PURPOSE: Protease-activated receptor 2 (PAR2) reportedly triggers the immune response in allergic asthma. We aimed to investigate the mechanism on allergic inflammation mediated by PAR2. METHODS: Human lung epithelial cells (A549 cells) were used for in vitro, and the German cockroach extract (GCE)-induced mouse model was developed for in vivo studies. RESULTS: In A549 cells, the levels of reactive oxygen species (ROS) and thymic stromal lymphopoietin (TSLP) were significantly increased by GCE treatment, but were suppressed by PAR2-antagonist (PAR2-ant) or N-acetylcysteine (NAC) treatment. Claudin-1 was degraded by GCE, and was restored by PAR2-ant or NAC in the cells. In the mouse model, the clinical appearance including bronchial hyperresponsiveness, bronchoalveolar lavage fluid analysis and total immunoglobulin E were significantly suppressed by PAR2-ant or NAC. Moreover, TSLP levels in the lung were suppressed by the same treatments in the lung. Claudin-1 was also degraded by GCE, and was restored by PAR2-ant or NAC. CONCLUSIONS: ROS generation and epidermal tight junction degradation are triggered by protease, followed by the induction of TSLP in allergic asthma. Our findings could suggest that PAR2-ant or anti-oxidants could be considered for allergic diseases as preventive alternatives.
Acetylcysteine ; Animals ; Asthma ; Blattellidae ; Bronchoalveolar Lavage Fluid ; Claudin-1 ; Epithelial Cells ; Humans ; Immunoglobulin E ; Immunoglobulins ; In Vitro Techniques ; Inflammation ; Lung ; Mice ; Oxygen ; Reactive Oxygen Species ; Receptor, PAR-2 ; Receptors, Proteinase-Activated ; Tight Junctions

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.
Airway Obstruction ; Asthma ; Blattellidae* ; Blotting, Western ; Cockroaches ; Collagenases ; DNA ; Electric Impedance ; Electrophoretic Mobility Shift Assay ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells* ; Epithelium ; Extracellular Matrix ; Fluorescent Antibody Technique ; Humans* ; Matrix Metalloproteinase 1* ; Matrix Metalloproteinases ; Phosphorylation ; Phosphotransferases ; Protein Kinases ; Real-Time Polymerase Chain Reaction ; RNA, Messenger ; RNA, Small Interfering ; Tight Junction Proteins ; Tight Junctions* ; Transcription Factors ; Transfection

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.
Abatacept ; Administration, Intranasal ; Allergens ; Antibodies ; Asthma ; Biological Products ; Blattellidae* ; Bronchi ; Collagen ; Cytoplasm ; Eosinophils ; Epithelium ; Family Characteristics ; Goblet Cells ; Inflammation* ; Interleukin-13 ; Interleukin-4 ; Interleukin-5 ; Lung ; Metaplasia ; Respiratory Hypersensitivity ; T-Lymphocytes ; T-Lymphocytes, Cytotoxic

PURPOSE: Cockroach feces are known to be rich in IgE-reactive components. Various protease allergens were identified by proteomic analysis of German cockroach fecal extract in a previous study. In this study, we characterized a novel allergen, a chymotrypsin-like serine protease. METHODS: A cDNA sequence homologous to chymotrypsin was obtained by analysis of German cockroach expressed sequence tag (EST) clones. The recombinant chymotrypsins from the German cockroach and house dust mite (Der f 6) were expressed in Escherichia coli using the pEXP5NT/TOPO vector system, and their allergenicity was investigated by ELISA. RESULTS: The deduced amino acid sequence of German cockroach chymotrypsin showed 32.7 to 43.1% identity with mite group 3 (trypsin) and group 6 (chymotrypsin) allergens. Sera from 8 of 28 German cockroach allergy subjects (28.6%) showed IgE binding to the recombinant protein. IgE binding to the recombinant cockroach chymotrypsin was inhibited by house dust mite chymotrypsin Der f 6, while it minimally inhibited the German cockroach whole body extract. CONCLUSIONS: A novel allergen homologous to chymotrypsin was identified from the German cockroach and was cross-reactive with Der f 6.
Allergens ; Amino Acid Sequence ; Blattellidae* ; Chymotrypsin* ; Clone Cells ; Cockroaches ; DNA, Complementary ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Expressed Sequence Tags ; Feces ; Hypersensitivity ; Immunoglobulin E ; Mites ; Pyroglyphidae ; Sequence Homology ; Serine Proteases

A study was performed to estimate the prevalence of the external bacterial flora of two domestic cockroaches (Blattella germanica and Blatta orientalis) collected from households in Tebessa (northeast Algeria). Three major bacterial groups were cultured (total aerobic, enterobacteria, and staphylococci) from 14 specimens of cockroaches, and antibiotic susceptibility was tested for both Staphylococcus and Pseudomonas isolates. Culturing showed that the total bacterial load of cockroaches from different households were comparable (P<0.001) and enterobacteria were the predominant colonizers of the insect surface, with a bacterial load of (2.1 × 10⁵ CFU/insect), whereas the staphylococci group was the minority. Twenty-eight bacterial species were isolated, and susceptibility patterns showed that most of the staphylococci isolates were highly susceptible to chloramphenicol, gentamycin, pristinamycin, ofloxacin, clindamycin, and vancomycin; however, Pseudomonas strains exhibited resistance to amoxicillin/clavulanic acid, imipenem, and the second-generation antibiotic cephalosporin cefuroxime.
Algeria ; Animals ; Blattellidae ; microbiology ; Drug Resistance, Bacterial ; Female ; Housing ; Male ; Microbiota ; Pseudomonas ; drug effects ; Staphylococcus ; drug effects

PURPOSE: Population studies have reported that sensitization to inhalant allergens is rare in young children; however, most subjects in those studies had little or no symptoms or signs highly suggestive of allergic diseases. The aim of the present study was to assess the prevalence of sensitization to inhalant allergens in young children with symptoms and/or signs of allergic disease. METHODS: We analyzed the results of all specific IgE tests performed at our hospital laboratory in children younger than 6 years presenting with symptoms and/or signs highly suggestive of allergic diseases between 2008 and 2013. Specific IgE tests for Dermatophagoides pteronyssinus, Dermatophagoides farinae, Alternaria alternata, German cockroach, cat dander, egg white or egg yolk, milk, peanut, and soybean were performed on 295 children; a specific IgE concentration > or =0.35 or > or =0.2 IU/mL was considered positive. We also compared allergen sensitization rates using the two cutoff values. RESULTS: One hundred eighty-one children (61.4%) were positive to at least 1 allergen tested and 53 children (18.9%) were positive to at least 1 inhalant allergen when a specific IgE concentration > or =0.35 IU/mL was considered positive. The children were more likely to have asthma or allergic rhinitis when they were sensitized to any inhalant allergen, particularly house dust mites. The prevalence of sensitization to inhalant allergens increased with age (P<0.001). There was no significant difference in the prevalence of polysensitization among different age groups, but sensitization to both inhalant and food allergens significantly increased with age. CONCLUSION: Our results suggest that specific IgE tests to common inhalant allergens, particularly the house dust mites, may be considered when performing blood screening tests for young children presenting with symptoms and/or signs of allergic diseases.
Allergens* ; Alternaria ; Animals ; Asthma ; Blattellidae ; Cats ; Child* ; Dander ; Dermatophagoides farinae ; Dermatophagoides pteronyssinus ; Egg White ; Egg Yolk ; Humans ; Immunoglobulin E ; Laboratories, Hospital ; Mass Screening ; Milk ; Prevalence ; Pyroglyphidae ; Rhinitis ; Soybeans

PURPOSE: The cockroach (CR) is an important cause of respiratory allergic disorders. We prepared a German CR extract in a standardized way and analyzed its allergenic properties. METHODS: The extract was prepared from German CR (Blattella germanica) obtained from a Korean colony, and its allergenic activity was compared with that of the commercial Hollister-Stier (HS) extract. The concentrations of Bla g 1 and Bla g 2 were measured, and an in vitro specific IgE binding inhibition assay was performed to assess IgE reactivity. Proteolytic activity was examined by gelatin zymography. RESULTS: Bla g 1 and Bla g 2 were detected at 405 U/mg and 273 ng/mg, respectively, in the Korean extract, and at 187 U/mg and 56 ng/mg, respectively, in the HS extract. The Korean extract showed 94.2% inhibition of IgE reactivity, as compared with the HS extract. A similar pattern of IgE-reactive bands was detected for the two extracts, indicating that their allergenic components are similar. The proteolytic activities of the Korean and HS extracts were found to be similar in gelatin zymography. The endotoxin levels in the Korean and HS extracts were 3,440 EU/mL and 6,580 EU/mL, respectively. CONCLUSIONS: The German CR extract was prepared in a standardized way. The extract produced in this study will be useful for the development of allergy diagnostics and immunotherapeutic agents.
Allergens ; Blattellidae ; Cockroaches ; Gelatin ; Hypersensitivity ; Immunoglobulin E ; Korea

The cockroach represents one of the most common sources of indoor allergens worldwide, and 40%-60% of patients with asthma in urban and inner-city areas possess IgE antibodies to cockroach allergens. In Korean homes, four cockroach species have been found, of which the most commonly encountered is the German cockroach. The pathogenic mechanism underlying the association between cockroach allergens and allergic diseases has not been fully elucidated. Allergenicity is associated with the cockroach allergens themselves, enzymatic protease activity, and ligands for pattern recognition receptors. Although allergen-specific adaptive immune responses orchestrate the cockroach allergic response, recent data suggest that the innate immune system is also a critical contributor to pathogenesis. We review the current evidence for the demographics of cockroach exposure and sensitization, characteristics of cockroach allergens, and inflammatory responses to cockroach allergens initiated through protease-dependent pathways.
Allergens ; Antibodies ; Asthma ; Blattellidae ; Cockroaches ; Demography ; Humans ; Hypersensitivity ; Immune System ; Immunoglobulin E ; Ligands ; Receptor, PAR-2 ; Receptors, Pattern Recognition

BACKGROUND:Recent studies have attempted to document the correlation of an atopy patch test (APT) and a skin prick test. The results have suggested a possible role of delayed allergic reaction in the development of eczematous skin lesions in patients with atopic dermatitis (AD) OBJECTIVE: The aim of this study was to evaluate the relationship of APT with skin prick test and specific IgE for cockroach antigen. And we attempted to find the optimal vehicle and concentration of APT for cockroach allergy in AD patient. METHODS: We performed APTs in 50 patients with AD with using German cockroach allergens (the extract with different concentrations and vehicles). The reactions were evaluated after 48 hours, and these were compared with the results of the skin prick test and the total and specific IgE levels. RESULTS: The AD patients who had cockroach allergy, according to a skin prick test or the specific IgE level, showed higher positive APT reactionsthan the non-allergic patients to cockroach. The APT reactions showed a significant relationship with the skin prick test and the specific IgE level (85.7%). With regard to the form of cockroach antigen, a positive APT was more frequent with using the powder form that with using the extract. Regarding the vehicle, petrolatum induced higher positive APT reactions than did distilled water. There was a close relationship between the antigen concentration in petrolatum base and a positive APT reaction. Ten and fifty percent cockroach antigen elicited positive APT reactions than did 1% cockroach antigen with using a petrolatum base. CONCLUSION: Petrolatum is an optimal vehicle for an APT for cockroach because an APT with a petrolatum base revealed the highest correlation with cockroach allergy. The dose-response relationship between antigen concentration and the frequencies of positive APT reactions demonstrated that 10% and 50% cockroach antigen is superior to 1% cockroach antigen.
Allergens ; Blattellidae ; Cockroaches ; Dermatitis, Atopic ; Dimaprit ; Humans ; Hypersensitivity ; Immunoglobulin E ; Patch Tests ; Petrolatum ; Skin ; Water

BACKGROUND AND OBJECTIVES: The serine protease allergens have been known as one of the most important inhalent allergens of insects as well as house dust mites. There is no known serine protease allergen of German cockroach, which is a well-known etiological agent of allergic diseases. We identified the serine protease genes of cockroach, Blattella germanica, and examined its possibility as an allergen. MATERIALS AND METHOD: We have isolated three partial genes, Bg3, Bg5 and Bg6, of serine protease from the Blattella germanica cDNA library using the degenerate oligonucleotide PCR primers specific for the conserved regions. RESULTS: The genes, Bg3, Bg5 and Bg6, were composed of 396 bp 131 amino acids, 513 bp 170 amino acids, and 410 bp 136 amino acids, respectively. Northern hybridization analysis indicated that the size of Bg5 and Bg6 transcripts was approximately 0.7 kb. The gene, Bg5, was also identified as a multiple gene by the Southern blot analysis. The gene, Bg3, showed significant homology to trypsin-like serine protease of various insects. The deduced amino acids sequence of the gene Bg5 was matched to the mite group III allergen as well as various species of insect serine protease sequences. The gene Bg6 also showed high homology to the amino acids sequences of insect serine proteases and mite group III allergens. Three Blattella germanica serine protease gene fragments revealed close genetic relationships with Dermatophagoides farinae group III allergen in the phylogenetic analysis. CONCLUSION: The high homology and close phylogenetic relationship raise the possibility of three serine protease genes as being an allergen of German cockroach.
Allergens ; Amino Acids ; Blattellidae ; Blotting, Southern ; Chimera ; Clone Cells ; Cloning, Organism ; Cockroaches ; Dermatophagoides farinae ; Gene Library ; Humans ; Hypersensitivity ; Insects ; Mites ; Polymerase Chain Reaction ; Pyroglyphidae ; Serine ; Serine Endopeptidases ; Serine Proteases