Cloning and purification of the first termicin-like peptide from the cockroach Eupolyphaga sinensis

Abstract Background Termicin is an antimicrobial peptide with six cysteines forming three disulfide bridges that was firstly isolated from the salivary glands and hemocytes of the termite Pseudacanthotermes spiniger. In contrast to many broad-spectrum antimicrobial peptides, termicin is most active against filamentous fungi. Although more than one hundred complementary DNAs (cDNAs) encoding termicin-like peptides have been reported to date, all these termicin-like peptides were obtained from Isoptera insects. Methods The cDNA was cloned by combination of cDNA library construction kit and DNA sequencing. The polypeptide was purified by gel filtration and reversed-phase high performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined by Edman degradation and mass spectrometry. Antimicrobial activity was tested against several bacterial and fungal strains. The minimum inhibitory concentration (MIC) was determined by microdilution tests. Results A novel termicin-like peptide with primary structure ACDFQQCWVTCQRQYSINFISARCNGDSCVCTFRT was purified from extracts of the cockroach Eupolyphaga sinensis (Insecta: Blattodea). The cDNA encoding Es-termicin was cloned by cDNA library screening. This cDNA encoded a 60 amino acid precursor which included a 25 amino acid signal peptide. Amino acid sequence deduced from the cDNA matched well with the result of protein Edman degradation. Susceptibility test indicated that Es-termicin showed strong ability to kill fungi with a MIC of 25 μg/mL against Candida albicans ATCC 90028. It only showed limited potency to affect the growth of Gram-positive bacteria with a MIC of 200 μg/mL against Enterococcus faecalis ATCC 29212. It was inactive against gram-negative bacteria at the highest concentration tested (400 μg/mL). Es-termicin showed high sequence similarity with termicins from many species of termites (Insecta: Isoptera). Conclusions This is the first report of a termicin-like peptide isolated from E. sinensis that belongs to the insect order Blattodea. Our results demonstrate the diversity of termicin-like peptides, as well as antimicrobial peptides in insects.(AU)

Endotoxin Is Not Essential for the Development of Cockroach Induced Allergic Airway Inflammation

PURPOSE: Cockroach (CR) is an important inhalant allergen and can induce allergic asthma. However, the mechanism by which CR induces airway allergic inflammation and the role of endotoxin in CR extract are not clearly understood in regards to the development of airway inflammation. In this study, we evaluated whether endotoxin is essential to the development of CR induced airway allergic inflammation in mice. MATERIALS AND METHODS: Airway allergic inflammation was induced by intranasal administration of either CR extract, CR with additional endotoxin, or endotoxin depleted CR extract, respectively, in BALB/c wild type mice. CR induced inflammation was also evaluated with toll like receptor-4 (TLR-4) mutant (C3H/HeJ) and wild type (C3H/HeN) mice. RESULTS: Intranasal administration of CR extracts significantly induced airway hyperresponsiveness (AHR), eosinophilic and neutrophilic airway inflammation, as well as goblet cell hyperplasia in a dose-dependent manner. The addition of endotoxin along with CR allergen attenuated eosinophilic inflammation, interleukin (IL)-13 level, and goblet cell hyperplasia of respiratory epithelium; however, it did not affect the development of AHR. Endotoxin depletion in CR extract did not attenuate eosinophilic inflammation and lymphocytosis in BAL fluid, AHR and IL-13 expression in the lungs compared to CR alone. The attenuation of AHR, eosinophilic inflammation, and goblet cell hyperplasia induced by CR extract alone was not different between TLR-4 mutant and the wild type mice. In addition, heat inactivated CR extract administration induced attenuated AHR and eosinophilic inflammation. CONCLUSION: Endotoxin in CR extracts may not be essential to the development of airway inflammation.

Sensibilidad a cucaracha y factores de riesgos en riníticos y asmáticos / Cockroach sensibility, socioeconomics factors and enviromental risks in rhinitic and asthmatic

El objetivo de la siguiente investigación es determinar si existe asociación entre sensibilidad a cucaracha y factores de riesgos socioeconómicos y ambientales en riníticos y asmáticos de la ciudad de Córdoba. La sensibilidad a cucaracha persiste desde el año 1960 con un porcentaje alrededor del 45%. No se pudo encontrar ninguna asociación entre sensibilidad a cucaracha y factores de riesgo en riníticos y asmáticos. Puede que el presente estudio no haya tenido un número suficiente de pacientes, como para encontrar la asociación que se planteó en la hipótesis.

The objective to determine if exists association among cockroach sensibility and factor of socioeconomic and environmental risks in rhinitic and asthmatic of the city of Córdoba. The sensibility to cockroach persists from the year 1990 with a percentage around 45%. No association among sensibility to cockroarch and risks factors in rhinitic and asthmatic, was found. May be the present study has not had an enough number of patients, like to find the association that we thought about in the hypothesis.

IgE Binding Reactivity of Peptide Fragments of Bla g 4, a Major German Cockroach Allergen

Cockroaches have been recognized as a major cause of asthma. Bla g 4 is one of the most important German cockroach allergens. The aim of this study is to investigate IgE reactivity to the recombinant Bla g 4 (rBla g 4) in the sera of allergic patients and identify linear IgE binding epitope. For protein expression, full-length Bla g 4 (EF202172) was divided into 5 overlapping peptide fragments (E1: aa 1-100, E2: aa 34-77, E3: aa 74-117, E4: aa 114-156, and E5: aa 153-182). The full-length and 5 peptide fragments of Bla g 4 was generated by PCR and over-expressed in E. coli BL21 (DE3). The IgE binding reactivities of the full-length and peptide fragments were measured by ELISA using 32 serum samples of cockroach allergy. The sera of 8 patients (25%) reacted with rBla g 4. Four sera (100%) showed IgE-binding reactivity to full-length and peptide fragment 4, and 2 sera (50%) reacted with peptide fragment 2. One (20%) serum reacted with peptide fragment 3. The results of ELISA using overlapping recombinant fragments indicated that the epitope region was located at amino acid sequences 34-73 and 78-113, and major IgE epitope of Bla g 4 was located at amino acid sequences 118-152 of C-terminal. B-cell epitope analysis of German cockroach allergen Bla g 4 could contribute to the strategic development of more specific and potentially efficacious immunotherapy.

Reactivity of German Cockroach Allergen, Bla g 2, Peptide Fragments to IgE Antibodies in Patients' Sera

Bla g 2 is a cockroach allergen of great importance. This study was conducted to identify IgE-binding epitope(s) of Bla g 2 using the recombinant protein technique. Approximately 50% of tested sera showed IgE reactivity to Pichiaexpressed Bla g 2 (PrBla g 2) and E. coli-expressed Bla g 2 (ErBla g 2). Only 5.3% of serum samples showed stronger reactivity to PrBla g 2 than ErBla g 2, indicating that serum was reactive to conformational or carbohydrate epitopes. The full-length and 5 peptide fragments of Bla g 2 were produced in E. coli. All fragments showed IgE-binding activity to the cockroach-allergy patients' sera. Specifically, peptide fragments of amino acid residue 1-75 and 146-225 appeared to be important for IgE-binding. The information about the IgE-binding epitope of Bla g 2 can aid in the diagnosis and treatment for cockroach allergies.

Diagnóstico de alergia a baratas no ambiente clínico: estudo comparativo entre o teste cutâneo e IgE específica / Use of the skin prick test and specific immunoglobulin E for the diagnosis of cockroach allergy

OBJETIVOS: Comparar a prevalência da sensibilização a baratas (Blattella germanica e Periplaneta americana) em crianças com e sem asma, verificando a concordância entre testes cutâneos e IgE específica bem como determinar a relação entre a sensibilização a baratas e níveis séricos de IgE total. MÉTODOS: Foi realizado um estudo caso-controle, envolvendo 76 crianças asmáticas e 42 não asmáticas, entre 6 e 14 anos de idade, em Recife, Brasil. Todas as crianças submeteram-se ao teste cutâneo e dosagem sérica de IgE específica para B. germanica e P. americana e determinação sérica da IgE total. RESULTADOS: As crianças asmáticas apresentaram maior positividade aos testes cutâneos para B. germanica (27,6 versus 4,8 por cento) e P. americana (27,6 versus 2,4 por cento) que aquelas do grupo controle. A concordância entre o teste cutâneo e a IgE específica foi razoável para B. germanica (Kappa = 0,25) e fraca para P. americana (Kappa = 0,17). A média geométrica da IgE total foi 591,70 kU/L entre pacientes asmáticos e 345,85 kU/L entre os controles, não havendo diferença estatisticamente significante. Em pacientes com testes positivos para baratas, a média geométrica da IgE total foi significativamente maior em comparação aos pacientes cujos exames foram negativos. CONCLUSÃO: A sensibilização a baratas foi associada à asma. O teste cutâneo pode refletir mais apropriadamente uma sensibilização a baratas clinicamente relevante que a IgE específica.

Aeroallergen sensitivity among Thai children with allergic respiratory diseases: a hospital-based study.

One hundred and twenty patients, aged 3-15 years, attending the Allergy Clinic, Thammasat University Hospital, with allergic rhinitis, asthma or both conditions were studied. Standardized skin prick tests were performed with 15 common aeroallergens. The subjects were 85 boys and 35 girls with the mean age of 8.1 years (SD = 3.4 years). There were 63 cases (53%), 22 cases (18%) and 35 cases (29%) diagnosed with allergic rhinitis only, asthma only and both conditions, respectively. The prevalence of asthma among the allergic rhinitis patients was 36 percent whereas the prevalence of allergic rhinitis among the asthmatic patients was 61 percent. The sensitization patterns for major aeroallergens among the three groups were similar with the most common sensitized allergens as D. pteronyssinus, followed by D. farinae, American cockroach, Kopok and German cockroach, respectively. Among the atopic patients, a greater number of allergen reactivity was significantly associated with the increased risk of having their coexistence of allergic rhinitis and asthma (p = 0.0046; score test for trend of odds ratio).

Sensitization to common allergens, especially pollens, among children with respiratory allergy in the Trakya region of Turkey.

Asthma and allergic rhinitis are common problems in children and the causative pollen allergens vary according to the geographical area. The aim of this study was to investigate patterns of sensitization to common inhalant allergens, especially pollens, in Turkish children living in the Trakya region and to determine differences between rural and urban areas. Allergen skin testing was prospectively performed on 539 children aged between 4 and 17 years with respiratory allergy. The reaction was considered to be positive if the mean wheal diameter was at least 3 mm greater than that of the negative controls. We detected positive skin reactions in 420 (77.9%) children. Two hundred and eighty-one (52.1%) mite, 277 (51.4%) pollen, 174 (32.3%) mold, 65 (12.1%) animal dander, 12 (2.2%) cockroach and 6 (1.1%) latex skin sensitivities were detected. Among the pollen allergies 173 were cereal pollen (32.1%), 170 grass pollen (31.5%) and 144 tree pollen allergies (26.7%). The most common positive skin test among the pollens was to cultivated wheat (Titicum vulgare) (n = 116, 21,5%), followed by rye grass (Lolium perenne) and orchard grass (Dactylis glomerata). Positive skin reactions to Alternaria, to Candida albicans, and to all pollens except Ulmus competris, Pinus sylvetris, Platanus vulgaris and Tilia platyphyllos, were higher in children with allergic rhinitis than in those with asthma. In children from rural areas, allergic skin reactivity was found to be more common against Candida albicans, sheep dander and all pollens except Corylus avellana, Fraxinus excelsior, Populus alba, Pinus sylvetris, Platanus vulgaris and Chenopodium album, than in urban children. Although Trakya is close to Greece and other Mediterranean countries, this study suggests that the pollens, which sensitize children, are not similar.

Allergen sensitization to aeroallergens including Blomia tropicalis among adult and childhood asthmatics in Thailand.

To study prevalence of allergen sensitization among asthmatics in Thailand, skin prick tests (SPT) were performed in 84 pediatric, 71 adult asthmatics and 71 adult volunteers. Allergen extracts used for testing included common allergens in Thailand and in Singapore. The incidence of positive SPT to any allergen among the three groups (childhood, adult patients and adult controls) were 64.3%, 43.7% and 35.2%, respectively. Dermatophagoides were the most common allergens sensitized by both pediatric (58.3%) and adult asthmatics (40.8%). Twenty-four children (28.6%) and 8 adult patients (11.3%) were sensitized to storage mites (Blomia tropicalis and/or Austroglyciphagus malaysiensis). All patients sensitized to Blomia tropicalis were sensitized to Dermatophagoides. Twenty-seven percent and 15.5% of childhood and adult asthmatics were sensitized to cockroach allergens. The rates of sensitization to oil palm pollen in childhood and adult asthmatics were 8.3% and 5.6%, respectively. Sensitization to other pollens and spores were less than 5%. This study confirms the importance of Dermatophagoides among Thai asthmatics.

Cockroach allergen detection and cockroach allergens of allergic Thai patients.

Hybridomas secreting monoclonal antibodies (MAb) specific to American cockroach (Periplaneta americana) were produced through a fusion of immune splenocytes of a BALB/c mouse immunized with crude cockroach (CR) extract and mouse myeloma cells. Two hybridomas namely 38G6 and 3C2 were established. These specific hybridomas secreted IgG1 monoclonal immunoglobulins with antigenic specificities to CR protein components of over 207 to 72 kDa and 45 to 40 kDa, respectively. The monoclonal antibodies were applied to select their specific epitopes out of the crude CR extract using affinity chromatography. A Prausnitz-Kustner test revealed that these epitopes were allergens which caused wheals and flares of the skin of a guinea-pig previously sensitized with a pool of serum samples from CR allergic patients. The monoclonal antibodies were also used in a capture ELISA to detect specific IgE in serum samples of allergic Thai patients. It was found that 72% and 76% of the patients had IgE antibodies to the epitopes of MAb 38G6 and MAb 3C2, respectively, indicating that the two epitopes are major CR allergens among the CR allergic Thai patients. An antibody-sandwich ELISA was developed for quantitative detection of CR allergens using the two monoclonal antibodies as a capture reagent and rabbit polyclonal antibodies to crude CR extract as a detection reagent. The assay could detect allergenic epitopes contained in as little as 122 pg of crude cockroach extract, and has high potential for direct measurement of the marker allergens in extracts of environmental samples.

Recombinant American cockroach component, Per a 1, reactive to IgE of allergic Thai patients.

Twelve similar recombinant Per a 1 clones were produced from an American cockroach (CR) cDNA library. The nucleotide sequence of a representative cline, i.e. clone A6, contained 579 base pairs (bp) and a 372 bp open reading frame (2-373) encoding 124 amino acids. A stop codon was found at position 374-376 followed by a 3' end untranslated region with an AATAAA polyadenylation signal and a poly (A) tail. The estimated molecular mass of the 24 amino acid residue protein was 13.8 kDa, with a predicted isoelectric point value of 4.74. Cysteine or N-linked glycosylation was not found. The deduced amino acid sequence of the A6 revealed 84.68-95.97% identity to other previously reported Per a 1 clones and 65.87-69.60% homology to the previously reported Bla g 1 clones. However, while previously reported Per a 1 clones showed homology to ANG12, a precursor protein in the midgut of the female Anopheles gambiae secreted after the blood meal, the A6 DNA sequence was found to have homology (37.1%) to DNA of G2, a putative protein in the midgut of Aedes aegypti (AY 050565). The deduced amino acid sequence of A6 contained a mitochondrial energy transfer protein signature, phosphorylation sites for the cAMP-and cGMP-dependent protein kinase C and casein kinase II. Hydrophobic and hydrophilic characteristics of the A6 deduced peptide indicated that it was a transmembrane protein. This is the first report that Per a 1 is a transmembrane protein. The deduced amino acid sequence of the A6, which contained the sequence LIRSLFGLP, differed in one amino acid from two previously reported epitopes, i.e. LIRALFGL and IRSWFGLP, of Per a 1.0104 which bound 80% and 100%, respectively, to IgE of the allergic patients tested. The A6 DNA sequence was deposited in the GenBank (Accession number AY 259514) and has been designated Per a 1.0105. The A6 expressed protein bound to monoclonal antibodies (MAb 3C2) specific to American cockroach and also bound to IgE of all (100%) of the 20 allergic Thai patients.

Allergic conjunctivitis.

The purpose of this research is to study the clinical features and risk factors of various types of allergic conjunctivitis. Four hundred and forty-five patients with a history of itching, foreign body sensation, lacrimation and red eyes were examined, and a skin test was performed and assessed to grade the severity. The mean age of the subjects was 24.5 +/- 16.3 years with female preponderance, except for vernal keratoconjunctivitis. The majority of the patients had perennial allergic conjunctivitis. Ninety-five percent of the patients had associated allergic diseases, especially allergic rhinitis. Sixty-six percent of the patients had a family history of atopy. Most patients had symptoms at night. Symptoms persisted throughout the year and were generally triggered by exposure to house dust. The allergy skin tests to common aero-allergens were positive in 95% of patients tested. Common allergens causing sensitization were house-dust mites, house dust, cockroaches, and grass pollen. Environmental control and avoidance of these allergens should be stressed in the management of these conditions.

Pattern of sensitization to common environmental allergens amongst atopic Singapore children in the first 3 years of life.

This study was aimed to investigate the sensitization pattern to a range of common allergens in young Singaporean children. A cross-sectional study involving 75 children aged below 3 years was carried out. They presented between December 1995 and April 2000 with symptoms of asthma, rhinitis, eczema, or food allergy. Their levels of allergen-specific serum IgE to a panel of foods (egg white, milk, soy protein, shrimp, wheat and peanut), pet dander, dust mites and cockroaches were measured with Pharmacia CAP System radioallergosorbent test kits. Serum IgE levels greater than 0.35 kU/l represented a positive result. Four children could not be tested with the complete panel because of insufficient serum. The prevalence of sensitization was highest for cow's milk (45.9%) followed by egg white (38.7%), dust mites Dermatophagoides pteronyssinus (31.4%) and Blomia tropicalis (25.5%). Sensitization to ingested allergens was significantly more prevalent in children aged 1 year or younger than in the older children (70.4% of those below 1 year, and 50% of those aged 1-3 years; p < 0.02). Sensitization to inhaled allergens, such as dust mites, was more likely to manifest as respiratory symptoms (allergic rhinitis and asthma), while ingested allergens were associated with gastrointestinal symptoms and eczema (p < 0.001). It was concluded that infants and young children are at high risk of sensitization to common environmental substances. Allergen avoidance is therefore important even in the very young. The prevalence of sensitization to food allergens is higher compared to inhalant allergens in young children.

Role of allergy in nasal polyps of Thai patients.

As distinct from many countries, allergy in Thailand is of the perennial type which may play a role in the formation of nasal polyps. Forty consecutive patients with nasal polyps and 30 normal subjects as control were studied at the Allergy Clinic, Department of Otolaryngology, Pramongkutklao Hospital. A positive clinical history and skin allergy testing are diagnostic criteria for allergy. In the nasal polyps group, these were 28 males and 12 females, aged between 12-65 years, with an average age of 38.5 years. In the control group, there were 18 males and 12 females, aged between 15-53 yeas, with an average age of 34 years. All had received prick skin testing with 6 common aeroallergens. The prick skin test was considered positive when the wheal was > or = 3 mm with surrounding erythema. Twenty-four of 40 patients (60%) with nasal polyps had a positive prick skin test, while 6 in the 30 control cases (20%) had a positive prick skin test. This difference was statistically significant (P = 0.0019), Odd's ratio = 6.0 which means allergic persons were 6 times more prone to have polyps form than normal persons.

An investigation of aeroallergens affecting urban Malaysian asthmatics.

We investigated the aeroallergens affecting 200 asthmatics from the University Hospital in Kuala Lumpur, Malaysia and found 164 (82%) patients with skin prick test (SPT) reactivity to one or more of a panel of 14 allergens, which included indoor and outdoor animal and plant aeroallergens. Reactivity was most frequent to the indoor airborne allergens, with 159 (79.5%) reacting to either or both house dust mite (Dermatophagoides) species and 87 (43.5%) to cockroach. The SPT reactivity to house dust mites corresponded with the finding that patients found house dust to be the main precipitant of asthmatic attacks.

IgE binding patterns to German cockroach whole body extract in Korean atopic asthmatic children

It is widely known that the cockroach is an inhalant allergen in atopic asthma and allergic rhinitis. Even though Bla g I and Bla g II are considered as the major allergens, several relatively high-molecular weight (MW) cockroach allergens have also been recently identified by IgE-immunoblot in western countries. However, the environmental control and diagnostic tests mainly focussed on Bla g I and Bla g II. Furthermore there is no data about major IgE-binding cockroach antigens in Korea. We performed this study to identify the major German cockroach allergens in Korean atopic children. By the results of allergy skin tests, 14 children with atopic asthma (9 were cockroach-sensitive and 5 were cockroach-nonsensitive atopics) were enrolled in this study. We conducted IgE immunoblot and autoradiographic analysis using Yonsei-extract of German cockroach antigen produced in our laboratory, individual sera from 9 cockroach- sensitive children, and the pooled sera of 5 house-dust-mites-only-sensitive children. We performed an allergic skin test to cockroach mix, and a radioallergosorbent test (RAST) using German cockroach crude extract on all subjects. German cockroach-specific IgE was detected in 6 out of 9 subjects by RAST. We identified at least 15 IgE-binding protein bands, and among them, the components of MWs of 76, 64, 50, 38, and <14 kilodaltons (kDa) were the major German cockroach allergens in study subjects. Therefore, Bla g I (25-30 kDa) and Bla g II (36 kDa) could not be the absolute indicators of German cockroach sensitization and parameters of environmental control.

Sensibilizaçäo alérgica à Blatella germanica (Insecta: Dyctyoptera) em pacientes com asma e rinite na cidade de Säo Paulo, Brasil / Allergic sensitization to Blatella germanica (Insecta: Dyctyoptera) in patients with asthma and rhinitis in Säo Paulo city, Brazil

Em 203 pacientes com história de asma e rinite e com crises respiratórias ao contato com pó domiciliar, foram realizados testes cutâneos com B. germanica, pesquisa de IgE específica pelo método ELISA ("Enzyme-Linked Immunosorbent Assay") e prova de Immunoblotting. Em 37, as provas cutâneas foram positivas, e em 14 foram demonstrados anticorpos IgE específicos em concentraçöes baixas ou moderadas e apenas dois com nível elevado. Pela prova de "Immunoblotting", em oito soros, os anticorpos reconheceram fraçöes alergênicas presentes no extrato total. Sete pacientes apresentaram também anticorpos para a P. americana, em concentraçöes semelhantes àquelas determinadas para a B. germanica.

Pruebas cutáneas con antígenos de cucaracha en niños asmáticos residentes en una ciudad mediterránea / Skin sensitivity to cockroach antigens in asthmatic children living in an inner-city

Objetivo: Establecer la frecuencia de pruebas cutáneas positivas a cucaracha en un grupo de niños con asma bronquial alérgico residentes en la ciudad de Río Cuarto, Córdoba. Material y métodos: Se estudiaron 40 niños asmáticos tomados al azar -26 varones y 14 mujeres- con edades comprendidas entre 5,3 y 12,4 años (edad media, 8,2 años). Todos eran asistidos en el servicio de Alergia del Hospital San Antonio; provenían de zonas urbanas y suburbanas, y de clase socioeconómica media-baja. El diagnóstico de asma se basó en la historia clínica, niveles de IgE sérica total y signos de obstrucción respiratoria baja. Se consignó si en sus viviendas había desagües cloacales. Se efectuaron pruebas cutáneas en la espalda mediante la técnica de Prick empleando, en dilución 1:10 p/v, extractos antígenos glicerinados de mezcla de cuerpo entero de Blatella germanica, Blatta orientalis y Periplaneta americana; ácaros, hongos anemófilos, pólenes, otros inhalantes y alimentos. La reacción fue leída a los 15 minutos y se consideró positiva cuando el diámetro de la pápula era ò 3mm. Resultados: La sensibilidad cutánea a por lo menos uno de los alérgenos probados fue positiva en el 90 por ciento de los casos. Para la cucaracha fue del 45 por ciento. No se observó sensibilidad exclusiva a dicho antígeno. No hubo relación con los niveles de IgE sérica total. La cucaracha fue positiva en el 50 por ciento de los niños de hogares que carecían de desagües cloacales, y en el 33 por ciento de los casos que contaban con ese servicio. Conclusiones: La frecuencia de sensibilidad cutánea a alérgenos de cucaracha estaría condicionada, entre otros factores, por la carencia de servicio cloacal domiciliario. Esta carencia incrementaría el grado de infestación por cucaracha del ambiente donde el niño desarrolla sus actividades aumentando, en consecuencia, el riesgo de sensibilización