Establishment of fluorescence multi-flow cytometric immunoassay for the simultaneous quantitative detection of six allergen-sIgE antibodies.

BACKGROUND: The in vitro specific IgE (sIgE) assays now commonly used in clinical laboratories are not only time-consuming and expensive but also require many serum samples. To address these limitations, a novel fluorescent microsphere-based multiplex flow cytometric immunoassay was developed. This innovative assay enables rapid and simultaneous quantitative detection of multiple allergen-sIgE antibodies. OBJECTIVE: To establish a new method for the simultaneous quantitative detection of 6 allergen-sIgE antibodies based on fluorescence multiplex flow cytometry. METHODS: Six different encoded fluorescent microspheres were selected to covalently couple 6 allergens, and their antigen-coupling activities were verified. After optimizing the multiplexing procedure and reaction conditions, including the concentration of microspheres encapsulated by allergens, reaction temperature, and reaction time, standard curves were established to quantify the 6 allergen-sIgE, and their performance was evaluated according to clinical guidelines. RESULTS: The chosen analytical mode was optimized for the detection of the 6 allergens-sIgE for 70 minutes. The established coefficients of variation for multiplex flow cytometry reproducibility and intermediate precision were less than 10%. Linear regression analysis showed a highly significant quantitative correlation between the results of the multiple analyses of Dermatophagoides pteronyssinus, Dermatophagoides farinae, Artemisia, and cat hair allergens and ImmunoCAP (Thermo Fisher Scientific): the r2 values ranged from 0.85 to 0.97 (P < .0001). In addition, there was a high correlation between the results of the multiplex analysis of dog hair allergens and the capture enzyme-linked immunosorbent assay (r2 = 0.92, P < .0001). CONCLUSION: A high-throughput system called multiplex flow cytometry has been developed for the simultaneous detection of 6 inhalant allergens. The method has the advantage of being rapid and using less serum. Furthermore, it has the potential to be expanded to include other allergens and biologic agents.

PlexinD1 Deficiency in Lung Interstitial Macrophages Exacerbates House Dust Mite-Induced Allergic Asthma.

Interstitial macrophages (IMs) are key regulators of allergic inflammation. We previously showed that the absence of semaphorin 3E (Sema3E) exacerbates asthma features in both acute and chronic asthma models. However, it has not been studied whether Sema3E, via its receptor plexinD1, regulates IM function in allergic asthma. Therefore, we investigated the role of plexinD1 deficiency on IMs in allergic asthma. We found that the absence of plexinD1 in IMs increased airway hyperresponsiveness, airway leukocyte numbers, allergen-specific IgE, goblet cell hyperplasia, and Th2/Th17 cytokine response in the house dust mite (HDM)-induced allergic asthma model. Muc5ac, Muc5b, and α-SMA genes were increased in mice with Plxnd1-deficient IMs compared with wild-type mice. Furthermore, plexinD1-deficient bone marrow-derived macrophages displayed reduced IL-10 mRNA expression, at both the baseline and following HDM challenge, compared with their wild-type counterpart mice. Our data suggest that Sema3E/plexinD1 signaling in IMs is a critical pathway that modulates airway inflammation, airway resistance, and tissue remodeling in the HDM murine model of allergic asthma. Reduced IL-10 expression by plexinD1-deficient macrophages may account for these enhanced allergic asthma features.

Cut-off value of D. pteronyssinus specific IgE in double negative patients Der p 1 and Der p 2 and its clinical repercussion.

Accessibility to more precise diagnostic techniques such as component resolved diagnostics (CRD), provides us with an important advance in diagnostic aspects as well as treatment. The subject of this study aims to better understand the profiles of sensitization to Der p 1, Der p 2 and Der p 23 and to know to what extent their use could help us in optimizing the decision-making for their treatment with Specific Immunotherapy. Cross-sectional study of subjects older than 5 years, diagnosed with allergy to HDM using skin prick test and sIgE, with symptoms of rhinitis and/or asthma. Total and specific IgE was determined to D. pteronyssinus, nDer p 1, rDer p 2 and rDer p 23 using ImmunoCAP. 240 patients were recruited (97.1% rhinitis and 46.25% rhinitis and asthma). Four different phenotypes were observed: positive or negative for sIgE nDer p 1 and/or IgE rDer p 2. 17% of these patients sIgE were double negative for Der p 1 and Der p 2 (increasing with age and with significantly lower sIgE levels than the rest of the groups). Using ROC curves, value less than 2.18 KUA/L for D. pteronyssinus sIgE gave us a sensitivity and specificity of 0.882 and 0.985, respectively, to double negative IgE nDer p 1 and IgE rDer p 2 group. Despite positive SPT and sIgE to D. pteronyssinus, 17% of the studied population is IgE nDer p 1 and IgE rDer p 2 double negative, with a cut-off value of 2.18 KU/L, which is very relevant for taking of decisions in prescription of AIT. The double positive population sIgE nDer p 1 and IgE rDer p 2 is associated with asthma compared to the other groups and this does not seem to be influenced by IgE rDer p 23.

Indonesia-Based Study of the Clinical and Cost-Saving Benefits of Subcutaneous Allergen Immunotherapy for Children with Allergic Rhinitis in Private Practice.

BACKGROUND: Until now, the cost of allergy treatment in the insured public health care system and the non-insured self-financing private health care system in Indonesia has not been well documented and published, as well as the cost of allergy treatment with subcutaneous immunotherapy. OBJECTIVE: To evaluate the clinical and cost benefits of allergic rhinitis treatment in children with subcutaneous immunotherapy in a non-insured self-financing private health care system. METHODS: A retrospective cohort study conducted from 2015 until 2020 that compared the clinical improvement and health care costs over 18 months in newly diagnosed AR children who received SCIT versus matched AR control subjects who did not receive SCIT, with each group consisting of 1098 subjects. RESULTS: A decrease in sp-HDM-IgE level (kU/mL) from 20.5 + 8.75 kU/mL to 12.1 + 3.07 kU/mL was observed in the SCIT group. To reduce the symptom score of allergic rhinitis by 1.0 with SCIT, it costs IDR 21,753,062.7 per child, and for non-SCIT, it costs IDR 104,147,878.0 per child. Meanwhile, to reduce the medication score (MS) by 1.0 with SCIT, it costs IDR 17,024,138.8, while with non-SCIT, it costs IDR 104,147,878.0. Meanwhile, to lower combination symptoms and medication score (CSMS) by 1.0, with SCIT, it costs IDR 9,550,126.6, while with non-SCIT, it costs IDR 52,073,938.9. CONCLUSIONS: In conclusion, this first Indonesia-based study demonstrates substantial health care cost savings associated with SCIT for children with AR in an uninsured private health care system and provides strong evidence for the clinical benefits and cost-savings benefits of AR treatment in children.

The IgE Blocking Activity Induced by Dermatophagoides pteronyssinus Subcutaneous Immunotherapy Does Not Correlate with Specific IgA but with IgG4 in both Serum and Saliva.

BACKGROUND: The role of salivary-specific IgG4 and IgA in subcutaneous immunotherapy (SCIT) is not well defined. We aimed to investigate the change of IgG4 and IgA in both serum and saliva and their correlations with IgE-blocking-factor (IgE-BF) during SCIT. METHOD: 307 Dermatophagoides pteronyssinus (DP) allergic rhinitis and/or asthma patients were recruited for this study. 286 patients received DP-SCIT for 1 year. Twenty-one patients received only symptomatic treatment. DP-, Der p 1-, and Der p 2-specific IgE in serum, specific-IgG4 and Der p 2-specific IgA1 and IgA2 in both serum and saliva were measured at timepoints 0, 4, and 12 months during DP-SCIT. Correlation between salivary and serological IgG4, IgA, and their correlation with DP-specific IgE-BF measured in serum was evaluated. RESULTS: During DP-SCIT, the allergen-specific IgG4 in both saliva and serum increased and correlated significantly, the correlation becomes stronger over the treatment time. DP-specific IgE-BF significantly correlated with DP-specific IgG4 in serum (p < 0.0001) at different timepoints and in saliva at 12 months of SCIT (p < 0.01). No change in Der p 2-specific IgA during DP-SCIT was observed, and the IgA in serum did not correlate with IgA in saliva. There was no correlation between DP IgE-BF and Der p 2-specific IgA in serum or saliva. The control group did not exhibit significant changes in any antibody level measured. CONCLUSION: The IgE blocking activity induced by DP-SCIT treatment correlated with specific IgG4 and not IgA. The IgG4 in saliva correlates with serum IgG4 and can be an alternative immunological marker beyond 1 year of SCIT treatment.

Blockade of GITRL/GITR signaling pathway attenuates house dust mite-induced allergic asthma in mice through inhibition of MAPKs and NF-κB signaling.

GITRL/GITR signaling pathway plays an important role in allergy, inflammation, transplantation and autoimmunity. However, its role in asthma remains unclear. Thus, the present study aimed to investigate changes in this pathway and observe the therapeutic effect of its blocking on asthma. By using house dust mite-induced asthma model, changes of GITRL/GITR and its downstream molecules MAPKs (e.g., p38 MAPK, JNK and Erk) and NF-κB were observed. After that, GITRL in lung of mice was knocked down by recombinant adeno-associated virus to observe the impact on its downstream molecules and assess the therapeutic effect on asthma. These results showed that GITRL/GITR and its downstream molecules MAPKs/NF-κB were activated in asthmatic mice. This activation was suppressed after GITRL knockdown, and allergic airway inflammation and airway hyperresponsiveness were alleviated. These results demonstrate that GITRL/GITR-MAPKs/NF-κB signaling pathway participates in the pathogenesis of asthma. Blockade of GITRL/GITR signaling pathway exhibits protective effects in a mouse model of house dust mite-induced allergic asthma.

In vivo reprogramming of pathogenic lung TNFR2+ cDC2s by IFNß inhibits HDM-induced asthma.

Asthma is a common inflammatory lung disease with no known cure. Previously, we uncovered a lung TNFR2+ conventional DC2 subset (cDC2s) that induces regulatory T cells (Tregs) maintaining lung tolerance at steady state but promotes TH2 response during house dust mite (HDM)-induced asthma. Lung IFNß is essential for TNFR2+ cDC2s-mediated lung tolerance. Here, we showed that exogenous IFNß reprogrammed TH2-promoting pathogenic TNFR2+ cDC2s back to tolerogenic DCs, alleviating eosinophilic asthma and preventing asthma exacerbation. Mechanistically, inhaled IFNß, not IFNα, activated ERK2 signaling in pathogenic lung TNFR2+ cDC2s, leading to enhanced fatty acid oxidation (FAO) and lung Treg induction. Last, human IFNß reprogrammed pathogenic human lung TNFR2+ cDC2s from patients with emphysema ex vivo. Thus, we identified an IFNß-specific ERK2-FAO pathway that might be harnessed for DC therapy.

Designing an epitope vaccine against Dermatophagoides pteronyssinus: An in silico study.

The house dust mite, Dermatophagoides pteronyssinus, is a major source of the inhaled allergen Der p 1, which causes immunoglobulin E (IgE)-mediated hypersensitivity reactions manifesting in allergic diseases. To date, no drugs or vaccines effectively treat or prevent Der p 1 sensitization. We applied in silico immunoinformatics to design T-cell and B-cell epitopes that were specified and developed from the allergen Der p 1 of D. pteronyssinus. We identified the conserved epitope areas by predicting the accessibility and flexibility of B-cell epitopes, and the percentage of human leukocyte antigen representing T cells. Molecular docking using HADDOCK software indicated three optimal clusters: cluster 6 (z-score: -2.1), cluster 1 (z-score: -1.2), and cluster 3 (z-score: -0.6). The most negative Z-score was found in cluster 6, which represented three epitopes. The interaction between A chain proteins (IgE protein residues) and B chains (Der p 1 protein residues) exhibited a knowledge-based FADE and contact value >1, suggesting the best protein interactions occurred in the conserved area. Molecular dynamic simulation further predicted the stable nature of Der p 1 protein. The IQRDNGYQP region is the best candidate to be utilized as a D. pteronyssinus epitope vaccine, which could be used in the development of allergen-specific immunotherapy.

Asthmatic Eosinophils Promote Contractility and Migration of Airway Smooth Muscle Cells and Pulmonary Fibroblasts In Vitro.

Enhanced contractility and migration of airway smooth muscle cells (ASMC) and pulmonary fibroblasts (PF) are part of airway remodeling in asthma. Eosinophils are the central inflammatory cells that participate in airway inflammation. However, the role of asthmatic eosinophils in ASMC and PF contractility, migration, and differentiation to contractile phenotype has not yet been precisely described. A total of 38 individuals were included in this study: 13 steroid-free non-severe allergic asthma (AA) patients, 11 severe non-allergic eosinophilic asthma (SNEA) patients, and 14 healthy subjects (HS). For AA patients and HS groups, a bronchial allergen challenge with D. pteronyssinus was performed. Individual combined cell cultures were prepared from isolated peripheral blood eosinophils and immortalized ASMC or commercial PF cell lines separately. The migration of ASMC and PF was evaluated using wound healing assay and contractility using collagen gel assay. Gene expression of contractile apparatus proteins, COL1A1, COL5A1, and FN, in ASMC and PF was evaluated using qRT-PCR. We found that contractility and migration of ASMC and PF significantly increased after incubation with asthmatic eosinophils compared to HS eosinophils, p < 0.05, and SNEA eosinophils demonstrated the highest effect on contractility of ASMC and migration of both cell lines, p < 0.05. AA and SNEA eosinophils significantly increased gene expression of contractile apparatus proteins, COL1A1 and FN, in both cell lines, p < 0.05. Furthermore, the allergen-activated AA eosinophils significantly increased the contractility of ASMC, and migration and gene expression in ASMC and PF, p < 0.05. Thus, asthmatic eosinophils change ASMC and PF behavior by increasing their contractility and migration, contributing to airway remodeling.

Helminth infection induces non-functional sensitization to house dust mites.

BACKGROUND: IgE characterizes the humoral response of allergic sensitization but less is known about what modulates its function and why some patients present clinical symptoms for a given IgE level and others do not. An IgE response also occurs during helminth diseases, independently of allergic symptoms. This response could be a model of non-functional IgE. OBJECTIVE: To study the IgE response against environmental allergens induced during natural helminth infection. METHODS: In 28 non allergic subjects from the periphery of Ho Chi Minh city with (H+, n = 18) and without helminth infection (H-, n = 10), we measured IgE and IgG4 against several components of Dermatophagoïdes pteronyssinus (Dpt) and Ascaris (a marker of immunization against nematodes), and determined the IgE component sensitization profile using microarray ISAC biochips. The functional ability of IgE to induce degranulation of cultured mast cells was evaluated in the presence of Dpt. RESULTS: Non allergic H+ subjects exhibited higher levels of IgE against Dpt compared to H- subjects. Dpt IgE were not functional in vitro and did not recognize usual Dpt major allergens. IgE recognized other component allergens that belong to different protein families, and most were glycosylated. Depletion of IgE recognizing carbohydrate cross-reactive determinant (CCD) did not induce a reduction in Dpt IgE. The Dpt IgG4 were not significantly different. CONCLUSION: Helminth infections induced IgE against allergens such as Dpt and molecular components that belong to different sources as well as against CCD (such as ß-1,2-xylose and/or ⍺-1,3-fucose substituted N-glycans). Dpt IgE were not able to induce degranulation of mast cells and were not explained by sensitization to usual major allergens or N-glycans.

Tissue factor promotes airway pathological features through epithelial-mesenchymal transition of bronchial epithelial cells in mice with house dust mite-induced asthma.

It has recently been shown that expression levels of tissue factor (TF) are high in the serum and peripheral blood mononuclear cells of patients with asthma. However, whether TF impacts airway inflammation and remodelling in asthma remains unknown. The aim of this study was to investigate the effect of TF in asthma airway inflammation and remodelling using a house dust mite (HDM)-induced chronic asthma model and human bronchial epithelial (16HBE) cells. A chronic asthma model was constructed in BALB/c mice by the intranasal instillation of HDM. Mice were treated with short hairpin TF (shTF), and airway inflammation and remodelling features of asthma and epithelial-mesenchymal transition (EMT) were assessed. 16HBE cells were induced by transforming growth factor-ß1 (TGF-ß1) and HDM in the presence or absence of shTF; then, EMT markers and invasion and migration ability were determined. TF expression increased in the lung tissue and 16HBE cells when exposed to HDM. TF downregulation in the lung significantly reduced airway hyperresponsiveness, eosinophil inflammation, the EMT process, and levels of interleukin (IL)-4, IL-6, IL-13, and TGF-ß1 in bronchoalveolar lavage fluid of asthmatic mice. Moreover, TF downregulation inhibited migration and incursion and decreased the expression levels of fibronectin 1 and TGF-ß1, but increased the expression of E-cadherin in HDM- and TGF-ß1-stimulated 16HBE cells. These results demonstrated that TF promoted airway pathological features by enhancing the EMT of bronchial epithelial cells both in vitro and in mice with house dust mite-induced asthma.

RNA viruses in the house dust mite Dermatophagoides pteronyssinus, detection in environmental samples and in commercial allergen extracts used for in vivo diagnosis.

BACKGROUND: Allergy to house dust mites (HDM), the most important source of indoor allergens worldwide, is diagnosed and treated using natural extracts from cultures that can contain immunoactive components from the HDM microbiome, including mite-infecting viruses. This study aimed to contribute to the discovery and characterization of RNA viruses from Dermatophagoides pteronyssinus, followed by their detection in different mite-derived sources. METHODS: Viruses were assembled after in silico metatranscriptomic analysis of D. pteronyssinus RNA samples, visualized by electron microscopy, and RNA detected by direct RT-PCR or data mining. Mite culture performance was evaluated in vivo. RESULTS: Seven RNA viruses were identified in our laboratory stock colony. Picornavirus-like viral particles were detected in epithelial cells of the digestive system and in fecal pellets. Most of these viruses could be persistently transmitted to an inbred virus-free colony by inoculating fecal material from the stock colony. Upon viral infection, no significant effect could be seen on mite population growth. Transcriptomic screening confirmed the presence of homolog sequences to these viruses in independent laboratory stocks of D. pteronyssinus and in other Astigmata mites. Noteworthy, RNA from most of the viruses could be detected by RT-PCR on house dust samples, reference standards, and/or commercial diagnostic D. pteronyssinus extracts. CONCLUSIONS: Our results show that viral infections are common and widespread in D. pteronyssinus, both in natural and culture-based growth conditions. Potential effects on the mites themselves and consequences toward allergenicity in humans whether exposed naturally or after immunotherapy are discussed.

Association between Temporary Housing Habitation after the 2011 Japan Earthquake and Mite Allergen Sensitization and Asthma Development.

INTRODUCTION: We previously reported an increased prevalence of asthma in adults who lived in temporary housing after the 2011 Great East Japan Earthquake. The goal of the current study was to investigate changes in asthma prevalence and mite-specific immunoglobulin E (IgE) titers in temporary housing residents during 2014-2019. METHODS: By using the Global Initiative for Asthma guidelines, we diagnosed asthma in Ishinomaki city temporary housing residents aged 15 years or older. We then analyzed serum antigen-specific IgE levels to Dermatophagoides farinae (Der f), Dermatophagoides pteronyssinus (Der p), and Aspergillus fumigatus. RESULTS: The prevalence of asthma exceeded 20% across all age-groups throughout the study period. The proportion of study participants with a "positive" antigen-specific IgE titer (i.e., ≥0.35 IUA/mL) was higher in asthmatics than in nonasthmatics for Der f and Der p but not for Aspergillus fumigatus. Among residents ≥50 years old who were diagnosed with asthma, the percentage with a Der f-specific IgE titer ≥0.10 IUA/mL was higher than the proportion with ≥0.35 IUA/mL. Among study participants, asthma onset occurred before the earthquake, during residence in shelters or temporary housing, and (starting in 2016) after moving out of temporary housing. The Der p-specific IgE level was positively correlated with the duration of temporary housing (p < 0.05, r = 0.41) and inversely correlated with the time elapsed since moving out of temporary housing (p < 0.05, r = -0.35). CONCLUSION: Mite allergen sensitization was found in both asthmatic and nonasthmatic temporary housing residents after the 2011 Japan earthquake and tsunami; asthma developed even after subjects moved out of temporary housing.

Analysis of the long-term efficacy and safety of subcutaneous immunotherapy for atopic dermatitis.

Introduction: Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease characterized by severe pruritus and eczematous skin lesions. Subcutaneous immunotherapy (SCIT) refers to repeated contact with gradually increasing doses of allergen extracts, which improve patient tolerance to such allergens and controls, or reduces allergic symptoms. This study aimed to explore the long-term efficacy and safety of SCIT for patients with AD sensitized to house-dust mite (HDM). Methods: We conducted a retrospective analysis of 378 patients with HDM-sensitized AD. Among these patients, 164 received SCIT plus pharmacotherapy for 3 years (SCIT group) and the other 214 patients received only pharmacotherapy (non-SCIT group). The scoring atopic dermatitis (SCORAD) and pruritus visual analog scale (VAS) scores, laboratory test results, and adverse effects were recorded. Results: The SCORAD and pruritus VAS scores significantly decreased in the SCIT group. Also, the SCIT group showed higher reduction ratios of SCORAD and pruritus VAS scores than those observed in the non-SCIT group at 3 years after treatment initiation. The risk of development of new sensitization was higher in the non-SCIT group than in the SCIT group (relative risk 1.92 [95% confidence interval {CI}, 1.30-2.85]; p < 0.05). The eosinophil count of the participants significantly differed in the complete response (CR) group (p < 0.05) but not in the non-CR group (p = 0.098). However, the serum total immunoglobulin E value was not significantly reduced (p = 0.204). Of 8421 injections given to the patients, 231 injections (2.74%) showed adverse effects during the treatment period. Conclusion: Three years of SCIT can significantly reduce the severity and pruritus of moderate-to-severe AD with HDM sensitization. Patients who are multisensitized can also benefit from HDM SCIT. Patients can achieve long-term effects, such as prevention of neoallergen sensitization and inhibition of the allergy march.

Immunoglobulin G1 subclass responses can be used to detect specific allergy to the house dust mites Dermatophagoides farinae and Dermatophagoides pteronyssinus in atopic dogs.

BACKGROUND: In dogs with atopic dermatitis, intradermal testing (IDT) or allergen specific IgE serological testing are routinely employed to identify causative allergens. These allergens can then be used for allergen-specific immunotherapy and allergy management. The clinical relevance of this testing is affected by the source of allergen, and other biomarkers that are more related to specific allergens still need to be identified. The aim of this study was to investigate levels of specific IgE, total IgG, and IgG1 and IgG2 subclasses against the local house dust mites (HDM) Dermatophagoides farinae (DF) and D. pteronyssinus (DP) as biomarkers by using in-house ELISAs in healthy (n = 33) and atopic dogs (AD) (n = 44) that were either positive or negative by IDT to HDM. RESULTS: Being over 3 years of age was a risk factor for AD (Odds Ratio (OD) = 4.10, 95% Confidence interval (CI) 1.57-10.75, p = 0.0049), but there was no relation to IDT outcomes (OR = 0.9091, 95% CI 0.22-3.74, p = 1.00). High levels of all antibody isotypes (IgE, IgG, IgG1 and IgG2) against HDM were found in aged healthy dogs (> 3 years old). In AD, HDM-IgE and IgG1 levels were higher in dogs that were IDT positive to HDM than in IDT negative animals. Levels of IgE and IgG1 could be used to distinguish the specific allergens, whereas total IgG and IgG2 levels were not different between IDT-positive and IDT-negative AD. By the receiver operating characteristic curve at a false-positive rate = 0.10, both IgE and IgG1 showed better sensitivity than IgG and IgG2. Similar to IgE, serum IgG1 concentration was also relevant to IDT outcomes. CONCLUSIONS: Our in-house ELISAs coated with local HDM were useful for evaluating antibody levels, and we propose use of the HDM-specific IgG1 subclass as a biomarker to detect HDM specific allergens in AD, potentially together with an IgE based platform.

Aqueous intradermal low-dose house dust mite immunotherapy in tropical settings: a valid cost-effective approach for developing nations?

INTRODUCTION: Aqueous allergen injections, an effective and century-old technique, is considered a second-line approach in daily clinical practice. Inconveniences still surround conventional subcutaneous immunotherapy (SCIT) administration, such as a need for frequent injections, prolonged up-dosing schedules, elevated costs, and the unlikely possibility of a systemic reaction. The intradermal immunotherapy route (IDR) might favorably impact many of the aforementioned issues (Table 1). House dust mite (HDM) allergens are the main perennial sensitizers in the tropics, and as such, are solely employed in immunotherapy treatments. METHODS: We carried out a year-long real-life study in 25 perennial allergic rhinitis children, symptomatic on exposure to house dust, employing an intradermal low-dose allergen mix consisting of 50 ng of Dermatophagoides pteronyssinus/Dermatophagoides farinae and 120 ng of Blomia tropicalis, under a unique cost-wise protocol. Basal symptoms/signs and face Visual Analog Scale (fVAS) scores were recorded for 2 weeks and later compared with those registered throughout the 1-year treatment. Serum-specific IgG4 and IL-10 levels were employed in the assessment of the immune responses. RESULTS: Symptoms/signs and fVAS scores were significantly reduced from days 42 and 49, respectively, and remained so until treatment completion. Increases in specific IgG4's and IL-10 levels reflected significant immune responses. Injections were well tolerated and families reported improved health status (quality of life, QoL). CONCLUSIONS: A unique cost-effective immunotherapy alternative for deprived allergic communities in tropical settings is depicted; further research is needed.

Antibodies to Der p 1 and Der p 2 in allergic patients.

INTRODUCTION AND OBJECTIVES: Atopic individuals are characterized by increased IgE production and Th2 response if exposed to certain antigens. It is known that the mother transfers anti-mite antibodies to the fetus and newborn, IgG thru the placenta, and IgA thru breastfeeding, but it is not clear whether there is a protective mechanism mediated by them concerning the development of future allergies. This study aimed to compare the levels of IgA, IgG, and IgE antibodies specific to Der p 1 and Der p 2 between atopic and healthy individuals. METHODS: Serum samples of 98 patients and 44 healthy controls were subjected to quantification for specific IgE, IgG, and IgA antibodies against Der p 1 and Der p 2 by ImmunoCap® and ELISA, and subjected to statistical analysis as indicated. RESULTS: Atopic patients had higher serum levels of IgE, IgG, and IgA specific to Der p 1 and Der p 2. The correlation was more robust between IgE and IgG antibodies. CONCLUSIONS: Allergic patients produce higher levels of antibodies against Der p 1 and Der p 2 compared with healthy individuals. The mechanisms involved still require detailed studies.

IgE-binding residues analysis of the house dust mite allergen Der p 23.

House dust mites (HDMs) are one of the major causes of allergies in the world. The group 23 allergen, Der p 23, from Dermatophagoides pteronyssinus, is a major allergen amongst HDM-sensitized individuals. This study aims to determine the specific immunoglobulin E (sIgE) binding frequency and IgE-binding residues of recombinant Der p 23 (rDer p 23) allergen amongst a cohort of consecutive atopic individuals in a tropical region. We performed site-directed mutagenesis and carried out immuno-dot blot assays using 65 atopic sera. The immuno-dot blot assays results indicated that the two residues K44 and E46 which are located at the N-terminal region are the major IgE-binding residues. The rDerp-23 sIgE titers are strongly correlated to the number of IgE-binding residues for rDer p 23 (P < 0.001). Atopic individuals who were only sensitized to HDM have a significantly higher number of IgE-binding residues than the individuals who were polysensitized to HDM and other crude allergens (P < 0.05). Individuals with allergic multimorbidity and moderate-to-severe allergic rhinitis also have a higher number of IgE-binding residues compared to those with single allergic disease and mild allergic rhinitis. The results prompt us to hypothesize that the individuals who have a higher number of IgE-binding residues may face a bigger challenge to be treated through immunotherapy due to the complexity in designing an effective hypoallergen with a high number of IgE-binding residues. We propose that the development of a refined molecular diagnostic assay, which includes alanine substitution of surface-exposed residues could be a more precise diagnostic strategy to identify all the IgE-binding residues of a major allergen for an atopic individual and the development could be another new dimension in allergy diagnosis and allergen immunotherapy treatment.