Association between skin prick test and serum specific immunoglobulin E to American cockroach allergens in allergic rhinitis patients.

INTRODUCTION AND OBJECTIVES: American cockroach is a common aeroallergen sensitization in allergic rhinitis (AR) patients. Association between skin prick test (SPT) and specific immunoglobulin E (sIgE) to American cockroach allergen remains uncertain. This study aimed to evaluate the association between SPT and sIgE to American cockroach allergen in patients with AR. MATERIALS AND METHODS: This cross-sectional study was conducted in Thai AR patients aged 6-25 years from September 2013 to October 2014. SPT and sIgE to American cockroach allergen were performed and the correlation was calculated using SPSS Statistics version 18. RESULTS: Sixty-seven AR patients, with median age of 15 years were included in this study. SPT and sIgE to American cockroach allergen showed a positive result in 68.7% and 58.2% cases, respectively. Positive SPT or positive sIgE to American cockroach was found in 79.1%. Thirty-two patients (47.8%) tested positive for both SPT and sIgE to American cockroach allergen. Fourteen from a total of 67 cases (20.9%) with negative sIgE had positive SPT to American cockroach, while seven cases (10.4%) with negative SPT had positive sIgE to American cockroach. Moderate correlation was observed between mean wheal diameter (MWD) and sIgE level to American cockroach (r=0.465, p=0.001). No significant correlation was found between MWD of SPT or sIgE level to American cockroach and AR severity. CONCLUSION: A moderate correlation was observed between MWD of SPT and sIgE level to American cockroach. If SPT is negative in allergic rhinitis patients highly suspected of having American cockroach allergy, serum sIgE should be considered and vice versa.

Concordance of skin prick test and serum-specific IgE to locally produced component-resolved diagnostics for cockroach allergy.

BACKGROUND: Diagnosis of Periplaneta americana (American cockroach, ACR) allergy is commonly performed based on clinical history and skin prick test (SPT) or specific serum IgE (sIgE) measurement. The concordance of the findings with the SPT and sIgE results has never been investigated. OBJECTIVE: To compare the results of SPT with commercial ACR-extract (C-ACE) and sIgE measurement, using commercial kit and in-house enzyme-linked immunosorbent assay (ELISA) to the locally produced ACR extract (L-ACE) and native Per a 1, Per a 5, Per a 7, and Per a 9. METHODS: Sera from 66 individuals clinically diagnosed with chronic allergic rhinitis were included; 46 were positive SPT to C-ACE, and 20 were negative. Specific serum IgE levels were established by using a commercial test kit (ImmunoCap) and an in-house IgE-ELISA RESULTS: The percentage the C-ACE SPT-positive cases that were positive by the ImmunoCap-sIgE was 32.6%, indicating low concordance of the 2 assays. With the in-house ELISA, Per a 9 gave the highest sensitivity (98.00%), positive predictive value (PPV; 95.74%), and negative predictive value (NPV; 94.74%) of the sIgE quantification. The correlation coefficients (R) of the L-ACE-SPT and sIgE to L-ACE, Per a 1, Per a 5, Per a 7, and Per a 9 and ImmunoCap sIgE were 0.133, 0.278, 0.419, 0.280, and 0.432, and 0.256, respectively. CONCLUSION: Skin prick test and sIgE measurement using commercial reagents have low concordance. Data of this study showed that sIgE to the native Per a 9 should be considered as an adjunct to the clinical history in diagnosis of ACR sensitization/allergy, particularly when the SPT and the nasal challenge, which is the gold standard method, cannot be performed.

Serine protease allergen favours Th2 responses via PAR-2 and STAT3 activation in murine model.

BACKGROUND: Protease activity of Per a 10 favours Th2 responses by differential regulation of IL-12p70 and IL-23 cytokine subunits. This study aimed to elucidate the underlying mechanism of differential regulation of IL-12p70 and IL-23. METHODS: PAR-2 activation was blocked in murine model by administering SAM11 before each sensitization. CD11c+ p-STAT3+ cells were measured in lungs by flow cytometry. BMDCs were pretreated with SAM11 or isotype control or stattic and stimulated with Per a 10. p-STAT3 levels were measured using Western blot. Transcript levels of IL-12p35, IL-12/23p40 and IL-23p19 were measured using RT-PCR. Cytokine levels were analysed using ELISA. RESULTS: Protease activity of Per a 10 increased p-STAT3 levels in mouse lungs, which was reduced upon PAR-2 blockage. Percentage of p-STAT3+ CD11c+ cells was higher in Per a 10-administered mice and was reduced upon PAR-2 blockage. IL-12p35 and IL-12p70 levels were higher, and IL-23p19 and IL-23 levels were lower in both SAM11-treated mice and BMDCs indicating a role of PAR-2-mediated signalling. IL-4, TSLP, IL-17A, EPO activity, total cell count and specific IgE and IgG1 levels were lower in SAM11-administered mice. Inhibiting STAT3 activation via stattic also leads to lower levels of IL-23p19 and IL-23 and higher levels of IL-12p35. CONCLUSIONS: Per a 10 leads to PAR-2 activation on BMDCs resulting in downstream activation of STAT3 to regulate the balance between IL-12/IL-23 subunits causing a cytokine milieu rich in IL-23 to favour Th2 polarization.

Protease activity of Per a 10 potentiates Th2 polarization by increasing IL-23 and OX40L.

Proteases are implicated in exacerbation of allergic diseases. In this study, the role of proteolytic activity of Per a 10 was evaluated on Th2 polarization. Intranasal administration of Per a 10 in mice led to allergic airway inflammation as seen by higher IgE levels, cellular infiltration, IL-17A, and Th2 cytokines, whereas, inactive (Δ)Per a 10 showed attenuated response. There was an increased OX40L expression on lung and lymph node dendritic cells in Per a 10 immunized group and on Per a 10 stimulated BMDCs. Reduction in CD40 expression without any change at transcript level in lungs of Per a 10 immunized mice suggested CD40 cleavage. BMDCs pulsed with Per a 10 showed reduced CD40 expression with lower IL-12p70 secretion as compared to heat inactivated Per a 10. IL-23, TNF-α, and IL-6 levels were significantly higher in Per a 10 stimulated BMDCs supernatant. In DC-T cell coculture studies, Per a 10 pulsed BMDCs showed higher levels of IL-4 and IL-13 that were reduced on blocking of either IL-23 or OX40L. In conclusion, the data suggests a critical role of protease activity of Per a 10 in promoting Th2 polarization by increasing IL-23 secretion and OX40L expression on dendritic cells.

Per a 10 protease activity modulates CD40 expression on dendritic cell surface by nuclear factor-kappaB pathway.

Serine protease activity of Per a 10 from Periplaneta americana modulates dendritic cell (DC) functions by a mechanism(s) that remains unclear. In the present study, Per a 10 protease activity on CD40 expression and downstream signalling was evaluated in DCs. Monocyte-derived DCs from cockroach-allergic patients were treated with proteolytically active/heat-inactivated Per a 10. Stimulation with active Per a 10 demonstrated low CD40 expression on DCs surface (P < 0·05), while enhanced soluble CD40 level in the culture supernatant (P < 0·05) compared to the heat-inactivated Per a 10, suggesting cleavage of CD40. Per a 10 activity reduced the interleukin (IL)-12 and interferon (IFN)-γ secretion by DCs (P < 0·05) compared to heat-inactivated Per a 10, indicating that low CD40 expression is associated with low levels of IL-12 secretion. Active Per a 10 stimulation caused low nuclear factor-kappa B (NF-κB) activation in DCs compared to heat-inactivated Per a 10. Inhibition of the NF-κB pathway suppressed the CD40 expression and IL-12 secretion by DCs, further indicating that NF-κB is required for CD40 up-regulation. CD40 expression activated the tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), thereby suggesting its involvement in NF-κB activation. Protease activity of Per a 10 induced p38 mitogen-activated protein kinase (MAPK) activation that showed no significant effect on CD40 expression by DCs. However, inhibiting p38 MAPK or NF-κB suppressed the secretion of IL-12, IFN-γ, IL-6 and TNF-α by DCs. Such DCs further reduced the secretion of IL-4, IL-6, IL-12 and TNF-α by CD4(+) T cells. In conclusion, protease activity of Per a 10 reduces CD40 expression on DCs. CD40 down-regulation leads to low NF-κB levels, thereby modulating DC-mediated immune responses.

Two new types of allergens from the cockroach, Periplaneta americana.

Periplaneta americana cockroach is an important source of inhalant indoor allergen resource, and there are more than twenty IgE-binding components identified in P. americana, but only nine allergens were characterized. Our knowledge about cockroach allergens remains poor. In this work, two novel allergen proteins Per a 11 (alpha-amylase) and Per a 12 (chitinase) with molecular weight around 55 and 45 kDa, respectively, were purified and characterized from the midgut of cockroaches. Their primary sequences were determined by Edman degradation, mass spectrometry, and cDNA cloning. Sera from 39 and 30 of 47 (83.0% and 63.8%) patients reacted to Per a 11 and Per a 12 on immunoblots, respectively. The allergenicity of Per a 11 and Per a 12 was further confirmed by competitive ELISA, basophil activation test (BAT), and skin prick test (SPT). They appear to be of importance for the allergic reactions induced by cockroach and have a potential for component-based diagnosis of allergy.

Mixing compatibilities of Aspergillus and American cockroach allergens with other high-protease fungal and insect extracts.

BACKGROUND: Recent studies have shown that Alternaria and German cockroach allergens can be degraded by endogenous proteases from other insect and fungal extracts when combined for immunotherapy, but data supporting the compatibilities of other high-protease products in comparable mixtures have not been reported. OBJECTIVE: To assess the stabilities and compatibilities of Aspergillus fumigatus and American cockroach allergens after mixing with protease-rich extracts from other insects or fungi at concentrations similar to those recommended for subcutaneous immunotherapy. METHODS: Mixtures containing A fumigatus, American cockroach, and other fungal or insect extracts were evaluated by quantitative (enzyme-linked immunosorbent assays) and qualitative (immunoblotting) methods. Test mixtures and control samples at 10% to 50% glycerin concentrations were analyzed after storage for up to 12 months at 2°C to 8°C. RESULTS: Moderate to high recoveries of Aspergillus extract activities were retained in control samples and extract mixtures under all conditions examined. American cockroach extract controls were partly degraded at 10% to 25% glycerin, and cockroach allergen compatibilities were decreased significantly in mixtures with several fungal extracts at 25% glycerin. Mixing with other insects did not compromise the stability of American cockroach allergens at 25% to 50% glycerin. CONCLUSION: Aspergillus extracts exhibited favorable stabilities after mixing with other high-protease products. American cockroach extract potencies were unstable in less than 50% glycerin, even in the absence of other protease-containing allergens, and were destabilized in mixtures with several fungal extracts. Addition of fungal and insect extracts to separate treatment vials or preparation of fungal-insect mixtures at elevated glycerin concentrations might be necessary to produce compatible patient formulations for allergen immunotherapy injections.

Bla g 3: a novel allergen of German cockroach identified using cockroach-specific avian single-chain variable fragment antibody.

BACKGROUND: The IgE response to cockroach allergens is thought to be associated with asthma. German cockroach (GCr) allergen extract is a complex mixture of allergens, and the identification and characterization of immunodominant allergens is important for the effective diagnosis and treatment of GCr-induced asthma. OBJECTIVE: To characterize a novel GCr allergen homologous to the American cockroach allergen Per a 3. METHODS: GCr-specific avian monoclonal antibodies were used for direct immunoprecipitation of specific targets from whole-body GCr extract. Precipitated protein was identified by mass spectrometry and sequence analysis. Putative recombinant protein also was expressed, purified, and used for determination of allergenicity, determined by IgE enzyme-linked immunosorbent assay with serum from 61 GCr-allergic patients. The identified target also was analyzed for heat stability using a bead-based assay. RESULTS: The immunoprecipitated target of monoclonal antibody 2A1 was identified as a novel allergen of GCr homologous to American cockroach allergen Per a 3. This homolog, designated Bla g 3, has an apparent mass of 78 kDa, can be measured in GCr extract using antibody 2A1, and is a heat-stable protein. Screening of 61 serum samples from GCr-allergic patients showed a 22% prevalence of Bla g 3-specific IgE. CONCLUSION: Bla g 3 is a GCr allergen with structural homology to American cockroach allergen Per a 3.

Efficacy of recombinant allergens for diagnosis of cockroach allergy in patients with asthma and/or rhinitis.

BACKGROUND: Immunoglobulin E (IgE) reactivity to individual allergens among cockroach-allergic patients has revealed wide variability. The aim of this study was to assess the effectiveness of recombinant cockroach allergens for skin testing, and to determine sensitization profiles among cockroach-allergic patients living in Brazil. METHODS: Fifty-seven cockroach-allergic patients with asthma and/or rhinitis were recruited. Skin testing with recombinant (r) allergens from Periplaneta americana (rPer a 1 and rPer a 7) and Blattella germanica (rBla g 2, rBla g 4 and rBla g 5) were performed at 10 µg/ml and 5 µg/ml (rPer a 1). IgE antibodies to rPer a 7 and rPer a 1 were quantitated by ELISA. RESULTS: Of 57 patients tested, 3 (5.3%), 24 (42.1%), 4 (7%), 3 (5.3%) and 4 (7%) showed positive reactions to rPer a 1, rPer a 7, rBla g 2, rBla g 4 and rBla g 5, respectively. Twenty-eight patients (49.1%) had positive tests to at least one allergen. In keeping with skin test results, 31/57 patients (54.4%) and 5/55 patients (9%) had detectable IgE to rPer a 7 and rPer a 1, respectively. Levels of IgE to rPer a 7 were higher in patients with positive tests to rPer a 7 than those with negative tests (geometric mean 13.2 and 1.8 IU/ml, p < 0.05). There was good concordance of results of skin tests and measurements of serum IgE to rPer a 7. CONCLUSION: IgE reactivity to rPer a 7 (P. americana tropomyosin) was dominant among patients in Brazil. However, 50% of the patients did not present reactivity to any of the recombinant allergens tested.

Intranasal, liposome-adjuvanted cockroach allergy vaccines made of refined major allergen and whole-body extract of Periplaneta americana.

BACKGROUND: Cockroach (CR) allergens frequently cause severe asthma in CR-sensitized subjects. Allergen-specific immunotherapy causes a shift of allergic Th2 responses towards Th1 and/or regulatory T cell (Treg) responses which reduce airway inflammation and prevent disease progression. Data are relatively limited on immunotherapy via CR allergy vaccine. METHODS: The therapeutic efficacy of an intranasal liposome-adjuvant vaccine made of a refined Periplaneta americana arginine kinase (AK) was compared to the liposome-entrapped P. americana crude extract (CRE) vaccine. Adult BALB/c mice were rendered allergic to CRE. Three allergic mouse groups were immunized intranasally on alternate days with 8 doses of liposome-entrapped CRE (L-CRE), liposome-entrapped AK and placebo, respectively. One week later, all mice received a nebulized CRE provocation. Evaluation of vaccine efficacy was performed 1 day after provocation. RESULTS: Liposome-entrapped native AK attenuated airway inflammation after the CRE provocation and caused a shift of allergic Th2 to Th1 and Treg responses. The L-CRE also induced a shift from the Th2 to the Th1 response but did not induce a Treg response and could not attenuate the airway inflammation upon allergen reexposure. CONCLUSIONS: Intranasal liposome-adjuvant CR allergy vaccine containing native AK (Per a 9) is better than L-CRE in attenuating allergic airway inflammation. The findings of this study not only document a more comprehensive and beneficial immune response induced by the refined allergen vaccine but also raise the point that the shift from the Th2 to the Th1 response alone might not correlate with improved airway histopathology, clinical outcome and quality of life.

Serine protease Per a 10 from Periplaneta americana bias dendritic cells towards type 2 by upregulating CD86 and low IL-12 secretions.

BACKGROUND: Serine protease activity of Per a 10 from Periplaneta americana induces airway inflammation and systemic Th2 response towards self and bystander allergen. OBJECTIVE: In the present study the effect of proteolytic activity of Per a 10 allergen on dendritic cells (DCs) polarization and consequent T cell response was investigated. METHODS: Non-atopic subjects with no family history of asthma/allergy were recruited for the study. CD14(+) peripheral blood monocytes were purified, differentiated to immature DCs and stimulated with proteolytically active/inactivated native or recombinant Per a 10. DCs phenotype was analysed with flow cytometry and antigen presenting function assessed by co-culturing with autologous CD4(+) T cells. Cytokine levels were determined using ELISA. RESULTS: Immature DCs differentiated into mature CD14(-)CD83(+)HLA-DR(+) cells after incubating with proteolytically active/inactivated or recombinant Per a 10. Proteolytically active Per a 10 induced significant CD86 up-regulation on DCs compared to inactivated or recombinant Per a 10 lacking enzymatic activity. Proteolytic activity of Per a 10 showed dose-dependent effect on expression of CD80, CD86, CD83, CD1a and HLA-DR. However, no significant differences were observed phenotypically in active or inactive forms except for CD86. Active Per a 10 stimulated DCs secreted significantly low IL-12 (P < 0.01) and high IL-6, compared to inactive forms of Per a 10. Naive CD4(+) T cells primed with active Per a 10 pulsed DCs also secreted significantly less IL-12 (P < 0.01) and high IL-4, IL-5 plus IL-6 (P < 0.01); in contrast to DCs pulsed with inactivated or recombinant Per a 10. CONCLUSION AND CLINICAL RELEVANCE: Proteolytic activity of Per a 10 modulates DCs towards type 2 by CD86 up-regulation, high IL-6 and reduced IL-12 secretions. Proteolytically inactive Per a 10 can be further explored for immunotherapy.

Sensitization to Per a 2 of the American cockroach correlates with more clinical severity among airway allergic patients in Taiwan.

BACKGROUND: In Taiwan, 57.5% of asthmatic patients are allergic to cockroaches, which are a major indoor allergen for immunoglobulin E (IgE)-mediated respiratory diseases. OBJECTIVE: To determine whether sensitization to different cockroach allergenic components correlates with different clinical manifestations and severities. METHODS: The complementary DNAs (cDNAs) encoding for Per a 1 through 7 and Per a 9 were generated by reverse transcription polymerase chain reaction and cloned into the Escherichia coli expression system. Sixty-four subjects were divided into 3 groups based on the clinical severity of their allergic reaction: those with persistent asthma and rhinitis (AS), those with allergic rhinitis only (AR), and the nonallergic controls (NA). Serum levels of interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), chemokine (C-C motif) ligand 20 (CCL-20), and granulocyte macrophage colony-stimulating factor (GM-CSF) were measured, and the binding frequencies to each recombinant allergen were examined. RESULTS: Serum levels of IL-8, MCP-1, and CCL-20 were significantly higher in the AS group than in the AR and NA groups. The numbers of IgE-binding allergens did not correlate with the clinical severity of airway allergy to cockroaches. However, 81% in the AS group had IgE-binding activity to Per a 2, which was significantly higher than that of the AR group (45%, P < .05). In contrast, 80% of AR patients had IgE-binding activity to Per a 9 compared with only 28.5% of AS patients (P < .01). CONCLUSION: Allergens from American cockroaches do not have equal importance in terms of pathogenicity. Sensitization to Per a 2 correlates with more severe airway allergy and elevated proinflammatory chemokines. This may help in selecting target allergens for component resolved diagnosis and immunotherapeutic agents.

Comparison of irritatant reactions between using lyophilized and commercial food allergen extracts in atopy patch tests in a normal population.

BACKGROUND: Several authors have investigated the use of the atopy patch tests (APT) for the diagnosis of non-IgE mediated food allergy, primarily in patients with atopic dermatitis and digestive disorders. However, one of the difficulties in atopy patch testing is the lack of standardization. Several commercial APTs containing freeze-dried food extracts are now available, but their diagnostic accuracy is still largely undefined. The objective of this study is to evaluate the irritant reactions and safety of atopy patch tests in healthy subjects by using lyophilized and commercial food allergen extracts. METHODS: A cross-sectional study was carried out in healthy volunteers. Atopy patches using lyophilized and commercial allergens, including cow's milk, egg, wheat, soy and shrimp, were assessed. Additionally, commercial extracts of house dust mite (D. pteronyssinus 10,000 AU/ml, D. farinae 10,000 AU/ml) and American cockroach were also evaluated. RESULTS: Eighteen healthy volunteers (13 women, median age 26 years) were enrolled. All APT results, both from using lyophilized and commercial allergen extracts, showed no reactions. There were no systemic allergic reactions or irritant reactions observed. CONCLUSION: APTs using lyophilized and commercial food allergen extracts and commercial extracts of house dust mite and American cockroach showed no irritant reactions in Thai non-atopic subjects.

Comparison of allergenic components and biopotency in whole body extracts of wild and laboratory reared American cockroaches, Periplaneta americana.

BACKGROUND AND OBJECTIVE: Most allergen extracts/vaccines used today in clinical practice are derived from natural allergen sources. Therefore, their allergic components may vary as these are prone to natural variation. This study aims to compare the allergenic components and biological potency of crude extracts from wild and laboratory reared American cockroaches. METHODS: Crude extracts of male and female of wild and laboratory reared American CR, were prepared by the same method. Their allergenic components were evaluated by in vitro assays such as protein contents, protein profiles, quantification of major allergens (Per a 1 and Per a 9) and IgE inhibition ELISA assay. RESULTS AND DISCUSSION: There was no statistically significant difference between the protein contents and the concentrations of Per a 1 in the crude extracts from both groups. However, the Per a 9 levels in extracts of wild CR were significantly higher than those from the extracts of laboratory reared CR. The protein patterns of the extracts of laboratory reared CR exhibited more consistency in the number of bands with higher intensity than those of wild CR. Pooled extracts of laboratory reared CR could inhibit IgE binding to that of wild CR up to 78%. The endotoxin content of extracts of laboratory reared CR were ten times less than those of the the wild CR. We have successfully determined the allergenic potency of the extracts of laboratory reared CR versus those of the wild CRs by in vitro assays. Further studies should be performed to determine the biological potency of CR extracts by in vivo assays for clinical application. CONCLUSION: Our finding indicates that the laboratory reared CR would be the better source of material in vaccine production than the wild CR.

Convenient, rapid and economic detection and semi-quantification of American cockroach allergen in the environment.

BACKGROUND: Measuring allergen levels in the environment provides useful information to guide the management of allergic patients. A laboratory-based test kit sandwich ELISA for quantification of Per a 9, the major allergen of Periplaneta americana was recently developed. However, it is not suitable for screening. OBJECTIVE: To develop a simple, rapid, and economic format for semi-quantification of Per a 9 assay using dot-blot ELISA technique. METHODS: The efficacy of direct dot-blot ELISA and sandwich dot-blot ELISA was evaluated. Direct dot-blot ELISA was selected for further modification into 6 protocols. The selected protocol of direct dot-blot was further compared with the laboratory-based test kit, sandwich ELISA. RESULTS: The lowest detection limits in protocols no. 1-6 were 3.9, 15.6, 15.6, 62.5, 125 and 62.5 microg/ml of native Per a 9 whereas time required for each protocol was 145, 45, 30, 26, 18 and 26 minutes, respectively. The sensitivity of direct dot-ELISA was 3.9 microg/ml of Per a 9. Protocol no. 3 was the most suitable assay because its detection limits were as low as 15.6 microg/ml of CR allergen and the total process took only 30 minutes. In comparison with the 2 days required for laboratory sandwich ELISA, the selected protocol provided a similar yield of allergen detection but it offers significant savings of time. Additionally, this method could be easily interpreted by various groups of people. CONCLUSION: This modified direct dot-blot ELISA is the first membrane ELISA which is a semiquantitative test appropriate for screening American cockroach allergen owing to its simplicity, speed and good yield.

Clinico-immunological changes post-immunotherapy with Periplaneta americana.

BACKGROUND: Cockroach proteins induce allergies including asthma in predisposed individuals. Well-designed controlled studies are required to show the effect of cockroach immunotherapy (IT). This study is aimed to assess changes in clinical and immunological parameters post-IT with Periplaneta americana extract. MATERIALS AND METHODS: A double-blind, placebo-controlled trial of cockroach IT was performed for 1year in 50 patients of asthma, rhinitis or both. The efficacy of IT was assessed by change in skin reactivity and clinical parameters such as symptom/drug score, airway reactivity and immunological parameters namely IgE, IgG1 and IgG4, IL-4 and IFN-γ by enzyme-linked immunosorbent assay and western blotting using patients' sera at baseline and after 1year of treatment. RESULTS: Immunotherapy with cockroach extract demonstrated significant improvement in clinical parameters of active group patients compared with baseline values and placebo group. Specific IgE levels showed a modest reduction, while IgG4 levels increased significantly in active IT group after 1year. IgE immunoblotting demonstrated reduction in intensity and number of specific bands, whereas IgG4 binding showed more number and distinct bands following IT. Active group patients showed correlation between increase in IgG4/IgG1 ratio and reduction in symptom score post-IT. CONCLUSIONS: Immunotherapy with cockroach extract improved clinical and immunological status of asthma and rhinitis patients. Clinical improvement in patients after IT is associated with immunological changes.

Allergy to cockroaches: challenges in diagnosis.

BACKGROUND: Blattella germanica and Periplaneta americana are the most common domestic species of cockroaches, found all over the world under favorable conditions. Allergen sensitivity can be detected through in vivo tests, such as skin prick tests (SPT) for immediate hypersensitivity and in vitro techniques, represented mainly by the sIgE determination. Nevertheless, there is no gold standard for the detection of hypersensitivity to cockroaches. This study aims to evaluate the agreement between skin prick test to different cockroach allergenic extracts with serum specific IgE determination in the diagnosis of cockroach allergy in asthmatic and non-asthmatic children. METHODS: A case-control study involving 74 asthmatic and 42 non-asthmatic children aged between 6 and 14 years was conducted in Recife, Brazil. All individuals were submitted to a skin prick test (SPT) with Dermatophagoides pteronyssinus, D. farinae, Blomia tropicalis (IPI-ASAC) and three different commercial cockroach extracts (Greer, Hollister-Stier, and IPI-ASAC Brasil) of B. germanica and P. americana, and to the quantification of total serum IgE and specific serum IgE to B. germanica and P. americana. RESULTS: The mean diameter of induced papule was considerably greater among the asthmatic patients when compared to non-asthmatic controls, regardless of the species or type of cockroach extract. The correlations between the various types of utilized extracts for the two species studied were not sufficiently strong. Hollister-Stier extract was the most sensible extract among asthmatics in this study for both B. germanica (54.1% [N = 40]) and P. americana (59.5% [N = 44]). A satisfactory correlation was found between the serum levels of specific IgE and total IgE for both species of cockroaches. The correlation of specific IgE serum level from each species with its respective SPT was not considered satisfactory. CONCLUSIONS: The weak correlation between the different extracts clearly indicates a need for standardization of the extracts for SPT for cockroach allergy diagnosis. According to this study, only a patient with high specific IgE serum levels and a positive SPT to a cockroach species should be truly classified as hypersensitive to B. germanica and/or P. americana.

Bioinformatic and immunological investigation of Per a 5 (delta class GST) allergen from Periplaneta americana.

INTRODUCTION: GSTs are multifunctional enzymes involved in cellular detoxification and present as potent allergens in several sources. Present study investigates allergenic relevance of GST from P. americana and determine its cross reactive potential with other indoor allergen sources. METHODS: Computational analysis with FASTA and ConSurf webserver was performed to determine potentially cross reactive allergens. Further, Per a 5 gene was cloned in pET 22b+ vector and expressed in E.coli BL21 cells and the rPer a 5 protein was purified using Ni-NTA affinity chromatography. Enzymatic activity of rPer a 5 was assessed using CDNB and cumene hydroperoxide. ELISA and immunoblot were performed using cockroach hypersensitive patient's sera. Functional activity of rPer a 5 was evaluated by basophil activation test. Inhibition studies were carried out with D. pteronyssinus, A. alternata and C. lunata extracts. RESULTS: Per a 5 demonstrates highest sequence similarity with delta class GST of Blattella germanica (94.9%). It also exhibits significant sequence similarity (50-58%) with mite, fungal and helminth allergenic GSTs. ConSurf analysis reveals high degree of evolutionary similarity in N terminal region of Per a 5, especially at GST dimerization interface. The purified rPer a 5 protein resolved at 27 kDa on SDS-PAGE. The rPer a 5 protein exhibits GST activity and possess upto 65% immunoreactivity with cockroach hypersensitive patient's sera in ELISA and immunoblot. It upregulates expression of CD203c on basophils signifying its biological ability to activate effector cells. rPer a 5 significantly inhibits corresponding GSTs in P. americana, D. pteronyssinus, A. alternata and C. lunata with EC50 values of 15.5 ng. 38.38 ng, 41.4 ng and 61.66 ng, respectively. CONCLUSION: Recombinant delta class GST of P. americana is a clinically relevant allergen showing upto 65% immunoreactivity with hypersensitive patient's sera. Per a 5 GST allergen showed phylogenetic similarity with dust mite, fungal and birch allergens thereby demonstrating allergen cross reactivity.