CM) allergic patients. OBJECTIVE AND METHODS: To biochemically and immunologically characterize PR samples at different maturation stage and to verify PR tolerability in CM allergic children. Seventy patients, with suspected CM allergy, were enrolled. IgE to CM, α-lactalbumin (ALA), β-lactoglobulin (BLG) and caseins (CAS) were tested using ImmunoCAP, ISAC103 and skin prick test. Patients underwent a double-blind, placebo-controlled food challenge with CM, and an open food challenge with 36 months-maturated PR. Extracts obtained from PR samples were biochemically analyzed in order to determine protein and peptide contents. Pepsin and trypsin-chymotrypsin-pepsin simulated digestions were applied to PR extracts. Each PR extract was investigated by IgE Single Point Highest Inhibition Achievable assay (SPHIAa). The efficiency analysis was carried out using CM and PR oral challenges as gold standards. RESULTS: The IgE binding to milk allergens was 100% inhibited by almost all PR preparations; the only difference was for CAS, mainly α(S1)-CAS. Sixteen patients sensitized to CM tolerated both CM and PR; 29 patients tolerated PR only; 21 patients, reacted to both CM and PR, whereas 4 patients reactive to CM refused to ingest PR. ROC analysis showed that the absence of IgE to BLG measured by ISAC could be a good marker of PR tolerance. The SPHIAa using digested PR preparations showed a marked effect on IgE binding to CAS and almost none on ALA and BLG. CONCLUSIONS: 58% of patients clinically reactive to CM tolerated fully maturated PR. The preliminary digestion of CAS induced by PR maturation process, facilitating a further loss of allergenic reactivity during gut digestion, might explain the tolerance. This hypothesis seems to work when no IgE sensitization to ISAC BLG is detected

Testing human sera containing specific IgE on allergen conjugated micro-beads.

<p>Three human individual serum samples (49812, 49141, 33020), each of them known to have specific IgE to one allergen and not to the others were tested on the three micro-beads present in the same tube. Serum 31851 was from a subject recognizing nDer s 1 and nPen m 1, but not nPru p 3. A non allergic donor was used for control purposes. # Serum sample numbers; ° Values are expressed as kU/l; * Values are expressed as Median Fluorescence Intensity [MFI]. Upper right corner: an example of micro-bead fluorescence clusters.</p

ABA <i>versus</i> ISAC correlation results on 137 serum samples selected on the basis of nDer s 1, nPen m 1, and nPru p 3 mutually exclusive IgE positivity are reported.

<p>Panel A: All 411 IgE values, obtained by testing the three allergens; Panel B: 137 IgE results obtained on nDer s 1 allergen; Panel C: 137 IgE results obtained on nPen m 1 allergen; Panel D: 137 IgE results obtained on nPru p 3 allergen. For graphical visualization needs on log scales, zero values for ABA were set at 10 MFI on the X axis, and at 0.01 kU/l for ISAC values on the Y axis. The Spearman r correlation coefficient, the χ² and the Fisher's exact tests were used where applicable.</p