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Background: Cockroach allergy contributes to morbidity among urban children with asthma. Few trials address the effect of subcutaneous immunotherapy (SCIT) with cockroach allergen among these at-risk children., Objectives: We sought to determine whether nasal allergen challenge (NAC) responses to cockroach allergen would improve following 1 year of SCIT., Methods: Urban children with asthma, who were cockroach-sensitized and reactive on NAC, participated in a year-long randomized double-blind placebo-controlled SCIT trial using German cockroach extract. The primary endpoint was the change in mean Total Nasal Symptom Score (TNSS) during NAC after 12 months of SCIT. Changes in nasal transcriptomic responses during NAC, skin prick test wheal size, serum allergen-specific antibody production, and T-cell responses to cockroach allergen were assessed., Results: Changes in mean NAC TNSS did not differ between SCIT-assigned (n = 28) versus placebo-assigned (n = 29) participants (P = .63). Nasal transcriptomic responses correlated with TNSS, but a treatment effect was not observed. Cockroach serum-specific IgE decreased to a similar extent in both groups, while decreased cockroach skin prick test wheal size was greater among SCIT participants (P = .04). A 200-fold increase in cockroach serum-specific IgG 4 was observed among subjects receiving SCIT (P < .001) but was unchanged in the placebo group. T-cell IL-4 responses following cockroach allergen stimulation decreased to a greater extent among SCIT versus placebo (P = .002), while no effect was observed for IL-10 or IFN-γ., Conclusions: A year of SCIT failed to alter NAC TNSS and nasal transcriptome responses to cockroach allergen challenge despite systemic effects on allergen-specific skin tests, induction of serum-specific IgG 4 serum production and down-modulation of allergen-stimulated T-cell responses., (Copyright © 2024 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Royal jelly (RJ), a creamy substance secreted by honeybees, is the exclusive diet for queen bee differentiation and life maintenance. RJ has been used in cosmetics, beverages, medicines, and supplements worldwide. However, allergy is a concerning issue for RJ, especially in atopic dermatitis (AD) and asthma patients. In some cases, allergic reactions are seen after the first intake of RJ, suggesting the existence of allergens cross-reactive with RJ. Information about the cross-reactive allergens is very important for the safe application of RJ; however, study of this cross-reactivity is quite limited. In this study, we attempted to identify allergens cross-reactive with RJ by using serum samples from 30 AD patients who had never been exposed to RJ. In an enzyme-linked immunosorbent assay (ELISA) experiment, RJ-binding IgE antibodies were detected in the serum of 10 out of 30 patients, and their antibody titers ranged from 4- to 2,048-fold dilution ratios. Additionally, 3 AD patients were determined to be positive in a skin-prick test (SPT) with an RJ solution. Significant correlations were observed between the anti-RJ antibody titer and nonspecific IgE and between the anti-RJ antibody titer and the Eczema Area and Severity Index score. We further examined the cross-reactivity between RJ and 14 typical allergens by using an ELISA-inhibition assay and demonstrated that the following 6 allergens showed cross-reactivity with RJ: the European house dust mite (HDM) (Dermatophagoides pteronyssinus), American HDM (Dermatophagoides farinae), snow crab (Chionocetes spp.), edible crab (Cancer pagurus), German cockroach (Blatella germanica), and honeybee venom (Apis mellifera). In conclusion, people with a history of allergic diseases, including AD, asthma, and allergic rhinitis, should be cautioned against consuming RJ products because of the potential for cross-reactive responses to ensure the safe and successful use of RJ supplements., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: One or more of the authors are/were employed by commercial company. Taketoshi Hata, Furusawa-Horie Takako, Yasuko Arai, and Kenji Ichihara are employed by API Co., Ltd. This does not alter the authors' adherence to all PLOS ONE policies on sharing data and materials.

The cockroach allergen Bla g 1 forms a novel fold consisting of 12 amphipathic alpha-helices enclosing an exceptionally large hydrophobic cavity which was previously demonstrated to bind a variety of lipids. Since lipid-dependent immunoactivity is observed in numerous allergens, understanding the structural basis of this interaction could yield insights into the molecular determinants of allergenicity. Here, we report atomic modelling of Bla g 1 bound to both fatty-acid and phospholipids ligands, with 8 acyl chains suggested to represent full stoichiometric binding. This unusually high occupancy was verified experimentally, though both modelling and circular dichroism indicate that the general alpha-helical structure is maintained regardless of cargo loading. Fatty-acid cargoes significantly enhanced thermostability while inhibiting cleavage by cathepsin S, an endosomal protease essential for antigen processing and presentation; the latter of which was found to correlate to a decreased production of known T-cell epitopes. Both effects were strongly dependent on acyl chain length, with 18-20 carbons providing the maximal increase in melting temperature (~20 °C) while completely abolishing proteolysis. Diacyl chain cargoes provided similar enhancements to thermostability, but yielded reduced levels of proteolytic resistance. This study describes how the biophysical properties of Bla g 1 ligand binding and digestion may relate to antigen processing, with potential downstream implications for immunogenicity.

Background: Allergy to German cockroach (CR) is common in urban environments and is an important allergen in children with asthma., Objective: We hypothesize that the evolution of allergic sensitization and clinical disease is associated with distinct patterns of allergen-specific T cell reactivity. To test this hypothesis, a subset of high-risk inner-city children participating in the URECA (Urban Environment and Childhood Asthma) birth cohort were selected to evaluate CR-specific T cell reactivity from three distinct groups based on acquisition of aeroallergen sensitivity from ages 2 to 10: low atopy with minimal to no sensitivity (n = 26), early-onset allergic sensitization (n = 25) and late-onset allergic sensitization (n = 25)., Methods: Using pools of previously identified CR-derived T cell epitopes, we characterized the allergen-specific T cell response in these 76 subjects from blood samples obtained at age 10. CR-specific production of IL-5, IFNγ and IL-10 was measured by ELISPOT following two-week in vitro culture with CR extract., Results: T cell responses were significantly higher in the early-onset atopy group compared to low atopy (P = 0.01), and a trend for higher cytokine production in the late onset compared to the low atopy cohort was also observed (P = 0.06). T cell responses were similar between early- and late-onset cohorts. Furthermore, a comparison of T cell reactivity between asthmatic and non-asthmatic individuals revealed significantly higher cytokine production in asthmatics compared to non-asthmatics (P = 0.02) within both the CR-allergic and non-allergic cohorts., Conclusions and Clinical Relevance: In conclusion, the present study reports that higher T cell reactivity is associated with allergen sensitization and asthma. Interestingly, no significant difference in T cell reactivity was observed in allergic children with early-onset versus late-onset atopy., (© 2019 John Wiley & Sons Ltd.)

Background: Cockroach allergens are an important cause of IgE-mediated sensitization in inner-city asthmatic patients. However, cockroach extracts used for diagnosis and immunotherapy are not standardized., Objective: We sought to determine the allergen content of nonstandardized German cockroach extracts and the levels of sensitization to an expanded set of cockroach allergens as determinants of in vitro extract potency for IgE reactivity., Methods: Twelve German cockroach extracts were compared for allergen content and potency of IgE reactivity. Bla g 1, Bla g 2, and Bla g 5 were measured by using immunoassays. IgE antibody levels to 8 purified recombinant allergens from groups 1, 2, 4, 5, 6, 7, 9, and 11 were measured by using ImmunoCAP. IgE antibody binding inhibition assays were performed to assess extract in vitro potencies (concentration inhibiting 30% of the total IgE antibody-binding inhibition) relative to an arbitrarily selected reference extract in 5 patients with cockroach allergy., Results: Allergen levels were highly variable. Three new major allergens (groups 6, 9, and 11), were identified among highly cockroach-sensitized subjects (CAP class ≥ 3). Sensitization profiles were unique per subject without immunodominant allergens. The sum of IgE to 8 allergen components showed a good correlation with cockroach-specific IgE levels (r = 0.88, P < .001). In vitro potencies varied among different extracts per subject and among subjects for each extract., Conclusions: The in vitro potency of German cockroach extracts for IgE reactivity depends on allergen content and allergen-specific IgE titers of patients with cockroach allergy. These factors are relevant for selection of potent extracts to be used for immunotherapy and for the design and interpretation of data from immunotherapy trials., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

German cockroach extract is used clinically to evaluate allergen-specific sensitization and for subcutaneous allergen-specific immunotherapy, though there are no guidelines for standardization in its manufacture. We performed an immunological evaluation of 12 different cockroach extracts prepared from different sources and their potency to induce allergen-specific T cell reactivity. PBMC from 13 cockroach allergic donors were expanded in vitro with 12 different German cockroach extracts. After culture expansion, cells were re-stimulated with the different extracts and T cell responses were assessed by FluoroSpot (IL-5, IFNγ and IL-10 production). In parallel to the extracts, single allergen peptide pools for allergens from groups 1, 2, 4, 5, and 11 were tested to determine allergen immunodominance. Furthermore, to assess allergy specificity, PBMC from 13 non-allergic donors were also tested with the most potent extract and T cell responses were compared to the allergic cohort. Dramatic variations in T cell reactivity were observed to the different cockroach extract batches. Response magnitudes varied over 3 logs within a single donor. IL-5 production in the allergic cohort was significantly higher compared to the non-allergic cohort (p=0.004). Allergen content determination by ELISA detected much lower concentrations of Bla g 5 compared to Bla g 1 and 2. Mass spectrometric analysis revealed that Bla g 5 was present in similar amounts to Bla g 1 and 2 in extracts made from whole body, whereas it was not detected in extracts made from fecal matter, suggesting that Bla g 5 is not excreted into feces. Different donors exhibit different response patterns to different extracts, potentially dependent on the donor-specific T cell allergen immunodominance pattern and the allergen content of the extract tested. These findings have dramatic implications for the selection of potent extracts used for diagnostic purposes or allergen-specific immunotherapy.

Purpose: Cockroach exposure is a pivotal cause of asthma. Tight junctions are intercellular structures required for maintenance of the barrier function of the airway epithelium, which is impaired in this disease. Matrix metalloproteinases (MMPs) digest extracellular matrix components and are involved in asthma pathogenesis: MMP1 is a collagenase with a direct influence on airway obstruction in asthmatics. This study aimed to investigate the mechanism by which German cockroach extract (GCE) induces MMP1 expression and whether MMP1 release alters cellular tight junctions in human airway epithelial cells (NCI-H292)., Materials and Methods: mRNA and protein levels were determined using real-time PCR and ELISA. Tight junction proteins were detected using immunofluorescence staining. Epithelial barrier function was measured by transepithelial electrical resistance (TEER). The binding of a transcription factor to DNA molecules was determined by electrophoretic mobility shift assay, while the levels of tight junction proteins and phosphorylation were determined using Western blotting., Results: GCE was shown to increase MMP1 expression, TEER, and tight junction degradation. Both an inhibitor and small interfering RNA (siRNA) of MMP1 significantly decreased GCE-induced tight junction disruption. Furthermore, transient transfection with ETS1 and SP1 siRNA, and anti-TLR2 antibody pretreatment prevented MMP1 expression and tight junction degradation. An extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) inhibitor also blocked MMP1 release, ETS1/SP1 DNA binding, and tight junction alteration., Conclusion: GCE treatment increases MMP1 expression, leading to tight junction disruption, which is transcriptionally regulated and influenced by the ERK/MAPK pathway in airway epithelial cells. These findings may contribute to developing novel therapeutic strategies for airway diseases., Competing Interests: The authors have no financial conflicts of interest., (© Copyright: Yonsei University College of Medicine 2018.)

Introduction: Mites are an important source of allergens in the tropics. Other potential sources of allergens prevalent in the region such as insects have been poorly studied. Objective: To determine the relationship between exposure and allergic sensitization to cockroaches, mosquitos, ants and the interaction with mite sensitization. Materials and methods: We included patients with allergy tests for Blatella germanica, Aedes aegypti, Solenopsis invicta, Blomia tropicalis, Dermatophagoides farinae and D. pteronyssinus. IgE sensitization was evaluated by intraepidermal tests. Exposure to insects in houses was evaluated using traps for crawling and flying insects. Results: A total amount of 186 patients were included; 73 (39.2%) of them were sensitized to an insect (cockroaches: 21%, mosquitoes: 29%, ants: 26,3%), 71 (97.2%) also had sensitization to mites. Of the 148 patients sensitized to mites, only 47.9% were sensitized to an insect. In total, 104 houses were evaluated: 74% had cockroaches, 22% ants, and 52% mosquitoes. Among insect-sensitized patients, the number of insects at home was directly related to the size of the weal generated during the skin test: Cockroaches, r=0.781, p<0.001; mosquitoes, r=0.811, p<0.001, and ants, r=0.840, p<0.001. Conclusion: Sensitization to insects is frequent in allergic populations of the tropics and is strongly associated with sensitization to mites.

The German cockroach, Blattella germanica, is a worldwide pest that infests buildings, including homes, restaurants, and hospitals, often living in unsanitary conditions. As a disease vector and producer of allergens, this species has major health and economic impacts on humans. Factors contributing to the success of the German cockroach include its resistance to a broad range of insecticides, immunity to many pathogens, and its ability, as an extreme generalist omnivore, to survive on most food sources. The recently published genome shows that B. germanica has an exceptionally high number of protein coding genes. In this study, we investigate the functions of the 93 significantly expanded gene families with the aim to better understand the success of B. germanica as a major pest despite such inhospitable conditions. We find major expansions in gene families with functions related to the detoxification of insecticides and allelochemicals, defense against pathogens, digestion, sensory perception, and gene regulation. These expansions might have allowed B. germanica to develop multiple resistance mechanisms to insecticides and pathogens, and enabled a broad, flexible diet, thus explaining its success in unsanitary conditions and under recurrent chemical control. The findings and resources presented here provide insights for better understanding molecular mechanisms that will facilitate more effective cockroach control., (© 2018 Wiley Periodicals, Inc.)

Background: German cockroach (GCr) allergen extracts are complex and heterogeneous products, and methods to better assess their potency and composition are needed for adequate studies of their safety and efficacy., Objective and Methods: The objective of this study was to develop an assay based on liquid chromatography and multiple reaction monitoring mass spectrometry (LC-MRM MS) for rapid, accurate, and reproducible quantification of 5 allergens (Bla g 1, Bla g 2, Bla g 3, Bla g 4, and Bla g 5) in crude GCr allergen extracts., Results: We first established a comprehensive peptide library of allergens from various commercial extracts as well as recombinant allergens. Peptide mapping was performed using high-resolution MS, and the peptide library was then used to identify prototypic and quantotypic peptides to proceed with MRM method development. Assay development included a systematic optimization of digestion conditions (buffer, digestion time, and trypsin concentration), chromatographic separation, and MS parameters. Robustness and suitability were assessed following ICH (Q2 [R1]) guidelines. The method is precise (RSD < 10%), linear over a wide range (r > 0.99, 0.01-1384 fmol/μL), and sensitive (LLOD and LLOQ <1 fmol/μL). Having established the parameters for LC-MRM MS, we quantified allergens from various commercial GCr extracts and showed considerable variability that may impact clinical efficacy., Conclusions and Clinical Relevance: Our data demonstrate that the LC-MRM MS method is valuable for absolute quantification of allergens in GCr extracts and likely has broader applicability to other complex allergen extracts. Definitive quantification provides a new standard for labelling of allergen extracts, which will inform patient care, enable personalized therapy, and enhance the efficacy of immunotherapy for environmental and food allergies., (© 2017 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

German cockroaches are major household allergens that can trigger allergic airway inflammatory diseases with sensitive T-cell responses. Although the use of immune modulatory biologics, such as antibodies, to mediate allergic responses has recently been examined, only systemic administration is available because of the size limitations on intranasal administration. Here we utilized a cell-permeable peptide, dNP2, to deliver the cytoplasmic domain of cytotoxic T-lymphocyte antigen-4 (ctCTLA-4) through the airway epithelium to modulate Th2 responses in a German cockroach extract (GCE)-induced allergic airway inflammation model. The intranasal delivery efficiency of the dNP2-dTomato protein to the lungs was higher in GCE-induced asthmatic lung parenchymal cells compared to the sham cells. Intranasal administration of the dNP2-ctCTLA-4 protein inhibited airway hyper-responsiveness and reduced airway inflammation and remodeling, including goblet cell metaplasia and collagen deposition around the bronchi. The number of infiltrated cells, including eosinophils, and the levels of IL-4, IL-5, IL-13 and IFN-γ in the lungs were significantly reduced, presumably owing to inhibition of Th2 differentiation. However, intranasal administration of CTLA4-Ig did not inhibit airway inflammation. These results collectively suggest that dNP2-ctCTLA-4 is an efficient intranasally applicable candidate biologic for treating allergic asthma.

Background: Allergy to the German cockroach (Blattella germanica) is a significant asthma risk factor for inner-city communities. Cockroach, like other allergens, contains trypsin-like enzyme activity that contributes to allergenicity and airway inflammation by activating proteinase-activated receptors (PARs). To date, the enzymes responsible for the proteolytic activity of German cockroach allergen have not been characterized., Objectives: We aimed to identify, isolate and characterize the trypsin-like proteinases in German cockroach allergen extracts used for clinical skin tests. For each enzyme, we sought to determine (1) its substrate and inhibitor enzyme kinetics (Km and IC50), (2) its amino acid sequence and (3) its ability to activate calcium signalling and/or ERK1/2 phosphorylation via PAR2., Methods: Using a trypsin-specific activity-based probe, we detected three distinct enzymes that were isolated using ion-exchange chromatography. Each enzyme was sequenced by mass spectometery (deconvoluted with an expressed sequence tag library), evaluated kinetically for its substrate/inhibitor profile and assessed for its ability to activate PAR2 signalling., Findings: Each of the three serine proteinase activity-based probe-labelled enzymes isolated was biochemically distinct, with different enzyme kinetic profiles and primary amino acid sequences. The three enzymes showed a 57%-71% sequence identity with a proteinase previously cloned from the American cockroach (Per a 10). Each enzyme was found to activate both Ca ++ and MAPK signalling via PAR2., Conclusions and Relevance: We have identified three different serine proteinases from the German cockroach that may, via PAR2 activation, play different roles for allergen sensitization in vivo and may represent attractive therapeutic targets for asthma., (© 2017 John Wiley & Sons Ltd.)

Background and Objective: The allergenicity of several German cockroach (Bla-g) antigens at the level of IgE responses is well established. However, less is known about the specificity of CD4+ TH responses, and whether differences exist in associated magnitude or cytokine profiles as a function of disease severity., Methods: Proteomic and transcriptomic techniques were used to identify novel antigens recognized by allergen-specific T cells. To characterize different TH functionalities of allergen-specific T cells, ELISPOT assays with sets of overlapping peptides covering the sequences of known allergens and novel antigens were employed to measure release of IL-5, IFNγ, IL-10, IL-17 and IL-21., Results: Using these techniques, we characterized TH responses in a cohort of adult Bla-g-sensitized subjects, either with (n = 55) or without (n = 17) asthma, and nonsensitized controls (n = 20). T cell responses were detected for ten known Bla-g allergens and an additional ten novel Bla-g antigens, representing in total a 5-fold increase in the number of antigens demonstrated to be targeted by allergen-specific T cells. Responses of sensitized individuals regardless of asthma status were predominantly TH 2, but higher in patients with diagnosed asthma. In asthmatic subjects, Bla-g 5, 9 and 11 were immunodominant, while, in contrast, nonasthmatic-sensitized subjects responded mostly to Bla-g 5 and 4 and the novel antigen NBGA5., Conclusions: Asthmatic and nonasthmatic cockroach-sensitized individuals exhibit similar TH 2-polarized responses. Compared with nonasthmatics, however, asthmatic individuals have responses of higher magnitude and different allergen specificity., (© 2015 John Wiley & Sons Ltd.)

Background: The receptor for advanced glycation end products (RAGE) shares common ligands and signaling pathways with TLR4, a key mediator of house dust mite (Dermatophagoides pteronyssinus) (HDM) sensitization. We hypothesized that RAGE and its ligand high-mobility group box-1 (HMGB1) cooperate with TLR4 to mediate HDM sensitization., Objectives: To determine the requirement for HMGB1 and RAGE, and their relationship with TLR4, in airway sensitization., Methods: TLR4(-/-), RAGE(-/-), and RAGE-TLR4(-/-) mice were intranasally exposed to HDM or cockroach (Blatella germanica) extracts, and features of allergic inflammation were measured during the sensitization or challenge phase. Anti-HMGB1 antibody and the IL-1 receptor antagonist Anakinra were used to inhibit HMGB1 and the IL-1 receptor, respectively., Results: The magnitude of allergic airway inflammation in response to either HDM or cockroach sensitization and/or challenge was significantly reduced in the absence of RAGE but not further diminished in the absence of both RAGE and TLR4. HDM sensitization induced the release of HMGB1 from the airway epithelium in a biphasic manner, which corresponded to the sequential activation of TLR4 then RAGE. Release of HMGB1 in response to cockroach sensitization also was RAGE dependent. Significantly, HMGB1 release occurred downstream of TLR4-induced IL-1α, and upstream of IL-25 and IL-33 production. Adoptive transfer of HDM-pulsed RAGE(+/+)dendritic cells to RAGE(-/-) mice recapitulated the allergic responses after HDM challenge. Immunoneutralization of HMGB1 attenuated HDM-induced allergic airway inflammation., Conclusion: The HMGB1-RAGE axis mediates allergic airway sensitization and airway inflammation. Activation of this axis in response to different allergens acts to amplify the allergic inflammatory response, which exposes it as an attractive target for therapeutic intervention., (Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

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., (Copyright © 2014 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: While fungal exposures are assumed to provoke wheeze through irritant or allergenic mechanisms, little is known about the differential effects of indoor and outdoor fungi on early-life wheeze., Methods: In a Boston prospective birth cohort of 499 at-risk infants, culturable fungi in bedroom air and dust and outdoor air were measured at the age of 2-3 months. Wheeze was determined using bimonthly telephone questionnaires. Odds ratios were estimated for an interquartile increase in fungal natural log-transformed concentrations, adjusting for predictors of wheeze and potential confounders., Results: Increased odds of 'any wheeze' (≥1 vs 0 episodes) by age one were positively associated with indoor dust Alternaria [odds ratio (OR) = 1.83; 95% confidence interval (CI), 1.07-3.14], Penicillium [OR = 1.18; (0.98-1.43)], and Cladosporium [OR = 1.47; (1.16-1.85)]; indoor air Penicillium [OR = 1.26; (0.92-1.74)]; and outdoor air Cladosporium [OR = 1.68; (1.04-2.72)]. In contrast, indoor dust yeasts were protective [OR = 0.78; (0.66-0.93)]. 'Frequent wheeze' (≥2 vs <2 episodes) by age one was borderline associated with dust yeasts [OR = 0.86; (0.70-1.04)] and indoor air yeasts [OR = 1.53; (0.93-2.53)]. Alternaria concentration was associated with any wheeze for children with maternal mold sensitization [OR = 9.16; (1.37-61.22)], but not for those without maternal mold sensitization [OR = 1.32; (0.79-2.20)]., Conclusions: While wheeze rates were higher with exposures to fungal taxa considered to be irritant or allergenic in sensitive subjects, yeasts in the home had a strong protective association with wheeze in infancy. Molecular microbiologic studies may elucidate specific components of innate microbiologic stimulants that lead to contrasting effects on wheeze development., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Endemic, low-virulence parasitic infections are common in nature. Such infections may deplete host resources, which in turn could affect the reproduction of other parasites during co-infection. We aimed to determine whether the reproduction, and therefore transmission potential, of an epidemic parasite was limited by energy costs imposed on the host by an endemic infection. Total lipids, triacylglycerols (TAG) and polar lipids were measured in cockroaches (Blattella germanica) that were fed ad libitum, starved or infected with an endemic parasite, Gregarina blattarum. Reproductive output of an epidemic parasite, Steinernema carpocapsae, was then assessed by counting the number of infective stages emerging from these three host groups. We found both starvation and gregarine infection reduced cockroach lipids, mainly through depletion of TAG. Further, both starvation and G. blattarum infection resulted in reduced emergence of nematode transmission stages. This is, to our knowledge, the first study to demonstrate directly that host resource depletion caused by endemic infection could affect epidemic disease transmission. In view of the ubiquity of endemic infections in nature, future studies of epidemic transmission should take greater account of endemic co-infections.

Background: Cockroaches produce potent allergens, and cockroach feces are known to be especially rich in allergens. In this study, we analyze the allergenic components from cockroach feces and evaluate allergenicity of recombinant α-amylase identified from fecal extract., Methods: IgE-reactive proteins from German cockroach fecal extract were analyzed by proteomic analysis and immunoblotting. Recombinant α-amylase was produced and its allergenicity was evaluated by ELISA., Results: Analysis of German cockroach fecal extracts identified 12 IgE-reactive components. Most of these allergens were found to be digestive enzymes such as α-amylase, trypsin, chymotrypsin, metalloprotease, and midgut carboxypeptidase A, but the identity of 3 IgE-reactive proteins is still unknown. Glycinin-like proteins, which were likely derived from the cockroach diet, were also identified. German cockroach α-amylase shares the highest identity with pig α-amylase (55.8%), followed by mite group 4 allergens (Blo t 4, 50.4%; Der p 4, 49.8%; Eur m 4, 47.4%). In this study, recombinant α-amylase from German cockroach was expressed, and its allergenicity was examined by ELISA. Specific IgE against recombinant amylase was detected in 41.4% (12/29) of serum samples from German cockroach-sensitized subjects. Recombinant α-amylase was able to inhibit 55% of specific IgE to German cockroach whole-body extract., Conclusions: Amylase was found to be an important novel allergen in cockroach feces. It is hoped that recombinant α-amylase will be useful for further studies and clinical applications., (Copyright © 2013 S. Karger AG, Basel.)

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., (Copyright © 2013 S. Karger AG, Basel.)

Asthma is a chronic condition with high morbidity and healthcare costs, and cockroach allergens are an established cause of urban pediatric asthma. A better understanding of cell types involved in promoting lung inflammation could provide new targets for the treatment of chronic pulmonary disease. Because of its role in regulating myeloid cell-dependent inflammatory processes, we examined A(2B) R expression by myeloid cells in a cockroach allergen model of murine asthma-like pulmonary inflammation. Both systemic and myeloid tissue-specific A(2B) R deletion significantly decreased pulmonary inflammatory cell recruitment, airway mucin production, and proinflammatory cytokine secretion after final allergen challenge in sensitized mice. A(2B) R deficiency resulted in a dramatic reduction on Th2-type airways responses with decreased pulmonary eosinophilia without augmenting neutrophilia, and decreased lung IL-4, IL-5, and IL-13 production. Chemokine analysis demonstrated that eotaxin 1 and 2 secretion in response to repeated allergen challenge is myeloid cell A(2B) R dependent. In contrast, there were no differences in the levels of the CXC chemokines keratinocyte-derived chemokine and MIP-2 in the myeloid cell A(2B) R-deficient mice, strengthening A(2B) R involvement in the development of Th2-type airways inflammation. Proinflammatory TNF-α, IFN-γ, and IL-17 secretion were also reduced in systemic and myeloid tissue-specific A(2B) R deletion mouse lines. Our results demonstrate Th2-type predominance for A(2B) R expression by myeloid cells as a mechanism of development of asthma-like pulmonary inflammation.

Dicer-2 is a ribonuclease involved in the insect RNAi pathway. On attempting to knockdown Dicer-2 expression in the insect Blattella germanica by RNAi, we found that treatment with Dicer-2 dsRNA upregulated the targeted mRNA. This unexpected result was also observed after treating with a nucleopolyhedrovirus dsRNA. Experiments with this alien dsRNA showed an all-or-none response with a threshold for inducing Dicer-2 upregulation between 0.4 and 0.04 μg in terms of dsRNA concentration and between 50 and 20 bp in terms of dsRNA length. The response seems specific of dsRNA given that equivalent experiments carried out with dsDNA did not affect Dicer-2 expression. In insects, Dicer-2 is postulated to be a sensor of viral infections and a key antiviral defense element. The upregulation of Dicer-2 expression after dsRNA administration fits well with this sensor role, and the occurrence of this mechanism in B. germanica, a phylogenetically basal insect, suggests that sensing alien RNAs might be an ancestral function of Dicer-2 proteins.

Mucosal dendritic cells (DC) are intimately associated with the airway epithelium and thus are ideally situated to be first responders to pathogens. We hypothesize that DC drive innate immune responses through early release of tumor necrosis factor (TNF) α, which drives airway epithelial cell responses. In a mouse model, TNFα release was significantly increased following a single exposure to German cockroach (GC) frass, an event independent of neutrophil recruitment into the airways. While lung epithelial cells and alveolar macrophages failed to release TNFα following GC frass exposure, bone marrow-derived DC (BMDC) produced substantial amounts of TNFα suggesting their importance as early responding cells. This was confirmed by flow cytometry of pulmonary myeloid DC. Addition of GC frass-pulsed BMDC or conditioned media from GC frass-pulsed BMDC to primary mouse tracheal epithelial cells (MTEC) or MLE-15 cells induced chemokine (C-C) motif ligand (CCL) 20 and granulocyte macrophage (GM) colony-stimulating factor (CSF), both of which are important for DC recruitment, survival and differentiation. Importantly, DC do not produce CCL20 or GM-CSF following allergen exposure. Blocking TNFα receptor 1 (TNFR1) completely abolished chemokine production, suggesting that BMDC-derived TNFα induced airway epithelial cell activation and enhancement of the innate immune response. Lastly, blocking TNFR1 in vivo resulted in significantly decreased CCL20 and GM-CSF production in the lungs of mice. Together, our data strongly suggest that DC-derived TNFα plays a crucial role in the initiation of innate immune responses through the modification of airway epithelial cell responses., (Copyright © 2012 S. Karger AG, Basel.)

Background: The extent to which the sensitization to the German cockroach Blattella germanica (BG) affects onset/presence of rhinoconjunctivitis (RC) in children is unknown., Objectives: The present work was aimed to assess the prevalence of BG sensitization in an outpatient pediatric population from an allergy clinic, the association with allergic diseases, and the effect of age in children with allergic sensitization., Methods: Five hundred and four consecutive children with at least one positive skin test to a panel of 17 food and inhalant allergens, including BG, and with personal history of atopic diseases, were enrolled in an Allergy Unit of Palermo, Mediterranean area of Southern Italy. A questionnaire was administered to obtain data on epidemiologic and clinical characteristics. Atopy index was computed as the number of the individual positive skin prick tests. Logistic regression was used to estimate the associations between age classes and BG sensitization and RC, as well as the population-attributable risk (PAR) for RC., Results: Prevalence of BG sensitization was 10.5% (5.2% and 15.8% in lower and upper age classes respectively, p=0.0001). Atopy index significantly increased from the lower to the higher age class (p<0.0001). The older age class (OR 3.12; 95% CI 1.57-6.19) and a higher atopy index (OR 37.16; 95% CI 5.04-274.13) were recognized as main risks factors for BG sensitization., Conclusion: In the upper age class, the PAR of BG sensitization for RC was 20.6%. BG sensitization increases in the higher ages, along with atopic index, and BG sensitization is associated with rhinoconjunctivis in older allergic children., (© 2011 John Wiley & Sons A/S.)

Objective: To clone and express the arginine kinase (AK) gene of Blattella germanica and analyze its immune activity., Methods: The cDNA of AK was cloned using specific primers from the total RNA of Blattella germanica The open reading frame (ORF) of AK was cloned into pET-28A vector, and expressed in Escherichia coli BL21(DE3) with IPTG induction. The recombinant protein was purified by Ni2+ chelating affinity chromatography. The recombinant protein was detected by SDS-PAGE, and its immune activity was analyzed by Western blotting., Results: The cloned cDNA ORF sequence (GenBank accession No. FJ514482) contained 1071bp and encoded 356 amino acids. Its sequence homology with the published one (GenBank accession No. EU429466) was 97.2% at nucleotide level. The recombinant containing recombinant plasmid pET-28a-AK expressed a soluble protein of AK (Mr 45 000) after being induced with IPTG. The recombinant AK protein was recognized by sera of allergic patients, indicating that the recombinant AK protein has an adequate response activity., Conclusion: The AK gene of Blattella germanica has been cloned and the recombinant AK protein has been confirmed with immune activity.

Background: A doctor's diagnosis of asthma is associated with increased morbidity (pain and acute chest syndrome [ACS]) among children with sickle cell disease (SCD). An association between IgE levels and asthma and morbidity has not been investigated in children with SCD., Objective: We tested the hypothesis that elevated total and allergen-specific IgE levels are associated with asthma and SCD morbidity in children with SCD., Methods: A cross-sectional study of children with SCD who participated in the Silent Cerebral Infarct Trial was conducted. Logistic regression and negative binomial regression were used to investigate potential associations of total and allergen-specific IgE levels with asthma diagnosis and SCD morbidity, both confirmed by medical record review. Elevation of total IgE level was defined as age-adjusted and sex-adjusted IgE level exceeding the 90th percentile compared with a nonatopic reference population. IgE antibody positivity to Alternaria alternata (mold), Blattellagermanica (cockroach), and Dermatophagoides pteronyssinus (dust mite) was assessed by ImmunoCAP analysis., Results: Children with SCD (140 with asthma; 381 without asthma) were evaluated. Elevations in total IgE level (P = .04) and IgE antibody specific for Alternaria alternata (P = .0003), Blattella germanica (P = .008), and Dermatophagoides pteronyssinus (P = .01) were associated with asthma. ACS (P = .048) but not pain (P = .20) was associated with total IgE level, but neither was associated with specific IgE levels., Conclusion: Significantly increased levels of total and allergen-specific IgE levels are associated with asthma in SCD. High IgE levels are a risk factor for ACS but not pain rates., (Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Chromogenic microplate assays in 96 wells were used to determine the stability of enzyme activity in frass of Blattella germanica (Blattodea: Blattellidae). Frass samples were exposed to controlled conditions [temperature 15-35 °C and/or 53-100% relative humidity (RH)] and to household conditions (apartment). Exposure times were 0 (control), 90, 183 and 276 days. Starch digestion and cellulolytic activities decreased during exposure. Non-specific proteolytic activities were affected by changes in selective proteolytic activities. Activities towards AAPpNA and SA(3) pNA strongly increased at 100% RH, indicating the possible influence of microorganisms growing on frass. Activities towards BApNA and ArgpNA decreased with increasing decomposition time, whereas activity towards ZRRpNA was not influenced by exposure time. The largest decrease in activities towards ArgpNA and BApNA occurred at temperatures of 15 °C, 30 °C and 35 °C and at 100% RH. Activities towards BApNA and ZRRpNA were very stable under different temperature and RH conditions; this was confirmed by findings showing that these activities were stable in the experimental apartment. In comparison with the control, activities towards ZRRpNA and BApNA after 276 days decreased by 1% and 19%, respectively. The longterm persistence of proteolytic activities in cockroach frass increases their allergenic hazard potential.

Background: Respiratory allergy is influenced and determined by genetic and environmental factors. Migration is a good model to indirectly evaluate the possible influence of environment., Objective: To assess the clinical characteristics of respiratory allergy in immigrants to Italy, in comparison with the Italian population., Methods: The clinical/demographic data of those immigrants stably living in Italy and referred for the first time to allergy services for respiratory allergy were collected in a multicenter survey. All the patients underwent a standard diagnostic workup. A matched Italian population was also examined., Results: Six hundred ninety-eight immigrants and 859 Italians had at least one positive skin test and were analyzed. Most of the patients were referred to the allergy units by their general practitioners. In those patients, the demographic characteristics were not different, except for family size. Immigrants had less family history of atopy. Only 16% had a clinical history of allergy before migration. The time elapsed between migration and onset of symptoms was 5.3 ± 3.1 years, with a minimum of 0.5 and a maximum of 7 years. A higher rate of monosensitization was seen among immigrants, and the severity of their asthma/rhinitis was greater than in Italians. No difference was seen in the pattern of sensitizations., Conclusion: In this population of immigrants, environmental factors play a relevant role in the onset of respiratory allergies., (Copyright © 2011 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

We have shown that proteinase-activated receptor-2 (PAR(2)) activation in the airways leads to allergic sensitization to concomitantly inhaled Ags, thus implicating PAR(2) in the pathogenesis of asthma. Many aeroallergens with proteinase activity activate PAR(2). To study the role of PAR(2) in allergic sensitization to aeroallergens, we developed a murine model of mucosal sensitization to cockroach proteins. We hypothesized that PAR(2) activation in the airways by natural allergens with serine proteinase activity plays an important role in allergic sensitization. Cockroach extract (CE) was administered to BALB/c mice intranasally on five consecutive days (sensitization phase) and a week later for four more days (challenge phase). Airway hyperresponsiveness (AHR) and allergic airway inflammation were assessed after the last challenge. To study the role of PAR(2), mice were exposed intranasally to a receptor-blocking anti-PAR(2) Ab before each administration of CE during the sensitization phase. Mucosal exposure to CE induced eosinophilic airway inflammation, AHR, and cockroach-specific IgG1. Heat-inactivated or soybean trypsin inhibitor-treated CE failed to induce these effects, indicating that proteinase activity plays an important role. The use of an anti-PAR(2) blocking Ab during the sensitization phase completely inhibited airway inflammation and also decreased AHR and the production of cockroach-specific IgG1. PAR(2) activation by CE acts as an adjuvant for allergic sensitization even in the absence of functional TLR4. We conclude that CE induces PAR(2)-dependent allergic airway sensitization in a mouse model of allergic airway inflammation. PAR(2) activation may be a general mechanism used by aeroallergens to induce allergic sensitization.

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.

Cockroach infestations have been linked with allergic diseases such as asthma in humans. Bla g 5, sigma class glutathione S-transferase (GST), is the major cockroach allergen which has the highest IgE response value of all cockroach allergens. Although several cockroach allergens have been identified and cloned, information regarding their B ell and T cell IgE-binding epitopes is limited. In order to analyze the IgE binding epitopes of Bla g 5, full-length and five peptide fragments (A, 1-100 amino acid residue; B, 91-200; Ba, 1-125; Bb, 1-150; Bc, 1-175) were expressed. Twelve (37.5%) of 32 sera from cockroach-sensitized subjects showed positive IgE reactivity to the recombinant Bla g 5 (rBla g 5). Six strong positive sera were selected for the epitope study. Recombinant proteins not containing 176-200 amino acid residues were unable to react to sera from cockroach sensitized individuals, suggesting that this region contains the IgE-binding epitope. Despite strong IgE reactivity to rBla g 5, the pooled serum from 5 cockroach-sensitized patients did not show IgE reactivity to all synthetic peptides consisting of 15 residues covering 161-200 amino acids. These results suggest the possibility that Bla g 5 may have a conformational epitope in the C-terminal region. GST is the important target for the development of vaccines and drugs against allergic diseases because of high cross-reactivity among insect species. This study will aid recombinant allergen research for immunotherapy of cockroach allergens and other insect allergens.

Unlabelled: Although vacuum cleaning is recommended to reduce allergen levels and improve asthma and allergic rhinitis symptoms, some studies suggest it may increase allergen load in homes. We conducted a cross-sectional study to determine if home floor vacuuming was associated with sensitization to dust-mites and cockroaches, and serum eosinophil cationic protein (ECP), a biomarker for atopy, in 102 physician-diagnosed spirometry-confirmed asthmatics. We collected data on floor type, floor cleaning method and frequency, asthma severity, allergy medications used, serum ECP and skin prick tests (SPT) to three dust-mites [Dermatophagoides pteronyssisinus (Der p), Dermatophagoides farinae (Der f) and Blomia tropicalis (Blo t)] and two cockroaches [Periplanata americana (Per a) and Blatella germanica (Bla g)]. Those who vacuumed had increased sensitization to three dust-mites [adjusted ORs (95%CI) = Der p: 26.6 (1.8-405.2); Der f: 44.8 (3.2-620.9); Blo t: 14.1 (1.8-108.1)] but not to cockroaches, adjusted for cleaning frequency and other methods of floor cleaning. Subjects who vacuumed their floor had higher levels of serum ECP than those who did not [adjusted median difference (95%CI): 9.4 (1.1-17.7)], adjusted for use of nasal corticosteroids among those with allergic rhinitis. Vacuuming is associated with increased sensitization to dust-mite allergens and higher serum ECP., Practical Implications: We found an association between floor vacuuming and increased sensitization to dust-mite allergens and higher levels of an atopy biomarker. Current recommendations to use vacuuming to control allergen exposure and allergic conditions may need to be reconsidered until further studies are performed.

Background: Home infestations from Harmonia axyridis (ladybug) occur throughout the United States. IgE-mediated sensitization with allergic disease has been reported. The prevalence of ladybug sensitization has been studied by questionnaire and allergy testing in patients diagnosed as having allergic disease. Cross-reactivity with cockroach exists., Objectives: To determine the prevalence of ladybug specific IgE in the general population by specific IgE immunoassay and to examine cross-reactivity to cockroach., Methods: An experimental solid phase for use in immunoassay was prepared using a ladybug extract, and performance characteristics were determined. Serum samples from 100 adult blood bank donors were tested using the ladybug specific IgE immunoassay. Known ladybug IgE-positive serum samples obtained from symptomatic patients were used to study crossreactivity with German cockroach by specific IgE immunoassay inhibition., Results: The mean background response of the assay solid phase was 51 fluorescent units with an analytical cutoff of 59 fluorescent units. It was estimated that a response of 88 fluorescent units corresponds to a specific IgE concentration of 0.1 kUa/L. The extinction dilution curve was linear to 0.1 kUa/L. The assay cutoff was set at 0.1 kUa/L. Of the 100 blood donor serum samples, 10 were positive for ladybug specific IgE. Inhibition assays revealed partial cross-reactivity with German cockroach., Conclusion: Although an immunoassay solid phase prepared with ladybug whole body extract will identify persons in a general population sensitized to epitopes found in the extract, clinicians performing allergy testing using whole body ladybug extract should be aware that a positive result may or may not indicate that exposure to actual ladybug allergens is causing sensitization.

The role of TLR2 in modulating experimentally induced asthma is not fully understood. We recently identified that German cockroach (GC) frass contains a TLR2 ligand allowing us to investigate the role of a TLR2 agonist in a complex real world allergen in mediating allergic airway inflammation. GC frass exposure significantly increased airway inflammation, airway hyperresponsiveness and serum IgE levels in wild-type mice; however the same exposure in TLR2-deficient mice resulted in greatly exaggerated serum IgE and eosinophilia but diminished airway neutrophilia, suggesting a protective role for TLR2. Since GC frass inhalation usually induces airway neutrophilia, we queried the effect of neutrophil depletion on airway responses. Inhibition of neutrophil recruitment into the airways of naive wild-type mice before intratracheal inhalation of GC frass resulted in significantly increased levels of serum IgE and eosinophilia. Neutrophils are a rich source of MMP-9, and we found that MMP-9 levels were significantly increased in the airways of mice following exposure to GC frass. Importantly the levels of MMP-9 were significantly decreased in neutrophil-depleted and TLR2-deficient mice after exposure to GC frass, suggesting that TLR2 regulated MMP-9 release from neutrophils. Functionally, MMP-9-deficient mice had more acute allergic inflammation than wild-type mice, suggesting that MMP-9 was protective against experimentally induced asthma. These data suggest that TLR2 activation of neutrophils leads to release of MMP-9 which decreases allergic responses to GC frass. This suggests a protective role for TLR2 activation and MMP-9 release in the context of experimentally induced asthma in mice.

Cockroach suppression is fundamental to cockroach allergen mitigation in infested homes. The effects of various cockroach control strategies on cockroach populations and allergen concentration have not been examined in schools. This study was conducted to compare the effectiveness of integrated pest management (IPM) and conventional pest control in controlling German cockroach (Blattella germanica L.) infestations and concentrations of the cockroach allergen Bla g 1 in public school buildings. Two school districts included six schools that used conventional pest control and one district included seven schools that used IPM to control pests. Cockroach traps were deployed to assess the level of infestation, settled dust samples were collected in food service areas, classrooms, and other school areas, and the Bla g 1 allergen was quantified by ELISA. Both cockroach counts and Bla g 1 concentrations were dependent on the pest control approach, with highly significant differences between IPM-treated schools and conventionally treated schools in both the cockroach mean trap counts (0 versus 82.6 +/- 17.3 cockroaches/trap/wk, respectively) and in the amount of Bla g 1 in dust samples (2.8 +/- 0.3 versus 30.6 +/- 3.4 U/g dust). Cockroaches and Bla g 1 were primarily associated with food preparation and food service areas and much less with classrooms and offices. Our data extend recent findings from studies in homes, showing that cockroach allergens can be reduced by cockroach elimination alone or by integrating several tactics including education, cleaning, and pest control. IPM is not only effective at controlling cockroaches but also can lead to long-term reductions in cockroach allergen concentrations, resulting in a healthier environment for students and school personnel.

It is becoming increasingly clear that innate immune mediators play a role in regulating adaptive immune responses in asthma pathogenesis. Cockroach exposure is a major risk factor for the development of asthma. In this study we asked whether German cockroach (GC) feces (frass) could initiate an innate immune response. Naive BALB/c mice were challenged with a single intratracheal inhalation of GC frass. Proinflammatory cytokines were significantly increased in the bronchoalveolar lavage fluid at 3 h and were maintained at higher than baseline levels for at least 24 h. Neutrophil migration into the airways was evident as early as 3 h but was maximal between 6 and 24 h postinhalation. The early increase in cytokine expression was independent of TLR2 or TLR4. Newly infiltrated airway neutrophils were responsible for maintaining high levels of cytokines in the airways. Using neutrophils as an early marker of the innate immune response, we show that show that neutrophils isolated from the airways following GC frass inhalation express TLR2 and release cytokines. GC frass directly affected neutrophil cytokine production via TLR2, but not TLR4, as evidenced by the use of TLR-neutralizing Abs and neutrophils from TLR-deficient mice. Activation of cytokine expression occurred via GC frass-induced NF-kappaB translocation and DNA binding. These data show that GC frass contains a TLR2 agonist and, to our knowledge, this is the first report of an allergen directly activating cells of the innate immune system via TLR2 and suggests an important link between innate and adaptive immunity.

Objective: To express the major allergen of Blattella germanica (Bla g 2) in Pichia pastoris and obtain the soluble protein., Methods: The known Bla g 2 gene was used to design the primers which had the restriction enzyme sites. PCR method was applied to obtain the Bla g 2 gene. The gene fragment was then cut and ligated with the Pichia expression vector pGAPZaA, resulting in a recombinant plasmid pGAPZaA-Bla g 2. The linearized pGAPZaA-Bla g 2 was transformed into Pichia pastoris GS115 through electroporation, then screened to positive transformants, and the protein was expressed in YPD medium. Purification of the recombinant protein was achieved by metal (Ni2+) chelating affinity chromatography and Western-blotting assay indicated its IgE binding capacity., Results: With the expressed recombinant protein, SDS-PAGE showed the presence of the product in the supernatant of the culture with Mr 45 000. After 3 days culture, the recombinant protein occupied 50% of the total proteins in the supernatant. The recombinant protein was purified and Western-blot demonstrated an adequate IgE binding capacity of the product., Conclusion: A recombinant protein of Bla g 2 has been obtained, which is soluble in the supernatant and therefore can avoid a process of denaturalization and renaturation of the recombinant.

A highly active glutathione S-transferase was purified from adult German cockroaches, Blattella germanica. The purified enzyme appeared as a single band of 24 kDa by SDS/PAGE, and had a different electrophoretic mobility than, a previously identified Sigma class glutathione S-transferase (Bla g 5). Kinetic study of 1-chloro-2,4-dinitrobenzene conjugation revealed a high catalytic rate but common substrate-binding and cosubstrate-binding affinities, with V(max), k(cat), K(m) for 1-chloro-2,4-dinitrobenzene and K(m) for glutathione estimated to be 664 micromol x mg(-1) x min(-1), 545 s(-1), 0.33 mm and 0.76 mm, respectively. Interestingly, this enzyme possessed the highest activity for cumene hydroperoxide among insect glutathione S-transferases reported to date. Along with the ability to metabolize 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane and 4-hydroxynonenal, this glutathione S-transferase may play a role in defense against insecticides as well as oxidative stress. On the basis of the amino acid sequences obtained from Edman degradation and MS analyses, a 987-nucleotide cDNA clone encoding a glutathione S-transferase (BggstD1) was isolated. The longest ORF encoded a 24 614 Da protein consisting of 216 amino acid residues. The sequence had close similarities ( approximately 45-60%) to that of Delta class glutathione S-transferases, but had only 14% identity to Bla g 5. The putative amino acid sequence contained matching peptide fragments of the purified glutathione S-transferase. ELISA showed that BgGSTD1 bound to serum IgE obtained from patients with cockroach allergy, indicating that the protein may be a cockroach allergen.

Background: Matrix metalloproteinases (MMPs) digest extracellular matrix proteins and may play a role in the pathogenesis of bronchial asthma. MMP-9 levels are increased in the bronchoalveolar lavage fluid and sputum of asthmatics compared with that of controls. As exposure to cockroaches is an environmental risk factor for asthma, we sought to investigate the role of German cockroach fecal remnants (frass) on MMP-9 expression., Methods: Human bronchial epithelial cells (16HBE14o-) and primary normal human bronchial epithelial cells were treated with cockroach frass in the absence or presence of tumor necrosis factor (TNF)alpha. MMP-9 mRNA, protein levels and pro-MMP-9 activity were determined using real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and zymogram assays. Pretreatment of frass with aprotinin abolished protease activity. PD98059, a chemical inhibitor of extracellular signal regulated kinase (ERK), and SLIGKV, an activator of protease-activated receptor (PAR)-2 were also used. AP-1DNA binding was determined by electrophoretic mobility shift assay (EMSA) and ERK phosphorylation by Western blot analysis., Results: Cockroach frass augmented TNFalpha-mediated MMP-9 mRNA and protein expression by a mechanism dependent on active serine proteases within frass and not on endogenous endotoxin. Frass increased ERK phosphorylation, and chemical inhibition of ERK attenuated cockroaches' effects on MMP-9. Serine proteases are known to activate the PAR-2 receptor. We found that selective activation of PAR-2 using the peptide SLIGKV augmented TNFalpha-induced MMP-9 protein levels and increased ERK phosphorylation. Frass and SLIGKV each increased AP-1 translocation and DNA binding., Conclusions: These data suggest that German cockroach frass contains active serine proteases which augment TNFalpha-induced MMP-9 expression by a mechanism involving PAR-2, ERK and AP-1.

Objective: To compare the prevalence of allergy to cockroaches (Blattella germanica and Periplaneta americana) in asthmatic and non-asthmatic children and to determine the degree of agreement between the skin prick test and serum specific IgE levels, as well as to establish the relationship between cockroach allergy and total IgE levels., Methods: A case-control study involving 76 asthmatic and 42 non-asthmatic children aged between 6 and 14 years was conducted in Recife, Brazil. All individuals were submitted to the skin prick test and to the measurement of specific IgE for B. germanica and P. americana, as well as to the determination of total IgE concentration., Results: Asthmatic children showed a higher frequency of positive skin reactions to B. germanica (27.6 vs. 4.8%) and P. americana (27.6 vs. 2.4%) than non-asthmatic ones. The agreement between the skin prick test and the specific IgE results was reasonable for B. germanica (kappa = 0.25) and weak for P. americana (kappa = 0.17). Those patients who tested positive for cockroaches had a significantly higher geometric mean for total IgE than those who tested negative. The agreement between specific IgE and the skin prick test increased as total IgE levels rose, although some skin tests were negative even when total IgE levels were as high as 5000 kU/l. All determinations of cockroach-specific IgE were positive for total IgE levels greater than 2500 kU/l, even among asymptomatic patients., Conclusion: Allergic sensitivity to cockroaches was a predictive factor for asthma severity. The skin prick test is more appropriate for the detection of clinically relevant sensitivity to cockroaches than specific IgE determination.

The German cockroach, Blattella germanica (Linnaeus) (Dictyoptera: Blattellidae), produces several potent human allergens, one of which, Blattella germanica allergen 1 (Bla g 1), is produced in the midgut and excreted in faeces. We tested with descriptive and experimental approaches the hypothesis that Bla g 1 production is related to food intake in adult males and females of the German cockroach. Bla g 1 mRNA expression in the female midgut (assayed by real time quantitative polymerase chain reaction), her Bla g 1 content (assayed by enzyme-linked immunosorbent assay), and the female's faeces production and its Bla g 1 content tracked a cyclic pattern in relation to the gonadotrophic cycle. All four measures rose as food intake increased, declined before oviposition in relation to diminishing food intake, and remained low while the female carried an egg case for 20 days. After her first clutch of embryos hatched, the female resumed feeding, and faeces and Bla g 1 production increased concomitantly. Both Bla g 1 mRNA expression and Bla g 1 protein levels remained low in experimentally starved females. However, when starved females were allowed to feed, Bla g 1 production elevated and the gonadotrophic cycle resumed. Bla g 1 mRNA expression also increased six-fold in response to feeding compared to starved females. By contrast, there were no apparent cycles in the pattern of Bla g 1 production in males, reflecting their low and non-cyclic food intake. Our results therefore demonstrate that Bla g 1 production in B. germanica is modulated in relation to food intake.

Background: Cockroach infestation may sensitize and elicit allergic responses to genetically predisposed individuals. Invertebrate tropomyosins are a frequent cause of allergy and highly cross-reactive in nature. In this study, we aimed to produce recombinant German cockroach tropomyosin and investigate its allergenicity., Methods: German cockroach tropomyosin (Bla g 7) was cloned by reverse transcriptase polymerase chain reaction (RT-PCR). The cloned cDNA was over-expressed in Escherichia coli and purified by affinity chromatography using Ni-nitrilotriacetic (NTA) acid resin. The allergenicity of the recombinant tropomyosin was examined by enzyme-linked immunosorbent assay (ELISA)., Results: The cloned Bla g 7 shared up to 91% amino acid sequence identity with other cockroach tropomyosins. ELISA showed a recombinant Bla g 7 sensitization rate of 16.2% to German cockroach allergic sera. Recombinant tropomyosin was able to inhibit 32.4% of the specific IgE binding to cockroach extract., Conclusions: Tropomyosin represents a minor allergen in cockroach extracts. It is hoped that recombinant tropomyosin will be useful for further studies and clinical applications.