Immune Polarization in Allergic Patients: Role of the Innate Immune System

Allergens come into contact with the immune system as components of a very diverse mixture. The most common sources are pollen grains, food, and waste. These sources contain a variety of immunomodulatory components that play a key role in the induction of allergic sensitization. The way allergen molecules bind to the cells of the immune system can determine the immune response. In order to better understand how allergic sensitization is triggered, we review the molecular mechanisms involved in the development of allergy and the role of immunomodulators in allergen recognition by innate cells.

Occupational allergic multiorgan disease induced by wheat flour

Bakers are repeatedly exposed to wheat flour (WF) and may develop sensitization and occupational rhinoconjunctivitis and/or asthma to WF allergens.1 Several wheat proteins have been identified as causative allergens of occupational respiratory allergy in bakery workers.1 Testing of IgE reactivity in patients with different clinical profiles of wheat allergy (food allergy, wheat-dependent exercise-induced anaphylaxis, and baker's asthma) to salt-soluble and salt-insoluble protein fractions from WF revealed a high degree of heterogeneity in the recognized allergens. However, mainly salt-soluble proteins (albumins, globulins) seem to be associated with baker's asthma, and prolamins (gliadins, glutenins) with wheat-dependent exercise-induced anaphylaxis, whereas both protein fractions reacted to IgE from food-allergic patients.1 Notwithstanding, gliadins have also been incriminated as causative allergens in baker's asthma.2 We report on a 31-year-old woman who had been exposed to WF practically since birth because her family owned a bakery housed in the same home where they lived. She moved from this house when she was 25 years, but she continued working every day in the family bakery. In the last 8 years she had suffered from work-related nasal and ocular symptoms such as itching, watery eyes, sneezing, nasal stuffiness, and rhinorrhea. These symptoms markedly improved when away from work and worsened at work. In the last 5 years, she had also experienced dysphagia with frequent choking, especially when ingesting meats or cephalopods, which had partially improved with omeprazole therapy. Two years before referral to our clinic, she began to have dry cough and breathlessness, which she also attributed to her work environment. Upper and lower respiratory tract symptoms increased when sifting the WF and making the dough. The patient did not experience gastrointestinal symptoms with ingestion of cereal products. Skin prick test results were positive to grass (mean wheal, 6 mm), cypress (5 mm) and Russian thistle pollen (4 mm), WF (4 mm), and peach lipid transfer protein (6 mm) and were negative to rice flour, corn flour, profilin, mites, molds, and animal dander. Skin prick test with a homemade WF extract (10% wt/vol) was strongly positive (15 mm). Serologic tests yielded the following results: eosinophil cationic protein, 47 ?g/L; total serum IgE, 74 kU/L; specific IgE (ImmunoCAP; ThermoFisher, Uppsala, Sweden) to WF, 7.4 kU/L; barley flour, 1.24 kU/L; and corn, gluten, alpha-amylase, peach, and apple, less than 0.35 kU/L. Specific IgE binding to microarrayed purified WF allergens (WDAI-0.19, WDAI-0.53, WTAI-CM1, WTAI-CM2, WTAI-CM3, WTAI-CM16, WTAI-CM17, Tri a 14, profilin, ?-5-gliadin, Tri a Bd 36 and Tri a TLP, and gliadin and glutamine fractions) was assessed as described elsewhere.3 The patient's serum specifically recognized ?-5-gliadin and the gliadin fraction, and no IgE reactivity was observed to other wheat allergens. Spirometry revealed a forced vital capacity of 3.88 L (88%), an FEV1 of 3.04 L (87%), and FEV1/forced vital capacity of 83%. A methacholine inhalation test was performed following an abbreviated protocol,4 and the results were expressed as PD20 in cumulative dose (mg) of methacholine. Methacholine inhalation challenge test result was positive (0.24 mg cumulative dose) when she was working, and after a 3-month period away from work and with no visits to the bakery house, it gave a negative result. A chest x-ray was normal. Specific inhalation challenge test was carried out in the hospital laboratory by tipping WF from one tray to another for 15 minutes. Spirometry was performed at baseline and at 2, 5, 10, 15, 20, 30, 45, and 60 minutes after the challenge with WF. Peak expiratory flow was measured at baseline and then hourly over 24 hours (respecting sleeping time). A 12% fall in FEV1 was observed at 20 minutes and a 26% drop in peak expiratory flow at 9 hours after exposure to WF,

Is the performance of ImmunoCAP ISAC 112 sufficient to diagnose peach and apple allergies?

Food allergies constitute a public health issue, with a reported overall estimated prevalence of 6% in Europe1 and Rosacea as the main allergenic fruits among adults.2 The commercial microarray ImmunoCAP ISAC 112 (Thermofisher, Uppsala, Sweden) is a semiquantitative and reproducible in vitro diagnostic tool used for the determination of specific IgE (sIgE).3 However, its panel of allergens does not have the best accuracy when it comes to determining fruit allergies in the Mediterranean area: the inclusion of the thaumatinlike protein (TLP) Pru p 2 or the apple lipid transfer protein (LTP) Mal d 3 has been proposed to improve the diagnosis of peach4 and apple5 allergies, respectively, in the Mediterranean basin. We sought to determine the usefulness of a component-resolved microarray for the diagnosis of peach and apple allergies in the Mediterranean area.

Challenges for Allergy Diagnosis in Regions with Complex Pollen Exposures

Over the past few decades, significant scientific progress has influenced clinical allergy practice. The biological standardization of extracts was followed by the massive identification and characterization of new allergens and their progressive use as diagnostic tools including allergen micro arrays that facilitate the simultaneous testing of more than 100 allergen components. Specific diagnosis is the basis of allergy practice and is always aiming to select the best therapeutic or avoidance intervention. As a consequence, redundant or irrelevant information might be adding unnecessary cost and complexity to daily clinical practice. A rational use of the different diagnostic alternatives would allow a significant improvement in the diagnosis and treatment of allergic patients, especially for those residing in complex pollen exposure areas.

Thioredoxin-linked mitigation of allergic responses to wheat

Thioredoxin, a ubiquitous 12-kDa regulatory disulfide protein, was found to reduce disulfide bonds of allergens (convert S—S to 2 SH) and thereby mitigate the allergenicity of commercial wheat preparations. Allergenic strength was determined by skin tests with a canine model for food allergy. Statistically significant mitigation was observed with 15 of 16 wheat-sensitive animals. The allergenicity of the protein fractions extracted from wheat flour with the indicated solvent was also assessed: the gliadins (ethanol) were the strongest allergens, followed by glutenins (acetic acid), albumins (water), and globulins (salt water). Of the gliadins, the α and β fractions were most potent, followed by the γ and ω types. Thioredoxin mitigated the allergenicity associated with the major protein fractions—i.e, the gliadins (including the α, β, and γ types) and the glutenins—but gave less consistent results with the minor fractions, the albumins and globulins. In all cases, mitigation was specific to thioredoxin that had been reduced either enzymically by NADPH and NADP–thioredoxin reductase or chemically by dithiothreitol; reduced glutathione was without significant effect. As in previous studies, thioredoxin was particularly effective in the reduction of intramolecular (intrachain) disulfide bonds. The present results demonstrate that the reduction of these disulfide bonds is accompanied by a statistically significant decrease in allergenicity of the active proteins. This decrease occurs alongside the changes identified previously—i.e., increased susceptibility to proteolysis and heat, and altered biochemical activity. The findings open the door to the testing of the thioredoxin system in the production of hypoallergenic, more-digestible foods.

A strategy for the identification of proteins targeted by thioredoxin

Thioredoxins are 12-kDa proteins functional in the regulation of cellular processes throughout the animal, plant, and microbial kingdoms. Growing evidence with seeds suggests that an h-type of thioredoxin, reduced by NADPH via NADP-thioredoxin reductase, reduces disulfide bonds of target proteins and thereby acts as a wakeup call in germination. A better understanding of the role of thioredoxin in seeds as well as other systems could be achieved if more were known about the target proteins. To this end, we have devised a strategy for the comprehensive identification of proteins targeted by thioredoxin. Tissue extracts incubated with reduced thioredoxin are treated with a fluorescent probe (monobromobimane) to label sulfhydryl groups. The newly labeled proteins are isolated by conventional two-dimensional electrophoresis: (i) nonreducing/reducing or (ii) isoelectric focusing/reducing SDS/PAGE. The isolated proteins are identified by amino acid sequencing. Each electrophoresis system offers an advantage: the first method reveals the specificity of thioredoxin in the reduction of intramolecular vs. intermolecular disulfide bonds, whereas the second method improves the separation of the labeled proteins. By application of both methods to peanut seed extracts, we isolated at least 20 thioredoxin targets and identified 5—three allergens (Ara h2, Ara h3, and Ara h6) and two proteins not known to occur in peanut (desiccation-related and seed maturation protein). These findings open the door to the identification of proteins targeted by thioredoxin in a wide range of systems, thereby enhancing our understanding of its function and extending its technological and medical applications.

Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization.

We compared the antigen-specific antibody isotypes and lymphokine secretion by CD4+ T cells in BALB/c mice immunized intradermally with either Escherichia coli beta-galactosidase (beta-gal) or plasmid DNA (pDNA) encoding beta-gal in a cytomegalovirus-based expression vector (pCMV-LacZ). pCMV-LacZ induced mainly IgG2a, whereas beta-gal in saline or alum induced IgG1 and IgE beta-gal-specific antibodies. In addition, splenic CD4+ T helper (Th) cells isolated from pDNA-immunized mice secreted interferon-gamma but not interleukin (IL)-4 and IL-5, whereas Th cells from beta-gal-injected mice secreted IL-4 and IL-5 but not interferon-gamma after in vitro stimulation with antigen. Together these data demonstrate that pDNA immunization induced a T helper type 1 (Th1) response, whereas protein immunization induced a T helper type 2 (Th2) response to the same antigen. Interestingly, priming of mice with pCMV-LacZ prevented IgE antibody formation to a subsequent i.p. beta-gal in alum injection. This effect was antigen-specific, because priming with pCMV-LacZ did not inhibit IgE anti-ovalbumin antibody formation. Most importantly, intradermal immunization with pCMV-LacZ (but not pCMV-OVA) of beta-gal in alum-primed mice caused a 66-75% reduction of the IgE anti-beta-gal titer in 6 weeks. Also, pCMV-LacZ induced specific IgG2a antibody titers and interferon-gamma secretion by Th cells in the beta-gal in alum-primed mice. The data demonstrate that gene immunization induces a Th1 response that dominates over an ongoing protein-induced Th2 response in an antigen-specific manner. This suggests that immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.

Sensibilização ao látex em pacientes com mielomeningocele na urologia do HC-FMUSP: prevalência e fatores associados

INTRODUÇÃO: A alergia ao látex é um importante problema de saúde pública, especialmente em grupos de risco que têm contato frequente com este potente alérgeno. Este estudo estimou a prevalência e os fatores de risco para sensibilização ao látex em pacientes com mielomeningocele (MMC) submetidos a procedimentos cirúrgicos urológicos no HC-FMUSP. MÉTODOS: Foram selecionados pacientes com MMC submetidos a pelo menos uma cirurgia urológica, entre 2009 e 2014.Todos foram entrevistados e seus prontuários revisados. Uma amostra de sangue permitiu que a IgE específica ao látex, a K82, e seus recombinantes fossem investigados pelo método lmmunoCAP100 (kUa/L -1). A associação entre a exposição e o desfecho foi avaliada por meio de regressão logística de Poisson, Quiquadrado ou o teste exato de Fischer, para variáveis categóricas. O teste t de Student foi utilizado para comparar variáveis contínuas (nível de significância de 5%). Foram calculados a razão de prevalência (RP) e o intervalo de confiança de 95%. RESULTADOS: Foram identificados Duzentos e doze pacientes (51% do sexo masculino, 20,4 ± 6,4 anos de idade), 68 foram submetidos a pelo menos um procedimento urológico e 51 aceitaram participar (87,9%). Vinte e nove pacientes foram considerados não-sensibilizados (IgE específica para o látex :: a 0,7 kUa/L) e 22 sensibilizados ao látex com IgE > 0,7 kUa/L. Quando comparados os dois grupos, o sensibilizado apresentou um número de procedimentos cirúrgicos maior (11,6 ± 5,9 vs 7,2 ± 5,6) e dentre eles 48,3% apresentaram alguma alergia anterior contra 27,6% no grupo não sensibilizado. A sensibilização ao látex foi independentemente associada com alergia a produtos de látex (p = 0,014) e com o número de cirurgias anteriores (p = 0,032). A alergia ao látex tinha uma razão de prevalência de 2,87 (95% Cl: 1,24 a 6,65) ajustado para o número de cirurgias. Para cada procedimento cirúrgico, ajustado à alergia a produtos que contém látex, aumentou o risco para sensibilização em 4% (PR = 1,04; 95% CI: 1,00-1,09). CONCLUSÕES: A história de alergia ao látex e o número de cirurgias foram fatores de risco independentes para sensibilização ao látex

Importância clínica do estudo molecular de alergénicos alimentares

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Modelos de regressão para identificação de marcadores preditivos de asma na descendência de mulheres com atopia

Trabalho de projecto de mestrado, Bioestatística, Universidade de Lisboa, Faculdade de Ciências, 2016

Multiple regulatory variants located in cell type-specific enhancers within the PKP2 locus form major risk and protective haplotypes for canine atopic dermatitis in German shepherd dogs.

BACKGROUND Canine atopic dermatitis (CAD) is a chronic inflammatory skin disease triggered by allergic reactions involving IgE antibodies directed towards environmental allergens. We previously identified a ~1.5 Mb locus on canine chromosome 27 associated with CAD in German shepherd dogs (GSDs). Fine-mapping indicated association closest to the PKP2 gene encoding plakophilin 2. RESULTS Additional genotyping and association analyses in GSDs combined with control dogs from five breeds with low-risk for CAD revealed the top SNP 27:19,086,778 (p = 1.4 × 10(-7)) and a rare ~48 kb risk haplotype overlapping the PKP2 gene and shared only with other high-risk CAD breeds. We selected altogether nine SNPs (four top-associated in GSDs and five within the ~48 kb risk haplotype) that spanned ~280 kb forming one risk haplotype carried by 35 % of the GSD cases and 10 % of the GSD controls (OR = 5.1, p = 5.9 × 10(-5)), and another haplotype present in 85 % of the GSD cases and 98 % of the GSD controls and conferring a protective effect against CAD in GSDs (OR = 0.14, p = 0.0032). Eight of these SNPs were analyzed for transcriptional regulation using reporter assays where all tested regions exerted regulatory effects on transcription in epithelial and/or immune cell lines, and seven SNPs showed allelic differences. The DNA fragment with the top-associated SNP 27:19,086,778 displayed the highest activity in keratinocytes with 11-fold induction of transcription by the risk allele versus 8-fold by the control allele (pdifference = 0.003), and also mapped close (~3 kb) to an ENCODE skin-specific enhancer region. CONCLUSIONS Our experiments indicate that multiple CAD-associated genetic variants located in cell type-specific enhancers are involved in gene regulation in different cells and tissues. No single causative variant alone, but rather multiple variants combined in a risk haplotype likely contribute to an altered expression of the PKP2 gene, and possibly nearby genes, in immune and epithelial cells, and predispose GSDs to CAD.

Variance components analyses of multiple asthma traits in a large sample of Australian families ascertained through a twin proband

Background: Intermediate phenotypes are often measured as a proxy for asthma. It is largely unclear to what extent the same set of environmental or genetic factors regulate these traits. Objective: Estimate the environmental and genetic correlations between self-reported and clinical asthma traits. Methods: A total of 3073 subjects from 802 families were ascertained through a twin proband. Traits measured included self-reported asthma, airway histamine responsiveness (AHR), skin prick response to common allergens including house dust mite (Dermatophagoides pteronyssinus [D. pter]), baseline lung function, total serum immunoglobulin E (IgE) and eosinophilia. Bivariate and multivariate analyses of eight traits were performed with adjustment for ascertainment and significant covariates. Results: Overall 2716 participants completed an asthma questionnaire and 2087 were clinically tested, including 1289 self-reported asthmatics (92% previously diagnosed by a doctor). Asthma, AHR, markers of allergic sensitization and eosinophilia had significant environmental correlations with each other (range: 0.23-0.89). Baseline forced expiratory volume in 1 s (FEV1) showed low environmental correlations with most traits. Fewer genetic correlations were significantly different from zero. Phenotypes with greatest genetic similarity were asthma and atopy (0.46), IgE and eosinophilia (0.44), AHR and D. pter (0.43) and AHR and airway obstruction (-0.43). Traits with greatest genetic dissimilarity were FEV1 and atopy (0.05), airway obstruction and IgE (0.07) and FEV1 and D. pter (0.11). Conclusion: These results suggest that the same set of environmental factors regulates the variation of many asthma traits. In addition, although most traits are regulated to great extent by specific genetic factors, there is still some degree of genetic overlap that could be exploited by multivariate linkage approaches.

Scabies mite inactivated serine protease paralogues are present both internally in the mite gut and externally in feces

The scabies mite, Sarcoptes scabiei, is the causative agent of scabies, a disease that is common among disadvantaged populations and facilitates streptococcal infections with serious sequelae. Previously, we encountered large families of genes encoding paralogues of house dust mite protease allergens with their catalytic sites inactivated by mutation (scabies mite inactivated protease paralogues [SMIPPs]). We postulated that SMIPPs have evolved as an adaptation to the parasitic lifestyle of the scabies mite, functioning as competitive inhibitors of proteases involved in the host–parasite interaction. To propose testable hypotheses for their functions, it is essential to know their locations in the mite. Here we show by immunohistochemistry that SMIPPs exist in two compartments: 1) internal to the mite in the gut and 2) external to the mite after excretion from the gut in scybala (fecal pellets). SMIPPs may well function in both of these compartments to evade host proteases.

Allergy - 5: Allergy and the skin: eczema and chronic urticaria

Eczema is common, occurring in 15%-20% of infants and young children. For some infants it can be a severe chronic illness with a major impact on the child's general health and on the family. A minority of children will continue to have eczema as adults. The exact cause of eczema is not clear, but precipitating or aggravating factors may include food allergens (most commonly, egg) or environmental allergens/irritants, climatic conditions, stress. and genetic predisposition. Management of eczema consists of education; avoidance of triggers and allergens; liberal use of emollients or topical steroids to control inflammation; use of antihistamines to reduce itch; and treatment of infection if present. Treatment with systemic agents may be required in severe cases, but must be supervised by an immunologist. Urticaria (hives) may affect up to a quarter of people at some time in their lives. Acute urticaria is more common in children, while chronic urticaria is more common in adults. Chronic urticaria is not life-threatening, but the associated pruritus and unsightly weals can cause patients much distress and significantly affect their daily lives. Angioedema coexists with urticaria in about 50% of patients. It typically affects the lips, eyelids, palms, soles and genitalia. Management of urticaria is through education; avoidance of triggers and allergens (where relevant); use of antihistamines to reduce itch; and short-term use of corticosteroids when antihistamine therapy is ineffective. Referral is indicated for patients with resistant disease.

Monitoring inflammation and airway remodeling by FMT in a chronic asthma model

Asthma is a multifactorial disease for which a variety of mouse models have been developed. A major drawback of these models is represented by the transient nature of the airway pathology peaking 24 to 72 hours after challenge and resolving in 1 to 2 weeks. The objective of this study is to characterize the temporal evolution of pulmonary inflammation and remodeling in a recently described mouse model of chronic asthma (8 week treatment with 3 allergens relevant for the human pathology: Dust mite, Ragweed, and Aspergillus; DRA). We studied the DRA model taking advantage of fluorescence molecular tomography (FMT) imaging using near-infrared probes to non-invasively evaluate lung inflammation and airway remodeling. At 4, 6, 8 or 11 weeks, cathepsin- and metalloproteinase-dependent fluorescence was evaluated in vivo. A subgroup of animals, after 4 weeks of DRA, was treated with Budesonide (100 µg/kg intranasally) daily for 4 weeks. Cathepsin-dependent fluorescence in DRA-sensitized mice resulted significantly increased at 6 and 8 weeks, and was markedly inhibited by budesonide. This fluorescent signal well correlated with ex vivo analysis such as bronchoalveolar lavage eosinophils and alveolar cell infiltration. Metalloproteinase-dependent fluorescence was significantly increased at 8 and 11 weeks, nicely correlated with collagen deposition, as evaluated histologically by Masson’s Trichrome staining, and airway epithelium hypertrophy, and was also partly inhibited by budesonide. In conclusion, FMT proved suitable for longitudinal study to evaluate asthma progression, both in terms of inflammatory cell infiltration and airway remodeling, allowing the determination of treatment efficacy in a chronic asthma model in mice.