Allergen Extracts Research Articles

A randomised, double-blinded trial to assess the effect of oclacitinib and prednisolone on intradermal allergen and prick tests in dogs with atopic dermatitis.

Intradermal (IDT) and prick (PT) tests are used to select allergens for allergen-specific immunotherapy in dogs with atopic dermatitis (cAD). However, the use of antipruritic drugs before performing these tests may influence the results. To evaluate the influence of the drugs oclacitinib and prednisolone on the immediate-phase reactions of IDT and PT. Thirty client-owned dogs with cAD with positive reactions to at least one allergen extract on IDT or PT. Dogs were randomly assigned to receive oclacitinib 0.4-0.58 mg/kg per os, every 12 h (n = 14), or prednisolone 0.37-0.65 mg/kg p.o., every 12 h (n = 16) for 14 days. IDT and PT were performed on Day (D)0 before treatment and on D14. At D14 there was no significant reduction in the means of the orthogonal diameters of the positive immediate-phase reactions of the IDT (p = 0.064) in the oclacitinib group; however, in the PT, the diameter of the positive reactions reduced significantly (p = 0.048). In both tests, there was no significant reduction in the total number of positive reactions (IDT, p > 0.999; PT, p = 0.735). In the prednisolone group, the means of the orthogonal diameters of positive immediate-phase reactions were significantly reduced in both tests (IDT, p = 0.001; PT, p ≤ 0.001) and there also was a reduction in the total number of positive reactions (IDT, p = 0.022; PT, p = 0.001). The use of oclacitinib 0.4-0.58 mg/kg twice daily for 14 days does not interfere with IDT results in dogs with cAD. However, oclacitinib may reduce PT reactivity. The use of prednisolone 0.37-0.65 mg/kg twice daily results in a reduction in both IDT and PT results.

Validation Procedures for Quantification of Food Allergens by Enzyme-Linked Immunosorbent Assay (ELISA).

Enzyme-linked immunosorbent assay (ELISA) is a widely used analytical technique for food allergen detection and quantification. Validating ELISA protocols is important for both assay developers and end users as it ensures method reliability. This chapter describes the protocols for validating the sensitivity, specificity, precision, accuracy, robustness, and ruggedness of an ELISA. Example procedures are also provided for sample preparation, allergen extraction, and ELISA operation.

Problems Encountered Using Fungal Extracts as Test Solutions for Fungal Allergy Diagnosis.

Fungal allergy is a worldwide public health burden, and problems associated with a reliable allergy diagnosis are far from being solved. Especially, the lack of high-quality standardized fungal extracts contributes to the underdiagnosis of fungal allergy. Compared to the manufacturing processes of extracts from other allergen sources, the processes used to manufacture extracts from fungi show the highest variability. The reasons for the high variability are manifold as the starting material, the growth conditions, the protein extraction methods, and the storage conditions all have an influence on the presence and quantity of individual allergens. Despite the vast variety of studies that have analyzed the impact of the different production steps on the allergenicity of fungal allergen extracts, much remains unknown. This review points to the need for further research in the field of fungal allergology, for standardization and for generally accepted guidelines on the preparation of fungal allergen extracts. In particular, the standardization of fungal extracts has been and will continue to be difficult, but it will be crucial for improving allergy diagnosis and therapy.

BALB/c and C57BL/6 mice differ in oxidant and antioxidant responses in innate and adaptive immune cells in an asthma model induced by cockroach allergens

Asthma is a complex and heterogenous disease affected by a multitude of factors. Several phenotypes of asthma exist which are influenced by various molecular mechanisms that include presence of antioxidant and oxidant enzymes in different immune cells such as dendritic cells (DCs), alveolar macrophages (AMs), neutrophils, and T cells. Close interaction between epithelial cells and dendritic cells initiates complex pathogenesis of asthma followed by involvement of other innate and adaptive immune cells. In chronic phase of the disease, these immune cells support each other in amplification of airway inflammation where oxidant-antioxidant balance is known to be an important contributing factor. Genetic variability in antioxidant response may influence the development of airway inflammation, however it has not been studied in mice yet. The two most studied mice strains, i.e. BALB/c and C57BL/6 are reported to have dissimilar airway responses to the same allergens due to their genetic makeup. In this investigation, we explored whether these strains had any differences in pulmonary oxidant-antioxidant system (Nrf2, SOD2, iNOS, HO-1, nitrotyrosine) in different immune cells (DCs, AMs, neutrophils, T cells), airway inflammation (presence of eosinophils and/or neutrophils) and mucus production in response to repeated cockroach allergen extract (CE) mouse model of asthma. Our data show that C57BL/6 mice had better induction of antioxidant system than BALB/c mice. Consequently, iNOS/nitrotyrosine levels were much exaggerated in BALB/c than C57BL/6 mice. As a result, BALB/c mice developed mixed granulocytic airway inflammation, whereas C57BL/6 developed mostly eosinophilic airway inflammation. Our data suggest that an exaggerated oxidant generation along with a weak antioxidant induction in response to a natural allergen on a susceptible genetic background may determine development of severe asthma phenotype such as mixed granulocyte inflammation.

Short-term efficacy and safety observation of standardized mite allergen extract rush subcutaneous immunotherapy for allergic rhinitis: a prospective study

Objective: To conduct a comparative analysis of the efficacy, safety, and cytokine changes associated with three distinct dose escalation regimens of allergen-specific immunotherapy (AIT), and to provide valuable insights into the implementation of safer and more effective accelerated immunotherapy in clinical practice. Methods: A prospective study of subcutaneous immunotherapy (SCIT) was conducted at Renmin Hospital of Wuhan University, involving patients with allergic rhinitis visited from 2019 to 2022. Participants were allocated to one of three treatment groups based on their preferences: conventional immunotherapy (CIT, 23 cases), cluster immunotherapy (CLIT, 25 cases), or rush immunotherapy (RIT, 18 cases). The RIT group received a single subcutaneous injection of 150 mg of omalizumab one week before commencing treatment. Subjective evaluation indices, including the Combined Symptom and Medication Score (CSMS), Visual Analogue Scale (VAS), and single symptom score, were recorded alongside objective evaluation indices (e.g., sIgE, tIgE, Th1/2 and Th17 cytokines) and adverse reactions. Assessments were conducted at baseline, and at 1, 7, and 15 weeks after treatment. SPSS 22.0 software was used for data processing and analysis. Results: The study included a total of 66 patients, comprising 37 males and 29 females, who completed the treatment regimen. In all three groups, CSMS and VAS scores showed significant reductions at 1, 7, and 15 weeks post-treatment (all P<0.05). Notably, the RIT group demonstrated a significantly lower VAS score (4.33±0.94) compared to the CIT (9.48±1.37) and CLIT (9.44±1.33) groups at 1 week post-treatment (P<0.05). Additionally, the RIT group (0.62±0.23) exhibited a lower CSMS score than the CIT (1.54±0.21) and CLIT (1.06±0.22) groups at 15 weeks post-treatment (P<0.05). Furthermore, at the point of reaching the maintenance dose, the RIT group (0.61±0.20) demonstrated superior improvement in nasal itching symptoms compared to the CIT (1.78±0.38) and CLIT groups (1.56±0.32), with P<0.05. The incidence of local adverse reactions in the RIT group (36/11.76%) was lower than that in the CIT (69/20.00%) and CLIT groups (62/16.53%), with P<0.05. Notably, none of the three groups reported grade 3/4 systemic adverse reactions, and there was no statistically significant difference in systemic adverse reactions among the three groups. Following treatment, IL-4, IL-5, IL-6, IL-17, sIgE, sIgE/tIgE, and Eos% exhibited varying degrees of decrease in all three groups, whereas IL-10, TNF, and IFN-γ did not show significant changes. Conclusions: All three distinct dose escalation regimens of SCIT demonstrated substantial clinical efficacy. Of note, the approach of combining a single injection of omalizumab with RIT significantly improved early-stage efficacy and exhibited the advantages of safety, effectiveness, and convenience, establishing it as a reliable immunotherapy method.

Research of the Protein Profile and Allergenic Properties of the Tropomyosin and Other Allergenic Components in the Composition of Water-Salt Extract of Giant Tiger Shrimp (&lt;i&gt;Penaeus monodon&lt;/i&gt;)

Despite the success in identifying new crustacean allergens other than tropomyosin, many potential allergens remain unidentified. At present there are no diagnostic and treatment crustaceans allergens on the Russian market, including tropomyosin of invertebrates. The aim of the research was to explore allergenic properties of tropomyosin and other proteins in water-salt extract from tiger shrimp (Penaeus monodon). Raw and boiled meat of giant tiger shrimp was used to prepare allergenic extracts. To identify the specific activity of experimental allergens, sera from patients allergic to crustaceans with an established degree of activity (class 3) were used. The levels of specific IgE were from 3.5 to 17.5 IU/mL. ELISA was performed with the allergen samples. Polyacrylamide gel electrophoresis of raw and boiled shrimp allergen preparations was followed by Western blotting with the most allergen-specific serum. It has been shown that the extracts from raw and boiled shrimp have allergenic activity (bind IgE). The heat treatment of shrimp had no effect on allergenic activity except for one sample, where a strong reaction with the antigens was found. It is proved that the selected extraction conditions make it possible to reveal the multifractional nature of the protein profile of raw and boiled shrimp extracts. In Western blotting experiments the raw shrimp extract showed a weak reaction; tropomyosin was not detected. At the same time five protein fractions were identified in the boiled shrimp samples which reacted with IgE antibodies of a patient with crustaceans’ allergy. The data obtained will allow us expanding the knowledge about crustacean allergy and continueing our research to determine the optimal conditions for cleaning shrimp allergens.

On-Chip Discovery of Allergens from the Exudate of Large Yellow Croaker (Larimichthys Crocea) Muscle Food by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

It is extremely crucial to establish facile, accurate, and fast methods for testing allergenic proteins (allergens) in seafood. The current study focuses on the evaluation of fish muscle exudate proteins in an effort to discover potential allergens in fish exudate for allergy tests. Large yellow croaker (Larimichthys crocea) was studied as a seafood model. Magnetic beads (MBs) modified with an IgE antibody were utilized to isolate allergens existing in the exudate sample. Immunoglobulin E (IgE) in blood is a class of antibodies that is mainly associated with allergic reactions. Potential allergens in the muscle exudate were fished by IgE-biofunctional MBs in microfluidic channels. The protein-attached MBs were isolated under a magnetic field, eluted, and collected. The collected eluent was digested and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to identify allergens. Eight allergens from large yellow croaker exudate were identified, i.e., parvalbumin beta, parvalbumin, protein S100, histone H4, cytochrome c, fatty acid binding protein 3 (FABP3), microsomal glutamate S-transfer 3 (MGST3), and C-C motif chemokine 21 (CCL21). The presently proposed microfluidic-magnetic-based allergen extraction protocol enables a facile and rapid test of potentials of seafood allergies, providing a solution to circumvent food safety issues, especially for allergic populations.

Impact of the standardization unit’s definition on the in vitro biological potency of allergen extracts

In Europe, allergen products from different manufacturers can be labeled using the same unit with yet different definitions of that unit, which may cause confusion, as is the case for the index of reactivity (IR). In this context, house dust mite (HDM) Staloral 300 IR/mL, from Stallergenes Greer, and HDM Osiris 300 IR/mL, from ALK-Abelló, were characterized in vitro. Qualitatively, namely in terms of protein and allergen profiles, the two products were similar. Quantitatively, and despite the same 300 IR/mL labeling, the two products were shown to have different biological potencies, with HDM Staloral 300 IR/mL displaying a 2.4 times higher total allergenic activity (TAA) than HDM Osiris 300 IR/mL. This higher biological potency of HDM Staloral 300 IR/mL was paralleled by higher allergen and protein contents, namely 1.5 times more Der p 1 and Der f 1, 3.0 times more group 2 allergens, 2.7 times more Der p 23, and 1.8 times more protein. In contrast, HDM Staloral 300 IR/mL was shown to contain far fewer culture medium-derived proteins than HDM Osiris 300 IR/mL.

Tolerability and Safety of Sublingual Immunotherapy in Patients with Tree Pollen Allergy in Daily Practice-An Open, Prospective, Non-Interventional Study.

To investigate the tolerability and safety of two sublingual tree pollen extracts approved in 2018, a non-interventional study (NIS) was performed. This NIS was an 8-month observational study conducted at 84 sites throughout Germany. Study participants received either a sublingual liquid allergen extract of birch pollen (SBPE) or a liquid allergen extract consisting of a mixture of birch, hazel, and alder tree pollen (STPE). Data from 432 patients were analyzed for the occurrence of adverse events and patient compliance. At least one local reaction occurred in 69 (22.2%) patients, whereas systemic reactions were only observed in 27 (6.3%) patients. STPE-treated patients developed systemic reactions more frequently than SBPE-treated patients (SBPE: 9 (4.3%) vs. STPE: 18 (8.0%)). Only one patient developed a systemic grade III reaction. Severe systemic grade IV reactions were not observed. A total of 348 (98.6%) of the patients who completed all visits were satisfied or very satisfied with the sublingual immunotherapy (SLIT), and 322 (71%) patients completed all visits. Both investigated products were well tolerated by the patients and demonstrated a good safety profile. AEs were observed less frequently than in the preceding clinical phase III trial, and no new safety concerns were identified.

Sensitization to Rice in Allergic Children Pediatric Allergy and Immunology Unit, Children's Hospital, Ain Shams University

Background The prevalence of food allergy among Egyptian children is largely unknown, more so for the different food allergens. Although there are several studies reporting on the common food allergens, yet none was done to explore the allergy to rice, a food allergen that is much less recognized both by pediatricians and allergic patients. Objectives To estimate the frequency of sensitization to rice in pediatric allergic patients in Egypt. Patients and Methods This analytical cross-sectional study was carried out in the period from April 2019 to September 2020. The study included 150 physician-diagnosed allergic patients aged 6 months to 18 years, enrolled consecutively from the Pediatric Allergy and Immunology Unit, Children's Hospital, Ain Shams university. They were 90 (60%) males and 60 (40%) females. SPT was done using a commercial rice allergen extract, Prick-to-prick test with cooked rice. Patients with clinical and /or SPT results suggestive of rice allergy were subjected to oral challenge test under medical supervision in hospital. Results None of the screened allergic children (n = 150) gave a self/parentally reported history of allergy to rice, nor after inquiry by the investigator. SPT using commercial rice extract revealed that only 8 reacted positively (&amp;gt;3mm) and were considered sensitized. PPT was done to all patients using a home prepared cooked rice extract and the test was positive in 5 patients; 2 of these were positive also by SPT. A total of 11 patients (7.3%) were considered sensitized and were subjected to OCT and none had a positive test. There was statistically significant difference in age between rice sensitized and non sensitized, being higher in rice-sensitized patients, with p-value = 0.043. Conclusion Our study is a pilot research which draws the attention to the existence of sensitization to rice in allergic children. PPT is a useful test with a reliable specificity which when positive, would aid in diagnosing rice allergy in settings with limited resources and unavailability of rice protein extracts.

Sodium Chloride versus Lactose as a Carrier for House Dust Mite Allergen in Allergen Chamber Studies: A Clinical Study to Assess Noninferiority

Introduction: In the Fraunhofer allergen challenge chamber (ACC), a standardized, universal, good manufacturing practice-conforming technology using a spray dried solution of lactose (L) and allergen extract has been established. In this study, we investigated the noninferiority of hypertonic sodium chloride (S) versus L as a carrier for house dust mite (HDM) allergen to simplify manufacturing, reduce costs, and allow for wider use. Methods: Using a participant-blinded, sham exposure-controlled, single-arm, sequential intervention study, we challenged adults with HDM allergic rhinitis five times in the ACC. Participants were first exposed to S, L, and clean air (block 1), followed by S + HDM and L + HDM (block 2). Primary endpoints were mean total nasal symptom score (TNSS) and mean nasal secretion weight. Results: 19 participants were enrolled in the study (10 females; mean age 32 years [22–49], 4 with mild allergic asthma). The safety profile of S + HDM and L + HDM was similar; eight participants experienced mild procedure-related adverse events including tiredness, cough, and dyspnea. Due to dropouts, 13 participants completed the study and were evaluated. Mean TNSS and nasal secretion were as follows: S 0.98, 0.28 g; L 1.1, 0.20 g; clean air 1.1, 0.23 g; S + HDM 5.7, 4.8 g; L + HDM 5.1, 5.1 g. Separate block 1/block 2 MANOVAs with TNSS and nasal secretion as dependent variables revealed no significant differences between the carriers, neither alone and compared with clean air (p = 0.2059, Wilk’s λ = 0.78) nor combined with HDM (p = 0.3474, Wilk’s λ = 0.89). Noninferiority of S was established using a meta-analysis-based minimal clinical important difference of −0.55: mean TNSS difference between S + HDM and L + HDM was 0.62 (90% CI: −0.51 to 1.74). Conclusion: S as an HDM carrier was safe and well tolerated. It was noninferior to L which makes it an adequate and easy-to-use carrier substitute.

Extraction, Purification, and Development of Sublingual Film (SLIT Films) Comprising Cockroach Allergen for Treatment of Allergy.

Allergy associated with cockroaches are mostly from the American cockroach (Periplaneta americana) and German cockroach (Blattella germanica). The effective and safe treatment for cockroach allergy is Sublingual immunotherapy (SLIT). In this study, SLIT Films containing purified allergen extract of Periplaneta americana were prepared by solvent casting and were evaluated for their efficiency in delivery. Cockroach allergen extract was prepared and purified by ultrafiltration and chromatography. The molecular weight of protein content was identified and estimated by SDS- PAGE and ELISA. SLIT films were developed by the Quality by Design (QbD) approach and were evaluated for allergen- excipient compatibility, swelling index, taste, diffusion, in vitro dissolution, local toxicity, and stability analysis. Cockroach allergen protein extracts (cut-off 25-71KDa) were identified by SDS-PAGE and quantified by indirect ELISA and further selected for sublingual film preparation. The indirect ELISA results show a higher optical density (OD) value compared to crude extract. The weight uniformity and thickness of the film were between 13-18 mg and 0.04-0.06 mm. The disintegration time was found to be less than 1 min. The cumulative percentage release was also found to be satisfactory. The local toxicity study indicated no signs of irritation in the buccal mucosa of rabbits. The optimised F3 film had uniform thickness, faster disintegration and drug content within pharmacopeial limits. Ex vivo study revealed better permeability with 90% release of allergen in 7 minutes. The formulation was also stable at room temperature during the study period. SLIT Film containing cockroach allergen from Periplaneta americana was successfully developed and evaluated. SLIT films of cockroach allergen could be more beneficial and convenient for emergency use in patients when compared to subcutaneous immunotherapy. SLIT films provide dose accuracy and are a promising alternative for SCIT and SLIT drops and tablets.

Growth, allergen profile and microbiome studies in Dermatophagoides pteronyssinus cultures

Mites are mass-cultured to manufacture allergen extracts for allergy diagnostics and therapeutic treatment. This study focused on characterizing the growth, the allergen profile, and the microbiome of Dermatophagoides pteronyssinus cultures. Mite population, protein profile, total protein content and major allergen levels (Der p 1, Der p 2, Der p 23) were monitored at different times of three independent cultures. The allergenicity was studied by immunoblot using a pool of sera from allergic patients. Mite microbiome was characterized by sequencing the 16S rRNA gene from 600 adult mites from the last day of the culture. Endotoxin content was also analyzed. The cultures had a fast and unrelenting evolution. Mite density, total protein content, major allergen levels and the allergenicity were increased progressively during the cultures. Regarding the microbiome studies, the results confirm the presence of non-pathogenic bacteria, being firmicutes and actinobacteria the most common bacterial taxa, with a very low content of Gram-negative bacteria and endotoxin content. The allergenicity and levels of the main allergens in the mite cultures are objective methods useful to monitor the mite culture that help to produce standardized allergen extracts. The high presence of Gram-positive bacteria found limits the possibility for vaccine contamination by bacterial endotoxins.

Selected Technical Aspects of Molecular Allergy Diagnostics.

Diagnosis of allergic diseases is a complex, multi-stage process. It often requires the use of various diagnostic tools. The in vitro diagnostics (IVD), which includes various laboratory tests, is one of the stages of this process. Standard laboratory tests include the measurement of the serum concentration of specific immunoglobulin E (sIgE) for selected allergens, full allergen extracts and/or single allergen components (molecules). The measurement of IgE sIgE to the allergen components is called molecular allergy diagnosis. During the standard laboratory diagnostic process, various models of immunochemical tests are used, which enable the measurement of sIgE for single allergens (one-parameter tests, singleplex) or IgE specific for many different allergens (multi-parameter tests, multiplex) in one test. Currently, there are many different test kits available, validated for IVD, which differ in the method type and allergen profile. The aim of the manuscript is to present various technical aspects related to modern allergy diagnostics, especially in the area of molecular allergy diagnostics.

The Art of Dosing for Subcutaneous Immunotherapy in North America

Subcutaneous immunotherapy (SCIT) is a long-established treatment option for allergic rhinoconjunctivitis. Proper dosing of the allergens is critical for the efficacy and safety of SCIT. Of the hundreds of liquid allergen extracts in the United States, effective and well-tolerated SCIT dosing has only been established for a small number. Thus, SCIT dosing remains largely empiric and continues to be, by necessity, an art. To highlight the complexity of SCIT dosing, this review summarizes the historical and current landscape of U.S. allergen extracts, differences among U.S. and European allergen extracts, allergen selection for SCIT, considerations for compounding of allergen extract mixtures, and recommended dosing. As of 2021, 18 standardized allergen extracts are available in the United States; all other extracts remain unstandardized without characterization of allergen content or potency. U.S. allergen extracts differ from European extracts in formulation and potency characterization. There is no standardized methodology for SCIT allergen selection, and interpretation of allergen sensitization is not straightforward. Compounding of SCIT mixtures requires consideration of potential dilution effects, allergen cross-reactivity, proteolytic activity, and additives. Probable effective dose ranges for SCIT are recommended in U.S. allergy immunotherapy practice parameters, although there are few studies using U.S. extracts supporting these doses as therapeutic. In contrast, optimized doses of sublingual immunotherapy tablets have been confirmed in North American phase 3 trials. The SCIT dosing for each patient remains an art that requires clinical experience and consideration of polysensitization, tolerability, compounding of allergen extract mixtures, and the range of recommended doses within the context of extract potency variability.

Efficacy and safety of subcutaneous immunotherapy in asthmatic children allergic to house dust mite: a meta-analysis and systematic review.

Subcutaneous immunotherapy (SCIT) has been proved to be effective and safe in adult asthma. But it is still controversial in children. To evaluate the efficacy and safety of SCIT in asthmatic children with allergy to house dust mite. We searched the databases of Cochrane Library, EMBASE and MEDLINE (from 1 January 1990 to 1 December 2022). Two reviewers independently screened studies, extracted data and critically appraised the risk of bias. We used the Revman 5 to synthesize the effect sizes. We finally selected 38 eligible studies including 21 randomized controlled trials to evaluate the efficacy and safety of SCIT and 17 observational studies to assess the safety. The results revealed that short-term asthma symptom scores were declined with a standardized mean difference (SMD) of -1.19 (95% CI: -1.87, -0.50) in 12 researches with high heterogeneity. Short-term asthma medication scores were decreased with SMD -1.04 (95% CI: -1.54, -0.54) in 12 heterogeneous researches. One study showed no significant reduction in combined symptom and medication scores without providing details. No studies we reviewed reported long-term efficacy. SCIT resulted in an obviously increased risk of adverse reactions compared with placebo. For secondary outcomes, SCIT improved life quality and reduced the numbers of annual asthma attacks and allergen-specific airway hyperreactivity, but without significant improvement in pulmonary function, asthma control or hospitalization. SCIT can reduce the short-term symptom scores and medication scores regardless of different treatment duration or mono/polysensitization, but with an increased incidence of local and systemic adverse effects. Further studies on pediatric asthma are needed to evaluate the long-term efficacy, and clarify the effectiveness of SCIT in specific population using mix allergen extracts or with severe asthma. Overall, it is recommended for children with mild-moderate HDM-driven allergic asthma.

Comparison of Intact Allergen Extracts and Allergoids For Subcutaneous Immunotherapy: The Effect of Chemical Modification Differs Both Between Species and Between Individual Allergen Molecules.

Allergen products for subcutaneous immunotherapy (SCIT) contain intact allergen extracts or chemically modified allergoids. Chemical modification was introduced to reduce allergenicity while retaining immunogenicity and thereby enable safer and more efficient allergy immunotherapy. Experimental allergoids were produced from intact allergen extract for birch, grass, and house dust mite (HDM) to evaluate the effects of chemical modification. Preparations were compared with commercial allergoids and analyzed using SDS-PAGE/immunoblotting, IgE-inhibition assays, and crossed immunoelectrophoresis (CIE). Dermatophagoides pteronyssinus (Der p) vaccines were also tested for protease activity and immunizing capacity in a mouse model. The composition of IgE-binding epitopes in allergoids differed from that of intact allergen vaccines. Birch and grass allergoids produced smears of protein aggregates on SDS-PAGE, whereas intact allergen preparations showed distinct protein bands as expected. Der p allergoid vaccines, however, showed a distinct protein band corresponding to major allergen Der p 1 in both SDS-PAGE and CIE analysis, and commercial Der p allergoid vaccines showed Der p 1-related cysteine protease activity. Allergoids and intact allergen preparations differ with respect to the composition of IgE-binding epitopes. However, chemical cross-linking does not affect every allergen molecule to the same degree. Der p 1, for example, remains largely unmodified. Furthermore, the investigational HDM allergoid vaccines showed reduced and delayed immune responses when used for immunization of mice.

Going over Fungal Allergy: Alternaria alternata and Its Allergens

Fungal allergy is the third most frequent cause of respiratory pathologies and the most related to a poor prognosis of asthma. The genera Alternaria and Cladosporium are the most frequently associated with allergic respiratory diseases, with Alternaria being the one with the highest prevalence of sensitization. Alternaria alternata is an outdoor fungus whose spores disseminate in warm and dry air, reaching peak levels in temperate summers. Alternaria can also be found in damp and insufficiently ventilated houses, causing what is known as sick building syndrome. Thus, exposure to fungal allergens can occur outdoors and indoors. However, not only spores but also fungal fragments contain detectable amounts of allergens and may function as aeroallergenic sources. Allergenic extracts of Alternaria hyphae and spores are still in use for the diagnosis and treatment of allergic diseases but are variable and insufficiently standardised, as they are often a random mixture of allergenic ingredients and casual impurities. Thus, diagnosis of fungal allergy has been difficult, and knowledge about new fungal allergens is stuck. The number of allergens described in Fungi remains almost constant while new allergens are being found in the Plantae and Animalia kingdoms. Given Alt a 1 is not the unique Alternaria allergen eliciting allergy symptoms, component-resolved diagnosis strategies should be applied to diagnose fungal allergy. To date, twelve A. alternata allergens are accepted in the WHO/IUIS Allergen Nomenclature Subcommittee, many of them are enzymes: Alt a 4 (disulfide isomerase), Alt a 6 (enolase), Alt a 8 (mannitol de-hydrogenase), Alt a 10 (aldehyde dehydrogenase), Alt a 13 (glutathione-S-transferase) and Alt a MnSOD (Mn superoxide dismutase), and others have structural and regulatory functions such as Alt a 5 and Alt a 12, Alt a 3, Alt a 7. The function of Alt a 1 and Alt a 9 remains unknown. Other four allergens are included in other medical databases (e.g., Allergome): Alt a NTF2, Alt a TCTP, and Alt a 70 kDa. Despite Alt a 1 being the A. alternata major allergen, other allergens, such as enolase, Alt a 6 or MnSOD, Alt a 14 have been suggested to be included in the diagnosis panel of fungal allergy.

Encapsulation of Allergens into Core–Shell Chitosan Microparticles for Allergen-Specific Subcutaneous Immunotherapy

IgE-mediated allergic reaction occurs in response to harmless environmental compounds, such as tree and grass pollen, fragments of household microorganisms, etc. To date, the only way to treat IgE-mediated allergy is allergen-specific immunotherapy (ASIT), which consists of a prolonged subcutaneous administration of allergen extracts or recombinant proteins. The long duration of the treatment, the cost and the risk of life-threatening adverse reactions are the main limiting factors for ASIT. The aim of this work was to develop allergen proteins encapsulated in chitosan-based microparticles that can be safely administered at high doses and in a rash protocol. The egg white allergen, Gal d 1 protein, was used as a model antigen. The protein was packed into core–shell type microparticles (MPs), in which the core was formed with succinyl chitosan conjugated to Gal d 1, subsequently coated with a shell formed by quaternized chitosan. The obtained core–shell MPs containing Gal d 1 in the core (Gal-MPs) were non-toxic to macrophage and fibroblast cell lines. At the same time, Gal-MPs were quickly engulfed by bone marrow-derived dendritic cells or RAW264.7 macrophage cells, as was visualized using flow cytometry and confocal microscopy. Encapsulated Gal d 1 was not recognized by Gal d 1-specific IgE in ELISA. Female BALB/c mice were immunized with Gal-MPs subcutaneously three times a week for 2 weeks. Immunization of mice resulted in IgG titers 1250 ± 200 without IgE production. Allergy in control and vaccinated mice was induced by low-dose Gal d 1 injections in the withers of mice. IgE was induced in control-sensitized but not in the vaccinated mice. Thus, preventive vaccination with the encapsulated allergens is safe and rapid; it significantly reduces the risk of IgE production induced by respiratory and oral allergens.

Biological activity of human IgE monoclonal antibodies targeting Der p 2, Fel d 1, Ara h 2 in basophil mediator release assays.

Human Immunoglobulin E monoclonal antibodies (hIgE mAb) are unique tools for investigating IgE responses. Here, the biological activity of hIgE mAb, derived from immortalized B cells harvested from the blood of allergic donors, targeting three allergens (Der p 2, Fel d 1 and Ara h 2) was investigated. Three Der p 2-, three Fel d 1- and five Ara h 2-specific hIgE mAb produced by human B cell hybridomas, were combined in pairs and used to passively sensitize humanized rat basophilic leukemia cells and compared with sensitization using serum pools. Sensitized cells were stimulated with corresponding allergens (recombinant or purified), allergen extracts or structural homologs, having 40-88% sequence similarity, and compared for mediator (β-hexosaminidase) release. One, two and eight pairs of Der p 2-, Fel d 1- and Ara h 2-specific hIgE mAb, respectively, produced significant mediator release (>50%). A minimum hIgE mAb concentration of 15-30 kU/L and a minimum antigen concentration between 0.01-0.1 µg/mL were sufficient to induce a pronounced mediator release. Individual sensitization with one Ara h 2-specific hIgE mAb was able to induce crosslinking independently of a second specific hIgE mAb. Der p 2- and Ara h 2-specific mAb showed a high allergen specificity when compared to homologs. Mediator release from cells sensitized with hIgE mAb was comparable to serum sensitization. The biological activity of hIgE mAb reported here provides the foundation for novel methods of standardization and quality control of allergen products and for mechanistic studies of IgE-mediated allergic diseases, using hIgE mAb.