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Lactococcus lactis-derived microparticles ad mixed with allergen shift immune responses towards a regulatory profile in tree-nut allergic patients Proliferation and cytokine production from supernatant or proliferating cells data after stimulation with TBP alone did not show any significant changes in tree-nut-AP and HC (Figures S4A-C and S6A-C). Sublingual immunotherapy (SLIT) is a disease-modifying intervention that offers a potential long-lasting therapeutic effect in food allergy (FA). [Extracted from the article]

Diagnosis of type I hypersensitivity reactions (IgE-mediated reactions) to penicillins is based on clinical history, skin tests (STs), and drug provocation tests (DPTs). Among in vitro complementary tests, the fluoro-enzyme immunoassay (FEIA) ImmunoCAP® (Thermo-Fisher, Waltham, MA, USA) is the most widely used commercial method for detecting drug-specific IgE (sIgE). In this study, we aimed to analyze the utility of ImmunoCAP® for detecting sIgE to penicillin G (PG) and amoxicillin (AX) in patients with confirmed penicillin allergy. The study includes 139 and 250 patients evaluated in Spain and Italy, respectively. All had experienced type I hypersensitivity reactions to penicillins confirmed by positive STs. Additionally, selective or cross-reactive reactions were confirmed by DPTs in a subgroup of patients for further analysis. Positive ImmunoCAP® results were 39.6% for PG and/or AX in Spanish subjects and 52.4% in Italian subjects. When only PG or AX sIgE where analyzed, the percentages were 15.1% and 30.4%, respectively, in Spanish patients; and 38.9% and 46% in Italian ones. The analysis of positive STs showed a statistically significant higher percentage of positive STs to PG determinants in Italian patients. False-positive results to PG (16%) were detected in selective AX patients with confirmed PG tolerance. Low and variable sensitivity values observed in a well-defined population with confirmed allergy diagnosis, as well as false-positive results to PG, suggest that ImmunoCAP® is a diagnostic tool with relevant limitations in the evaluation of subjects with type I hypersensitivity reactions to penicillins. [ABSTRACT FROM AUTHOR]

Nanotechnology is science, engineering and technology conducted at the nanoscale, which is about 1–100 nm. It has led to the development of nanomaterials, which behave very differently from materials with larger scales and can have a wide range of applications in biomedicine. The physical and chemical properties of materials of such small compounds depend mainly on the size, shape, composition and functionalization of the system. Nanoparticles, carbon nanotubes, liposomes, polymers, dendrimers and nanogels, among others, can be nanoengineeried for controlling all parameters, including their functionalization with ligands, which provide the desired interaction with the immunological system, that is dendritic cell receptors to activate and/or modulate the response, as well as specific IgE, or effector cell receptors. However, undesired issues related to toxicity and hypersensitivity responses can also happen and would need evaluation. There are wide panels of accessible structures, and controlling their physico‐chemical properties would permit obtaining safer and more efficient compounds for clinical applications goals, either in diagnosis or treatment. The application of dendrimeric antigens, nanoallergens and nanoparticles in allergy diagnosis is very promising since it can improve sensitivity by increasing specific IgE binding, mimicking carrier proteins or enhancing signal detection. Additionally, in the case of immunotherapy, glycodendrimers, liposomes, polymers and nanoparticles have shown interest, behaving as platforms of allergenic structures, adjuvants or protectors of allergen from degradation or having a depot capacity. Taken together, the application of nanotechnology to allergy shows promising facts facing important goals related to the improvement of diagnosis as well as specific immunotherapy. [ABSTRACT FROM AUTHOR]

Fluoroquinolones (FQs) are photoreactive drugs, but it is not known whether laboratory light exposure can influence the induction of photoproducts and modify in vitro test results. The basophil activation test (BAT) has proven to be useful for evaluating immunoglobulin E (IgE)-mediated hypersensitivity to FQs, with a higher percentage of positive responders with ciprofloxacin (CIP) than with moxifloxacin (MOX). We studied the effect of laboratory light on CIP and MOX degradation, and drug-protein conjugate formation, and its influence on the BAT for evaluating IgE-mediated hypersensitivity to FQs. The results showed an important decrease in fluorescence emission intensity under light compared to dark conditions for MOX, and that BAT positivity was lower in light (17.9%) than in dark (35.7%) conditions. No changes were found for CIP in either fluorescence emission intensity or BAT results (46.4% in both conditions). We can conclude that light exposure is a critical factor in BAT results when photolabile drugs like MOX are used. Therefore, light is important when interpreting in vitro results. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Cephalosporins, after penicillins, are the most widely used antibacterial agents in infectious diseases and the cause of adverse immune reactions in the world. Whether a patient with a suspected allergy to a β-lactam can safely take a cephalosporin is often a matter of debate. However, there are no tests with enough sensitivity to detect allergy to cephalosporins. Understanding the way in which the drug metabolizes after protein conjugation is important if we are to make advances in the diagnosis of clinical allergy. Structural studies of cephalosporin−protein adducts have never been addressed successfully and are difficult to investigate. Our approach to determine the requirements involved in antigenic determinant structures consisted of designing and synthesizing a proposed skeleton that remains linked to the carrier protein after chemical degradation in cephalosporin conjugated to carrier proteins. In this study, a series of proposed epitopes were efficiently synthesized following a versatile methodology, involving the condensation of the R1acyl side chains of native cephalosporins, with the nuclear fragment structures (derived from amino acids or other aminofunctionalized molecules). The final well-defined structures 1−4 (a−f), representing a fragment from the proposed cephalosporin−Lys(protein) adduct intermediate, consist of closely related low-molecular-weight molecules, differing only in the functional group at C-3 and the R1side chains. They were assessed with sera from patients allergic to cephalosporins to study structure−IgE molecular recognition relationships. These IgE showed an enhanced recognition to proposed new skeleton epitopes with adequate functionality at C-3, with the specifities mainly related to the R1acyl side chain. Thus, this study led us to refine the model haptenic structures of cephalosporins and gain insight into the chemical mechanism of epitope formation. [ABSTRACT FROM AUTHOR]

Performance of basophil activation test and specific IgG4 as diagnostic tools in nonspecific lipid transfer protein allergy: Antwerp-Barcelona comparison. Nonspecific lipid transfer proteins (nsLTP) are commonly responsible for plant-food allergy, in some cases life-threatening. MAT and BAT can be used as complementary tools in the diagnosis of LTP-allergy, as the combination of both increased the sensitivity up to 95%. [Extracted from the article]

Diagnosing Drug Hypersensitivity Reactions (DHRs) could be a complicated process especially in children, since allergic‐like manifestation at this age is more often the expression of concomitant infections rather than a actual DHRs. In vivo tests are usually suggested as a first step; however, prick and intradermal tests could be painful and have shown different sensitivity and specificity among published studies. In some cases, in vivo tests such as Drug Provocation test (DPT) could be even contraindicated. Therefore, the need for in vitro testing is compelling, to add useful information along the diagnostic pathway and to limit the need of DPT. In this review, we analyze the different types of in vitro tests, focusing on those used more widely such as specific IgE and on those that are still for research settings, such as basophil activation test and lymphocyte transformation test, but that have shown some useful diagnostic potential. [ABSTRACT FROM AUTHOR]

Background: The involvement of allergen‐specific (s)IgE in local allergic rhinitis (LAR) has been debated. Here, we investigate the effect of nasal allergen challenge with Dermatophagoides pteronyssinus (NAC‐DP) in mucosal and peripheral B‐cell subpopulations in LAR patients. Methods: Nine LAR, 5 allergic rhinitis (AR), and 5 non‐atopic healthy control (HC) individuals were subjected to a 3‐day NAC‐DP protocol, and nasal biopsies and blood samples were collected before and after provocation. Nasal biopsies were used for immunohistochemistry and gene expression studies, whereas the frequency of lymphocyte subsets and basophil activation test (BAT) were analyzed in blood samples by flow cytometry. sIgG was measured in sera. Results: NAC‐DP induced an increase in IgE+CD38+ plasmablasts in the nasal mucosa of LAR patients, but not in AR or HC individuals. Markers of sequential recombination to IgE (εCSR) (from IgG) were observed in 33% of LAR, 20% of AR, and 0% of HC subjects. NAC‐DP increased the proportion of peripheral CD19+CD20+CD38+ plasmablasts in AR and LAR patients, but not in HC. Expression of the mucosal homing receptor CXCR3 in peripheral CD19+CD20+CD38+ plasmablasts from LAR, AR, and HC individuals was 7%, 5%, and 0.5%, respectively. In vitro DP stimulation increased proliferating CD19+CD20+CD38+ plasmablasts in LAR and AR patients, but not in HC. Serum DP‐sIgG was higher in LAR and AR patients as compared to HC. BAT was positive in 33%, 100%, and 0% of LAR, AR, and HC subjects, respectively. Conclusion: These results suggest that allergen exposure induces the sequential εCSR of IgG+CD19+CD20+CD38+ plasmablasts in the nasal mucosa of LAR patients. [ABSTRACT FROM AUTHOR]

Background: Amoxicillin (AX) is nowadays the β‐lactam that more frequently induces immediate allergic reactions. Nevertheless, diagnosis of AX allergy is occasionally challenging due to risky in vivo tests and non‐optimal sensitivity of in vitro tests. AX requires protein haptenation to form multivalent conjugates with increased size to be immunogenic. Knowing adduct structural features for promoting effector cell activation would help to improve in vitro tests. We aimed to identify the optimal structural requirement in specific cellular degranulation to AX using well‐precised nanoarchitectures of different lengths. Method: We constructed eight Bidendron Antigens (BiAns) based on polyethylene glycol (PEG) linkers of different lengths (600–12,000 Da), end‐coupled with polyamidoamine dendrons that were terminally multi‐functionalized with amoxicilloyl (AXO). In vitro IgE recognition was studied by competitive radioallergosorbent test (RAST) and antibody–nanoarchitecture complexes by transmission electron microscopy (TEM). Their allergenic activity was evaluated using bone marrow‐derived mast cells (MCs) passively sensitized with mouse monoclonal IgE against AX and humanized RBL‐2H3 cells sensitized with polyclonal antibodies from sera of AX‐allergic patients. Results: All BiAns were recognized by AX‐sIgE. Dose‐dependent activation responses were observed in both cellular assays, only with longer structures, containing spacers in the range of PEG 6000–12,000 Da. Consistently, greater proportion of immunocomplexes and number of antibodies per complex for longer BiAns were visualized by TEM. Conclusions: BiAns are valuable platforms to study the mechanism of effector cell activation. These nanomolecular tools have demonstrated the importance of the adduct size to promote effector cell activation in AX allergy, which will impact for improving in vitro diagnostics. [ABSTRACT FROM AUTHOR]

ABSTRACTDrugs can induce IgE mediated or T cell dependent immunological reactions. T cell dependent reactions are poorly understood, although T lymphocytes have been proposed as a protagonist in a number of non-immediate immunological reactions (NIR). The objective was to study in vivo different regulatory and proinflammatory cytokines and cytotoxic markers in patients with NIR to drugs. Twenty patients with NIR after drug intake were classified into two groups: Group A (severe), Stevens–Johnson syndrome and toxic epidermal necrolysis; and Group B (mild), maculopapular exanthema and desquamative exanthema. Another 25 subjects taking the same drugs but without reactions formed a control group. Samples were obtained within 24 hours of the reaction and 30 days later. IL-2, IL-4, IFN, TNF, perforin, granzyme B (GrB), and FasL mRNA expression levels were determined in peripheral blood mononuclear cells by competitive RT-PCR. There were 9 patients in Group A and 11 in Group B. The drugs involved were betalactams (8), anticonvulsants (6), allopurinol (1), sulfamethoxazole (1), amiodarone (1) dypirone (2), and erythromicine+paracetamol (1). At the acute stage there was a high increase of IL-2, IFN, and TNF mRNA expression in both groups vs. controls, perforin and GrB varied in each group with patients in Group A having the highest values, and FasL was only expressed in Group A. Relationships between the cytokines were only significant in Group B (p < 0.05). Only the relation between IFN-γ and TNF-α was significant in Group A. There was a significant correlation between cytotoxic markers in both groups (A: p < 0.001, B: p < 0.01). These data demonstrate the complexity of the Th1 phenotype in NIR after drug intake. In patients with mild NIR, cytokines appear to play a closely related role, whereas cytotoxic markers appear more relevant in severe reactions. [Copyright &y& Elsevier]

Immunotoxins appear as promising therapeutic molecules, alternative to allergen-specific-immunotherapy. In this work, we achieved the development of a protein chimera able to promote specific cell death on effector cells involved in the allergic reaction. Der p 1 allergen was chosen as cell-targeting domain and the powerful ribotoxin α-sarcin as the toxic moiety. The resultant construction, named proDerp1αS, was produced and purified from the yeast Pichia pastoris. Der p 1-protease activity and α-sarcin ribonucleolytic action were effectively conserved in proDerp1αS. Immunotoxin impact was assayed by using effector cells sensitized with house dust mite-allergic sera. Cell degranulation and death, triggered by proDerp1αS, was exclusively observed on Der p 1 sera sensitized-humRBL-2H3 cells, but not when treated with non-allergic sera. Most notably, equivalent IgE-binding and degranulation were observed with both proDerp1αS construct and native Der p 1 when using purified basophils from sensitized patients. However, proDerp1αS did not cause any cytotoxic effect on these cells, apparently due to its lack of internalization after their surface IgE-binding, showing the complex in vivo panorama governing allergic reactions. In conclusion, herein we present proDerp1αS as a proof of concept for a potential and alternative new designs of therapeutic tools for allergies. Development of new, and more specific, second-generation of immunotoxins following proDerp1αS, is further discussed. [ABSTRACT FROM AUTHOR]

Background: The use of hypoallergenic derivatives is considered beneficial to promote the safety and efficacy of allergen-specific immunotherapy. We aimed to assess the efficacy of reduced and alkylated (R/A) Pru p 3, a hypoallergenic folding variant of the major peach allergen, in subcutaneous immunotherapy (SCIT) using a murine model of peach allergy.Methods and Results: After sensitization with Pru p 3, BALB/c mice received SCIT with Pru p 3 or R/A Pru p 3 and were challenged with Pru p 3. SCIT with Pru p 3, but not with R/A Pru p 3, suppressed anaphylaxis upon the challenge significantly. SCIT with Pru p 3 did not suppress Pru p 3-specific IgE and IgG1 production, but enhanced IgG2a production. In contrast, SCIT with R/A Pru p 3 suppressed IgE and IgG1 production, but enhanced IgG2a production only moderately. The therapeutic efficacy of SCIT with Pru p 3 was associated with induction of IL-10 and IFN-γ.Conclusion: Hypoallergenic folding variant of Pru p 3 is not likely an efficacious therapeutic component in SCIT of peach allergy. The lower efficacy of R/A Pru p 3 might be attributed to poor antigenicity and/or weak stability due to its unfolded conformation. [ABSTRACT FROM AUTHOR]

Background: The measurement of nasal specific IgE (NsIgE) in local allergic rhinitis (LAR) patients is challenging and shows variability. The objective of this work was to evaluate a minimally‐invasive method of direct detection of NsIgE in patients with LAR to Dermatophagoides pteronyssinus (DP) using an automated immunoassay. Methods: Fifty patients participated (LAR, n = 14; allergic rhinitis (AR), n = 20; healthy controls [HC], n = 16). Detection of NsIgE was performed by direct application of the solid phase of a commercial DP ImmunoCAP® test 24 hours after DP nasal provocation. Results: There was no difference in the median volume of secretion absorbed by the solid phase of the ImmunoCAP test in the 3 studied groups (p = 0.17). According to receiver operating characteristic (ROC) curve analysis, NsIgE ≥0.1450 was the optimal cutoff point, obtaining in LAR patients 42.86% sensitivity with the highest specificity (100%), and 75% sensitivity and 100% specificity for AR. Conclusion: This study demonstrates the detection of NsIgE to DP in LAR by using a simple, commercial device with high specificity. [ABSTRACT FROM AUTHOR]

Introduction Hypersensitivity reactions to beta-lactams ( BLs) are often reported in children, with amoxicillin and, to a lesser extent, cephalosporins being the most frequent drugs involved. Although many of these children are considered to be allergic, a careful evaluation only confirms a low percentage. Objectives To analyse the clinical data, sensitization profile and diagnostic methods used in a large group of children with a clinical history of hypersensitivity reactions to BLs. Methods The study included children aged 1-14 yr with symptoms suggestive of hypersensitivity to BLs from January 2006-December 2012. Diagnosis was confirmed from a clinical history, specific Ig E determination, skin testing and, if necessary, a drug provocation test ( DPT). Results Of a total of 783 patients studied, only 62 (7.92%) were confirmed as being allergic, 9 (14.52%) with immediate and 53 (85.48%) with non-immediate reactions. In those with immediate reactions, 2 (22.22%) were diagnosed by in vitro test, 2 (22.22%) by skin testing and 5 (55.56%) by DPT; in those with non-immediate reactions, 2 (3.77%) were diagnosed by skin testing and 51 (96.23%) by DPT. In all cases, DPT was positive to the culprit drug (29 AX- CLV, 26 AX, 1 cefixime and 1 cefaclor), and the most usual symptoms were exanthema in 43 cases, urticaria in 12, urticaria-angio-oedema in 1 and erythema in 1 case. Conclusion After an allergological work-up, over 90% of the children evaluated were finally confirmed as tolerant to BLs. Most reactions were of the non-immediate type, and DPT was an essential tool for diagnosis. [ABSTRACT FROM AUTHOR]