Component-Resolved Diagnosis Based on a Recombinant Variant of Mus m 1 Lipocalin Allergen.

Exposure to the Mus m 1 aeroallergen is a significant risk factor for laboratory animal allergy. This allergen, primarily expressed in mouse urine where it is characterized by a marked and dynamic polymorphism, is also present in epithelium and dander. Considering the relevance of sequence/structure assessment in protein antigenic reactivity, we compared the sequence of the variant Mus m 1.0102 to other members of the Mus m 1 allergen, and used Discotope 2.0 to predict conformational epitopes based on its 3D-structure. Conventional diagnosis of mouse allergy is based on serum IgE testing, using an epithelial extract as the antigen source. Given the heterogeneous and variable composition of extracts, we developed an indirect ELISA assay based on the recombinant component Mus m 1.0102. The assay performed with adequate precision and reasonable diagnostic accuracy (AUC = 0.87) compared to a routine clinical diagnostic test that exploits the native allergen. Recombinant Mus m 1.0102 turned out to be a valuable tool to study the fine epitope mapping of specific IgE reactivity to the major allergen responsible for mouse allergy. We believe that advancing in its functional characterization will lead to the standardization of murine lipocalins and to the development of allergen-specific immunotherapy.

The allergen Mus m 1.0102: Dissecting the relationship between molecular conformation and allergenic potency.

BACKGROUND: The species Mus musculus experiences an obligate proteinuria: predominant are the Major Urinary Proteins (MUPs), that, collectively known as the major mouse allergen Mus m 1, are among the most important aeroallergens for mouse allergic patients. The production of a soluble and stable hypoallergenic form of Mus m 1 is essential for the development of immunotherapeutic protocols to treat allergic symptoms. METHODS: We introduced the substitution C138S in recombinant Mus m 1.0102, an allergenic isoform of Mus m 1. Solubility, conformation, stability and ability to refold after chemical denaturation were investigated with dynamic light scattering, circular dichroism, fluorescence and NMR spectroscopy. An in vitro degranulation assay was used to evaluate the protein allergenic potential, and compare it with Mus m 1.0102 and with an hypoallergenic variant bearing the substitution Y120A. RESULTS: Mus m 1.0102-C138S retains a native-like fold revealing, however, local conformational alterations that influence some of its physical and allergenic properties: it is monodispersed, thermostable up to 56°C, able to reversibly unfold and it exhibits an enhanced allergenicity. CONCLUSIONS: The unique free thiol group affects the solution structural stability of the native protein. Because the mutant C138S does not aggregate over time it is a good lead protein to develop diagnostic and therapeutic applications. GENERAL SIGNIFICANCE: We elucidated the relationship between unfolding reversibility and sulphydryl reactivity. We ascribed the enhanced allergenicity of the mutant C138S to an increased accessibility of its allergenic determinants, an enticing feature to further investigate the structural elements of the allergen-IgE interface.

Serological proteome analysis identifies crustacean myosin heavy chain type 1 protein and house dust mite Der p 14 as cross-reacting allergens.

BACKGROUND: Allergies to house dust mite (HDM) and to crustaceans are clinically and pathogenically linked. Several homologous allergenic proteins have been identified, among which tropomyosin is the prototype, expressing epitopes endowed with variable levels of immunoglobulin E (IgE) cross-reactivity. Component-resolved diagnosis (CRD) does not allow a thorough characterization of all relevant IgE reactivities to these allergen sources. OBJECTIVES: We studied 1 patient allergic to shrimp with positive skin prick test to HDM and negative scores for IgE to HDM allergen components routinely used in CRD (group 1 and 2 allergens, Der p 23 and tropomyosin). MATERIAL AND METHODS: In order to identify the allergen(s) involved in IgE reactivity, we used serological proteome analysis (SERPA), which utilizes two-dimensional gel electrophoresis (2DE), immunoblotting and mass spectrometry (MS). The identified allergenic proteins were tested with sera from 20 crustacean-allergic patients and 19 grass-allergic patients serving as controls. RESULTS: Der p 14 and myosin heavy chain type 1 (MHC1) were identified as the components recognized by patient's IgE in the proteome of Dermatophagoides pteronyssinus and Penaeus monodon, respectively. The MHC1 protein shows about 30% sequence identity with Der p 14 in specific domains, and cross-reactivity against epitopes shared by the 2 proteins was demonstrated by reduced reactivity to shrimp extract following pre-incubation with Der p 14. Serum IgE from 5 out of 20 patients allergic to crustaceans reacted with MHC1, compared to none among 19 controls (p < 0.05). CONCLUSION: We identified MHC1 as a relevant allergic component in the proteome of Penaeus monodon, the prototypic allergen source used in diagnosis of allergy to crustaceans. Our data demonstrate MHC1 cross-reactivity between MHC1 and Der p 14 from Dermatophagoides pteronyssinus.

Periostin expression in uninvolved skin as a potential biomarker for rapid cutaneous progression in systemic sclerosis patients: a preliminary explorative study.

Objectives: This study aimed to evaluate periostin serum levels and skin expression in patients with systemic sclerosis (SSc). Methods: We enrolled 35 patients with diffuse (d-SSc) or limited (l-SSc) SSc, 15 patients with very early diagnosis of systemic sclerosis (VEDOSS), and 30 sex-matched healthy controls. Periostin serum levels were determined by an enzyme-linked immunosorbent assay (ELISA). Periostin skin expression was determined by immunohistochemistry (IHC) on paired involved and uninvolved 5-mm skin biopsy samples in a subgroup of 10 d-SSc and 10 L-SSc patients. A 12-month follow-up was considered. Results: We included 50 patients (mean age 53.1 ± 16.1 years; women 94%; mean disease duration 38.2 ± 45.1 months; anti-centromere 50%; anti-Scl70 40%), 35 of them with a definite SSc (68.8% l-SSc; 31.4% d-SSc; mean mRSS 9.0 ± 7.2) and 15 with VEDOSS; 30 controls were also included in this study. Periostin serum levels were higher in SSc patients compared to controls (32.7 ± 8.0 ng/mL vs. 27.7 ± 7.3 ng/mL; p < 0.001), while these levels were comparable among different groups of patients (29.7 ± 6.9 ng/mL in VEDOSS, 33.4 ± 7.8 ng/mL in lc-SSc; and 34.0 ± 8.5 in dc-SSc; p = ns). SSc patients with digital ulcers had higher periostin serum levels (36.2 ± 7.9 ng/mL vs. 30.6 ± 7.3 ng/mL, p < 0.02). Samples from the involved skin of l-SSc and d-SSc patients showed a significant dermal expression of periostin; an identical periostin expression was evident in the uninvolved skin of patients with d-SSc. In 7 out of 10 L-SSc patients, periostin expression was absent on uninvolved skin. In the remaining three l-SSc patients, a mild periostin expression on IHC was detectable on uninvolved skin and all of these three l-SSc patients presented a dramatic skin progression. Conclusion: Periostin skin expression may be a useful biomarker to indicate the presence of a disease at a higher risk of rapid cutaneous involvement.

Hypersensitivity reactions to anti-SARS-CoV-2 vaccines: Basophil reactivity to excipients.

Basophil activation test (BAT) can tackle multiple mechanisms underlying acute and delayed hypersensitivity to drugs and vaccines and might complement conventional allergy diagnostics but its role in anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine-related hypersensitivity is ill-defined. Therefore, 89 patients with possible hypersensitivity (56 % with delayed mucocutaneous manifestations) to anti-SARS-CoV-2 vaccines were tested with BAT for Macrogol 3350, DMG-PEG 2000, PEG 20000, polysorbate-80 and trometamol and compared to 156 subjects undergoing pre-vaccine BAT. A positive BAT was associated with delayed reaction onset (p = 0.010) and resolution (p = 0.011). BAT was more frequently positive to DMG-PEG 2000 than to other excipients in both groups (p < 0.001). DMG-PEG 2000 reactivity was less frequent in vaccine-naïve (6 %) than vaccinated subjects (35 %, p < 0.001) and associated with mRNA-1273 vaccination. DMG-PEG 2000 BAT might therefore have a diagnostic role in subjects with delayed hypersensitivity reactions. Natural immunity might be a key player in basophil activation.

In search of a vaccine for mouse allergy: significant reduction of Mus m 1 allergenicity by structure-guided single-point mutations.

BACKGROUND: Mouse urinary proteins are relevant allergens from mice urine. We used the recombinant protein Mus m 1 as an allergen model to identify if, by altering Mus m 1 architecture via single-point mutations, we could effectively modify its allergenicity. METHODS: Based on structural considerations, we synthesized two single-point mutants, Mus m 1-Y120A and Mus m 1-Y120F, which were expected to harbor large structural alterations. Circular dichroism and fluorescence analysis showed significant conformational rearrangements of the aromatic side chains in the internal cavity of Mus m 1-Y120A when compared to Mus m 1-Y120F and Mus m 1. Evaluation of the allergenic potential of the recombinant molecules was performed in vitro with both immunochemical approaches and assays based on the measurement of basophil degranulation. Moreover, to assess the integrity of the T cell epitopes and as an in vitro measure of immunogenicity, we tested the reactivity of T lymphocytes from subjects allergic to mouse urine against proteins and synthetic peptides encompassing the immunodominant linear epitope containing the mutation. RESULTS: We found that the selected point mutation was able to modulate the protein allergenicity, and to severely impair the recognition of Mus m 1 by IgE, while T cell reactivity was fully maintained. CONCLUSIONS: In silico predicted, minimum selected structural modifications allowed to design one protein with reduced allergenicity and preserved immunogenicity. Structurally guided mutations can direct the design of proteins with reduced allergenicity which can be used as vaccines for a safer and more effective immunotherapy of allergic disorders.

Basal Serum Diamine Oxidase Levels as a Biomarker of Histamine Intolerance: A Retrospective Cohort Study.

Background: Histamine Intolerance (HIT) is a multifaceted pseudoallergic disorder possibly due to defective histamine metabolism. Diamine oxidase (DAO) contributes to histamine degradation and can be measured in the serum. The role of DAO measurement in the diagnostic work-up of HIT still remains unclear, and conflicting results have been reported in the literature. Therefore, we aimed to evaluate the possible clinical usefulness and consistency of DAO value ranges as provided by the assay manufacturer and verify whether they could predict the response to treatment. Methods: We retrospectively analyzed 192 outpatients with HIT symptoms and measured serum DAO values at baseline. Patients were prescribed either with low-histamine diet and/or enzymatic supplementation according to symptom severity and re-evaluated six to eight months later. Patients were stratified into three groups according to DAO levels: <3 U/mL, 3−10 U/mL, and >10 U/mL. HIT severity was assessed on a scale of 1 to 5 before and after treatment. Results: A total of 146 patients completed the study. Gastrointestinal and cutaneous symptoms, often associated with headache, were more frequent in subjects with DAO < 10 U/mL. Symptom severity and DAO ranges were correlated. Patients with intermediate DAO levels (3−10 U/mL) showed a more complex clinical phenotype but also a more significant improvement in symptom severity (score reduction 50%, interquartile range (IQR) = 33−60%) when compared to patients with low DAO (40%, IQR = 20−60%; p = 0.045) or high DAO (33%, IQR = 0−50%; p < 0.001). Complex clinical phenotypes were also more frequent in patients with intermediate DAO levels. Conclusions: HIT is characterized by typical symptoms and low levels of DAO activity. Symptom severity was associated with the degree of DAO deficiency. Patients with DAO values between 3 and 10 U/mL show the best response to treatment (low-histamine diet and/or DAO supplementation). DAO value could arguably be considered as a predictor of clinical response to treatment. Prospective studies are needed to confirm these data.

Allergen microarrays on high-sensitivity silicon slides.

We have recently introduced a silicon substrate for high-sensitivity microarrays, coated with a functional polymer named copoly(DMA-NAS-MAPS). The silicon dioxide thickness has been optimized to produce a fluorescence intensification due to the optical constructive interference between the incident and reflected lights of the fluorescent radiation. The polymeric coating efficiently suppresses aspecific interaction, making the low background a distinctive feature of these slides. Here, we used the new silicon microarray substrate for allergy diagnosis, in the detection of specific IgE in serum samples of subjects with sensitizations to inhalant allergens. We compared the performance of silicon versus glass substrates. Reproducibility data were measured. Moreover, receiver-operating characteristic (ROC) curves were plotted to discriminate between the allergy and no allergy status in 30 well-characterized serum samples. We found that reproducibility of the microarray on glass supports was not different from available data on allergen arrays, whereas the reproducibility on the silicon substrate was consistently better than on glass. Moreover, silicon significantly enhanced the performance of the allergen microarray as compared to glass in accurately identifying allergic patients spanning a wide range of specific IgE titers to the considered allergens.

Thermal stability, ligand binding and allergenicity data of Mus m 1.0102 allergen and its cysteine mutants.

The presented data were obtained with the lipocalin allergen Mus m 1.0102 and its cysteine mutants MM-C138A, MM-C157A and MM-C138,157A, whose structural features and unfold reversibility investigations are presented in the research article entitled "The allergen Mus m 1.0102: cysteine residues and molecular allergology" [1]. The data were obtained by means of a Dynamic Light Scattering-based thermal stability assay, a Fluorescence-based ligand-binding assay and a basophil degranulation test, and describe proteins' fold stability, ligand binding ability and allergenic potential, respectively. Analysis of the collected data produced the temperatures corresponding to the onset of the protein unfolding, the dissociation constants for N-Phenyl-1-naphthylamine ligand and the profiles of ß-hexosaminidase release from RBL SX-38 cells, sensitized with the serum of selected allergic patients and incubated with increasing antigens concentrations. These data allow for comparison of the lipocalin allergen Mus m 1.0102 with its conserved cysteines mutants and, with regard to their potential application in allergy diagnostics and immunotherapy, they contribute to the process of recombinant allergen characterization and standardization.

The allergen Mus m 1.0102: Cysteine residues and molecular allergology.

Mus m 1.0102 is a member of the mouse Major Urinary Protein family, belonging to the Lipocalins superfamily. Major Urinary Proteins (MUPs) are characterized by highly conserved structural motifs. These include a disulphide bond, involved in protein oxidative folding and protein structure stabilization, and a free cysteine residue, substituted by serine only in the pheromonal protein Darcin (MUP20). The free cysteine is recognized as responsible for the onset of inter- or intramolecular thiol/disulphide exchange, an event that favours protein aggregation. Here we show that the substitution of selected cysteine residues modulates Mus m 1.0102 protein folding, fold stability and unfolding reversibility, while maintaining its allergenic potency. Recombinant allergens used for immunotherapy or employed in allergy diagnostic kits require, as essential features, conformational stability, sample homogeneity and proper immunogenicity. In this perspective, recombinant Mus m 1.0102 might appear reasonably adequate as lead molecule because of its allergenic potential and thermal stability. However, its modest resistance to aggregation renders the protein unsuitable for pharmacological preparations. Point mutation is considered a winning strategy. We report that, among the tested mutants, C138A mutant acquires a structure more resistant to thermal stress and less prone to aggregation, two events that act positively on the protein shelf life. Those features make that MUP variant an attractive lead molecule for the development of a diagnostic kit and/or a vaccine.

Protective versus pathogenic anti-CD4 immunity: insights from the study of natural resistance to HIV infection.

HIV-1 exposure causes several dramatic unbalances in the immune system homeostasis. Here, we will focus on the paradox whereby CD4 specific autoimmune responses, which are expected to contribute to the catastrophic loss of most part of the T helper lymphocyte subset in infected patients, may display the characteristics of an unconventional protective immunity in individuals naturally resistant to HIV-1 infection. Reference to differences in fine epitope mapping of these two oppositely polarized outcomes will be presented, with particular reference to partially or totally CD4-gp120 complex-specific antibodies. The fine tuning of the anti-self immune response to the HIV-1 receptor may determine whether viral exposure will result in infection or, alternatively, protective immunity.Along this line, an efficacious anti-HIV strategy can rely on the active (i.e., through immunization) or passive targeting of cryptic epitopes of the CD4-gp120 complex, including those harboured within the CD4 molecule. Such epitopes are expected to be safe from genetic drift and thus allow for broad spectrum of efficacy. Moreover, since these epitopes are not routinely exposed in uninfected individuals, they are expected to become targets of neutralizing antibodies or other specifically designed molecules only after viral exposure, with a predictable low impact in terms of potentially harmful anti-CD4 self-reactivity.The experimentum naturae of naturally resistant individuals indicates a strategy to design innovative strategies to neutralize HIV-1 by acting on the sharp edge between harmful and protective self-reactivity.

A method for the analysis of milk and egg allergens for the atopy patch test.

The patch test with food antigens (atopy patch test, APT) has been reported as a more specific method than prick or RAST for the early detection of cow's milk and/or egg sensitizations in children. Standardization of APT extracts is a major issue on the road towards full clinical exploitation of this assay. Here, we used sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to characterize sensitivity and specificity of commercial preparations of APT for milk and egg allergies, which are expected to improve the reliability of this test, when compared with fresh food allergen sources. We found that: (i) SDS-PAGE is an appropriate technique for quality control of APT and (ii) commercial milk and egg APT are equivalent to fresh food preparations in terms of allergen content. Clinical trials aimed at characterizing sensitivity and specificity of APT in the diagnosis of food allergy in children will benefit from this technique.

HIV-1 and the self-nonself connection: how to sleep with the enemy and be much better off.

Envelope-based immunogens capable of generating high titers of neutralizing antibodies have until now been difficult to generate, or failed to act as useful vaccines to prevent HIV-1 infection and disease progression. On the other hand, humoral immune responses to self and allogeneic cellular antigens involved in HIV-1 docking and entry are present both in infected patients and in subjects with natural resistance to HIV-1 infection, where they share similarities but also display definite differences. By dissecting these subtle differences, crucial cellular and molecular markers, possibly correlated with natural resistance to HIV-1 and with the modulation of clinical progression in stably infected patients, have been identified. Here, state-of-the art knowledge on anti-self immune responses following infection or exposure to HIV will be reviewed. The possible implications of these mechanisms in the design of unconventional therapies aimed to counteract the peculiar HIV-1 capability to circumvent the immune system will be discussed.

Identification by serological proteome analysis of paramyosin as prominent allergen in dust mite allergy.

Component-resolved diagnosis (CRD) of IgE-mediated hypersensitivities is challenged by the possibility that single patients are sensitized to components not commercially available to the clinical lab. Here, we studied a patient with positive extract-based diagnosis of house dust mite (HDM) allergy based on routine in vivo (prick test) and in vitro (serum specific IgE) tests, whose serum scored negative for IgE to the three recombinant allergens routinely used in CRD (group 1 allergens, group 2 allergens and tropomyosin). By means of serological proteome analysis via two-dimensional gel electrophoresis combined with immunoblotting and mass spectrometry, paramyosin (group 11 allergen: Der f 11 and Der p 11) was identified as the allergen component recognized by serum IgE from this patient in a raw allergen extract. Nine patients (64%) had IgE to Der p 11 in a group of 14 HDM allergic patients. BIOLOGICAL SIGNIFICANCE: Our results add up to previous reports indicating that paramyosin is a clinically relevant HDM allergen and highlight that it can represent, in some patients, the first sensitizing component of this allergen source. This suggests that, at the moment, the use of allergen extract for the purpose of measuring IgE reactivity cannot be replaced by component resolved diagnosis and that group 11 allergens should be included among allergen components routinely tested in the clinical laboratory.

Selective increase in serum IgE following enfuvirtide administration in HIV-1 infected multidrug resistant patients.

Enfuvirtide (ENF) is the first HIV-1 entry inhibitor used in clinical practice and is currently administered via the subcutaneous route. We here evaluated whether ENF administration leads to a change in humoral parameters, including IgE, possibly related to ENF-associated injection site reactions (ISRs). A 24-week prospective study was conducted in multidrug resistant patients enrolled in the ENF Early Access Program characterized by CD4 counts < or =100 cells/microlitre and no other active antiretroviral drug. Licensed commercial laboratory assays were used to measure the parameters considered in this study. Results are reported as medians (interquartiles IQR). Statistical analyses were performed using Wilcoxon sign rank, Wilcoxon rank sum and Kruskall Wallis tests. Total IgM, IgA and IgG did not change significantly throughout the study. Conversely, a significant increase in IgE was observed in all patients, in those with normal as well as in those with altered IgE at baseline (BL). ISRs such as induration and number of nodules were more frequent in patients with altered BL IgE. IgE increased significantly in all patients, regardless of the different stratifications in their BL CD4 counts. Of note, the ENF-induced increase in CD4 occurred significantly in all patients, independently of their BL IgE levels. The immunological response associated with ENF treatment is accompanied by a selective increase in IgE levels. Determination of IgE could be used in the monitoring ISRs associated with ENE

Soothing and anti-itch effect of quercetin phytosome in human subjects: a single-blind study.

BACKGROUND: We evaluated the ability of quercetin, a natural antioxidant formulated in a specific delivery system, to reduce skin inflammation induced by a variety of stimuli, including UV radiation, stimulation with a histamine solution, or contact with chemical irritants. In particular, we tested the soothing and anti-itch effect of Quercevita(®), 1% cream for external use, a formulation characterized by a phospholipids-based delivery system. PATIENTS AND METHODS: The study was a monocentric, single blind trial that enrolled a group of 30 healthy volunteers. The back of each subject was examined to identify four quadrants with no previous skin damage or naevi that were treated in order to induce a controlled and reversible form of skin stress. The areas were treated as follows: no product; Quercevita(®) 1% cream, 2 mg/cm(2); placebo; positive control (a commercially available topical formulation containing 1% dexchlorpheniramine). RESULTS: Only quercetin phospholipids 1% and dexchlorpheniramine 1% achieved a significant reduction in erythema with comparable results: (-10.05% [P=0.00329] for quercetin phospholipids 1% vs -14.05% [P=0.00046] for the positive control). Moreover, quercetin phospholipids 1% and dexchlorpheniramine 1% were both associated with a significant decrease in mean wheal diameter: (-13.25% and -12.23% for dexchlorpheniramine 1%, respectively). Similar findings were reported for the other tested parameters. CONCLUSION: Quercetin has a skin protective effect against damage caused by a variety of insults, including UV radiation, histamine, or contact with toxic chemical compounds. Indeed, quercetin is able to reduce redness, itching, and inflammation of damaged skin; it may also help restore skin barrier function, increasing hydration, and reducing water loss.