OBJECTIVETo identify and isolate the genes encoding the allergens of Psilgramma menephorn by screening the cDNA expression library.

METHODSThe cDNA expression library of Psilgramma menephorn was constructed in lambdaZAPIIphage, and the library was screened using the sera from the patients allergic to Psilgramma menephorn and those from the rabbits immunized with Psilgramma menephorn extracts. The positive clones were subcloned into pBluescript plas, and the cDNA in the positive clones were amplified with PCR and sequenced.

RESULTS AND CONCLUSIONFive positive clones were obtained by immunological screening of 5 x 10(4) recombinants. Sequence analysis showed that the positive clones contained the new genes of Psilgramma menephorn allergens. This success in isolating these genes may facilitate the development of specific immunotherapy against Psilgramma menephorn allergy and further research of allergic diseases.

Adult ; Allergens ; genetics ; immunology ; Animals ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Female ; Gene Library ; Genes, Insect ; Humans ; Immunization ; Lepidoptera ; genetics ; Male ; Rabbits ; Sequence Analysis, DNA

OBJECTIVETo investigate the alteration of inflammasome and receptor during IgG promoter transfected to HepG2 cells.

METHODBy assay of Elisa to evaluate the secretion of IL-1 beta, IL-8, TNF-alpha and MCP-1 after puerarine and LPS administration, and by assay of real time PCR to evaluate the expression of mRNA of IL-1 beta, IL-8,TNF-alpha and MCP-1, as well as the receptors of TLR2, 4 and NOD2, MyD88.

RESULTIgG promoter did not active innate immunity and enhance the expression and secretion of inflammasome in HepG2. Puerarine did not active the inflammasome either. LPS activated the innate immunity and increased the secretion of IL-8, TNF-alpha and MCP-1.

CONCLUSIONIgG-HepG2 cells could be used specifically as the model of allergy type II for ingredients screening. It is suggested that puerarine was suite for the activator for this type of allergy as positive control.

Allergens ; analysis ; immunology ; Drugs, Chinese Herbal ; chemistry ; Gene Expression Regulation ; immunology ; Hep G2 Cells ; Humans ; Immunity, Innate ; immunology ; Immunoglobulin G ; genetics ; Inflammasomes ; immunology ; Promoter Regions, Genetic ; genetics ; Transfection

Reviewing the progress on study about the major allergen of Shuanghuanglian injection in recent years, resulted in that individual differences of anaphylactic shock are closely related with HLA gene polymorphism. Basing on this, we put forward the research strategy on susceptibility gene of important allergen of Shuanghuanglian injection based on the theory of genetic fingerprints, in order to make sure about the relationship the major allergen of Shuanghuanglian injection and HLA-DRB gene polymorphism and specificity IgE antibody, and to clarify the allergic reaction loci reduced allergic reactions, which can provide the reference data for the study on mechanisms for anaphylactic reaction of Shuanghuanglian injection, and research ideas for the sensitization mechanism of traditional Chinese medicine injection study.
Allergens ; adverse effects ; Anaphylaxis ; chemically induced ; genetics ; immunology ; Drugs, Chinese Herbal ; adverse effects ; Genetic Predisposition to Disease ; genetics ; HLA-DR Antigens ; genetics ; HLA-DRB1 Chains ; Humans ; Immunoglobulin E ; immunology ; Injections ; Polymorphism, Genetic ; genetics

BACKGROUNDThe dust mites, which are mostly represented by Dermatophagoides spp. (Acari: Pyroglyphidae), are the major sources of indoor allergens. Identification and characterization of these mite allergen molecules are an important step in the development of new effective diagnostic procedures and possible therapeutic strategies for allergic disorders associated with dust mites.

METHODSTotal RNA was extracted from Dermatophagoides farinae. The gene coding for Der f 3 was amplified by RT-PCR with the primers designed based on previous sequence published in GenBank. The target gene was cloned intermediately into pMD19-T plasmid and finally into plasmid pET28a (+), expressed in E. coli BL21 at the aid of the inducer isopropyl-D-thiogalactopyranoside (IPTG). The physicochemical properties, spatial structure of the allergen were analyzed with bioinformatics software.

RESULTSThe cDNA coding for group 3 allergen of Dermatophagoides farinae from China was cloned and expressed successfully. Sequencing analysis showed that there were nineteen mismatched nucleotides in five Der f 3 cDNA clones in comparison with the reference (GenBank Accession No. AY283291), which resulted in deduced amino acid sequence incompatibility in eleven residues. Bioinformatics analysis revealed that the Der f 3 pro-protein was an extracellular hydrophobic protein, consisting of 259 amino acids with a 16 amino acid signal peptide. The protein was deduced to have three chymotrypsin active sites (53-68 AA, 108-122 AA and 205-217 AA), one N-glycosylation site, one cAMP- and cGMP-dependent protein kinase phosphorylation site, four protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, and five N-myristoylation sites.

CONCLUSIONSDer f 3 is an extracellular hydrophobic protein which possesses multiple activation and phosphorylation sites. Polymorphism may exist in the Der f 3 gene but this needs to be further confirmed in the future.

Allergens ; chemistry ; genetics ; metabolism ; Amino Acid Sequence ; Animals ; Antigens, Dermatophagoides ; chemistry ; genetics ; metabolism ; Blotting, Western ; Cloning, Molecular ; Computational Biology ; Dermatophagoides farinae ; genetics ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Homology, Amino Acid

Over the past three decades, a large number of genetic studies have been aimed at finding genetic variants associated with the risk of asthma, applying various genetic and genomic approaches including linkage analysis, candidate gene polymorphism studies, and genome-wide association studies (GWAS). However, contrary to general expectation, even single nucleotide polymorphisms (SNPs) discovered by GWAS failed to fully explain the heritability of asthma. Thus, application of rare allele polymorphisms in well defined phenotypes and clarification of environmental factors have been suggested to overcome the problem of 'missing' heritability. Such factors include allergens, cigarette smoke, air pollutants, and infectious agents during pre- and post-natal periods. The first and simplest interaction between a gene and the environment is a candidate interaction of both a well known gene and environmental factor in a direct physical or chemical interaction such as between CD14 and endotoxin or between HLA and allergens. Several GWAS have found environmental interactions with occupational asthma, aspirin exacerbated respiratory disease, tobacco smoke-related airway dysfunction, and farm-related atopic diseases. As one of the mechanisms behind gene-environment interaction is epigenetics, a few studies on DNA CpG methylation have been reported on subphenotypes of asthma, pitching the exciting idea that it may be possible to intervene at the junction between the genome and the environment. Epigenetic studies are starting to include data from clinical samples, which will make them another powerful tool for research on gene-environment interactions in asthma.
Alleles ; Allergens ; Asthma/*genetics ; Endotoxins ; Environment ; *Epigenesis, Genetic ; *Gene-Environment Interaction ; Genome-Wide Association Study ; Humans ; Phenotype ; *Polymorphism, Genetic ; Polymorphism, Single Nucleotide

BACKGROUND: Many young children suffer from wheezing illness during viral respiratory infection, and some of them experience wheezing many years later and ultimately develop bronchial asthma. It is not clear whether atopy or bronchial hyperresponsiveness in the family is a significant risk factor for asthma in this clinical setting. Objective : To examine the genetic basis for the development of asthma after early childhood wheezing. Materials and METHODS: A measurement of serum total IgE concentration, skin prick test to common inhalant allergens, and methacholine bronchial provocation test were performed in 29 asthmatic children and their parents, and 22 non-asthmatic children with the past history of wheezing illness during the first three years of age and their parents. A questionnaire was performed to assess the presence of asthma and allergic rhinitis in the parents. RESULTS: Positive skin test response to common inhalant allergens was more prevalent in asthmatics than in non-asthmatics(67.8% vs. 27.2%). Serum total IgE concentration was significantly higher in asthmatics than in non-asthmatics(geometric mean: 173 vs. 83 IU/ ml). Positive skin test response to comman inhalant allergens was more prevalent in parents of asthmatics than in thoae of non-asthmatics(51.7% vs. 25.0%), but serum total IgE level was not different between the two groups(geometric mean: 132 vs. 120 IU/ml). Positive rate of methacholine bronchial provocation test, geometric mean of PC20-methacholine, and BR index were not different between the parents of asthmatics and non-asthmatics (18.1% vs. 13.9%; 164 vs. 180 mg/ml; 1.154+-0.077 vs. 1.055+-0.068, respectively). CONCLUSION: It is suggested that personal atopy is important in the development of asthma after early childhood wheezing, and parental atopy rather than bronchial hyperresponsiveness is a risk factor for the development of childhood asthma in this clinical setting.
Allergens ; Asthma* ; Bronchial Provocation Tests ; Child ; Genetics ; Humans ; Immunoglobulin E ; Methacholine Chloride ; Parents* ; Respiratory Sounds* ; Rhinitis ; Risk Factors ; Skin ; Skin Tests ; Surveys and Questionnaires

BACKGROUNDAllergen micro-arrays are powerful tools for screening of serum IgE-reactivity. In this study allergen micro-arrays were used to identify dominating IgE-binding allergens and cross-reactivity patterns among selected Chinese allergy patients.

METHODSThe study was conducted using patient sera from the cities of Guangzhou, Nanjing, Chengdu and Shenyang. In total 100 sera with Dermatophagoides pteronyssinus (Der p) specific IgE-levels higher than 50 kU/L were selected for testing against 103 individual allergens.

RESULTSAmong 100 selected patients, 95% showed IgE-reactivity towards house-dust mite allergens Dermatophagoides farinae (Der f) 1, Der f 2 and Der p 2 and 94% were IgE positive against Der p 1, and 60% of sera contained IgE reacting against allergen Euroglyphus maynei (Eur m) 2. IgE against cat allergen, Felisdomesticus (Fel d) 1, was seen in 20%. Only 2% showed specific IgE-reactivity to Der p 10, a panallergen belonging to the tropomyosin family. Serum IgE-reactivity towards other allergens was in general low. IgE-reactivity against pollen allergens showed geographic differences.

CONCLUSIONSThis study clearly confirms that group 1 and group 2 are major allergens of house dust mites. These selected house-dust mite allergy patients are close to being mono-sensitized. Der p 10 is not an important allergen for cross-reactivity. Specific IgE-sensitization towards pollen allergens is low in southern China compared to other regions. The prevalence of food and stinging insect allergens known to give rise to IgE-mediated cross-reactivity is 2% or less.

Adolescent ; Adult ; Allergens ; immunology ; Asian Continental Ancestry Group ; Child ; Child, Preschool ; Female ; Humans ; Hypersensitivity ; blood ; immunology ; Immunoglobulin E ; blood ; genetics ; immunology ; Male ; Middle Aged ; Young Adult

Clinical cases of type-1 hypersensitive reaction to rice (Oryza sativa) have been reported in western countries as well as in Japan. Among rice proteins, 14-16 kD globulin proteins encoded by multiple gene family have been identified as major rice allergens. In this study, a rice cDNA library was constructed using lambda UniZap vector and screened with a rat anti-16 kD globulin protein polyclonal antibody in order to isolate Korean rice allergenic cDNA clones. Five independent cDNA clones, termed RAK1-5, were obtained after second rounds of plaque assay and immunoblot analysis. These clones encoded 13-19 kD recombinant proteins upon IPTG induction, which were identified by the polyclonal antibody in immunoblot analysis. DNA sequencing analysis showed that RAK1-4 have 99% sequence homology with RA5b, and RAK5 is closely related with RA14c. This result indicated that RA5b gene is widely distributed in our cDNA library among other possible rice allergenic genes, and more study is needed to isolate heterogeneous or novel rice allergen genes. Copyright 2000 Academic Press.
Allergens/immunology ; Allergens/genetics* ; Amino Acid Sequence ; Animal ; Antibodies/immunology ; Antibody Specificity ; Cloning, Molecular ; Cross Reactions ; DNA, Complementary ; Female ; Gene Library ; Genetic Vectors ; Immunoblotting/methods* ; Molecular Sequence Data ; Plant Proteins/immunology ; Plant Proteins/genetics* ; Rats ; Rats, Inbred Strains ; Rice/genetics* ; Sequence Analysis, DNA

Lower respiratory symptoms in bakery workers may be induced by wheat flour and endotoxins. We hypothesized that endotoxins from wheat flour may stimulate innate immunity and that interleukin-18 (IL-18) gene polymorphisms may affect their regulatory role in innate immune responses to endotoxins. To investigate the genetic contribution of IL-18 to sensitization to wheat flour, we performed a genetic association study of IL-18 in Korean bakery workers. A total of 373 bakery workers undertook a questionnaire regarding work-related symptoms. Skin prick tests with common and occupational allergens were performed and specific antibodies to wheat flour were measured by ELISA. Three polymorphisms of the IL-18 gene (-607A/C, -137G/C, 8674C/G) were genotyped, and the functional effects of the polymorphisms were analyzed using the luciferase reporter assay. Genotypes of -137G/C (GC or CC) and haplotype ht3 [ACC] showed a significant association with the rate of sensitization to wheat flour. Luciferase activity assay indicated ht3 [AC] as a low transcript haplotype. In conclusion, the regulatory role of IL-18 in lipopolysaccharide-induced responses in bakery workers may be affected by this polymorphism, thus contributing to the development of sensitization to wheat flour and work-related respiratory symptoms.
Adult ; Alleles ; Allergens/immunology ; Antibodies/analysis/immunology ; Female ; Genes, Reporter ; Genotype ; Haplotypes ; Humans ; Interleukin-18/*genetics ; Male ; Middle Aged ; Occupational Diseases/*genetics/immunology ; *Polymorphism, Single Nucleotide ; Questionnaires ; Respiratory Hypersensitivity/*genetics/immunology ; Skin Tests ; Triticum/*immunology

BACKGROUNDExpression of murine calcium-activated chloride channel family member 3 (mCLCA3) has been reported to be increased in the airway epithelium of asthmatic mice challenged with ovalbumin (OVA). However, its role in asthmatic airway inflammation under no OVA exposure has not yet been clarified.

METHODSmCLCA3 plasmids were transfected into the airways of normal BALB/c mice. mCLCA3 expression and airway inflammation in mouse lung tissue were evaluated. Cell differentials and cytokines in bronchoalveolar lavage fluid (BALF) were analyzed. The expression of mCLCA3 protein and mucus protein mucin-5 subtype AC (MUC5AC) were analyzed by Western blotting. The mRNA levels of mCLCA3, MUC5AC and interleukin-13 (IL-13) were determined quantitatively.

RESULTSmCLCA3 expression was not detected in the control group while strong immunoreactivity was detected in the OVA and mCLCA3 plasmid groups, and was strictly localized to the airway epithelium. The numbers of inflammatory cells in lung tissue and BALF were increased in both mCLCA3 plasmid and OVA groups. The protein and mRNA levels of mCLCA3 and MUC5AC in the lung tissue were significantly increased in the mCLCA3 plasmid and OVA groups compared to the control group. The level of IL-13, but not IL-4, IL-5, IFN-γ, CCL2, CCL5 or CCL11, was significantly increased compared with control group in BALF in the mCLCA3 plasmid and OVA groups. The level of IL-13 in the BALF in the mCLCA3 plasmid group was much higher than that in the OVA group (P < 0.05). The level of mCLCA3 mRNA in lung tissue was positively correlated with the levels of MUC5AC mRNA in lung tissue, IL-13 mRNA in lung tissue, the number of eosinophils in BALF, and the content of IL-13 protein in BALF. The level of IL-13 mRNA in lung tissue was positively correlated with the number of eosinophils in BALF and the level of MUC5AC mRNA in lung tissue.

CONCLUSIONThese findings suggest that increased expression of a single-gene, mCLCA3, could simulate an asthma attack, and its mechanism may involve mCLCA3 overexpression up-regulating IL-13 expression.

Allergens ; Animals ; Asthma ; Chloride Channels ; Female ; Inflammation ; chemically induced ; metabolism ; Interleukin-13 ; metabolism ; Interleukin-4 ; genetics ; metabolism ; Interleukin-5 ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Mucin 5AC ; genetics ; metabolism ; Ovalbumin ; pharmacology