Quantification of Der f 1 in houses of patients allergic to house dust mite, Dermatophagoides farinae, using a locally produced detection reagents.

Background: House dust mite (HDM) allergen quantification in house dust samples before and after the allergen elimination is one means of convincing the target population about the health benefits of allergen removal from their environment. Objective: To produce local reagents for quantification of Der f 1 (major allergen of Dermatophagoides farinae) in dust samples from houses of HDM allergic Thai patients. Methods: Recombinant Der f 1 was used for immunization of a BALB/c mouse for hybridoma production. Polyclonal antibody (PAb) to whole body extract of D. farinae was prepared from an immunized rabbit. A sandwich ELISA (MAb-allergen-PAb) was used, in comparison with the commercialized reagents (Indoor Biotechnology, UK), to quantify Der f 1 in dust samples. Results: Two hybridoma clones, Df1-1 and Df1-2, were established. Their secreted MAbs (MAbDf1-1 and MAbDf1-2, respectively) bound to the homologous antigen as well as native Der f 1 and a crude extract of D. farinae. Epitopes of MAbDf1-1 and MAbDf1-2 were located at amino acid residues 206NSQHYGISNYCQ217 and 283DYW---NSWD-WGDSG298 of Der f 1. MAbDf1-1 had higher affinity to Der f 1 than the MAbDf1-2. A sandwich ELISA (MAbDf1-1-allergen-PAb) and commercialized reagents (MAb1-allergen-MAb2 sandwich ELISA) were used in comparison for quantification of Der f 1 in 42 dust samples collected from bedrooms and living rooms of 21 houses of the HDM allergic patients. All of the 42 dust samples measured by both ELISAs had the Der f 1 levels higher than 2 mg per gram of fine dust which is the HDM allergy sensitizing level. In addition, Der f 1 levels in 41 samples (except 1 sample from a living room) measured by the MAbDf1-1-PAb and MAb1-MAb2 sandwich ELISAs were higher than 10 mg per g of dust which is the morbidity level of HDM allergen. The local sandwich ELISA showed a high coefficient correlation (r = 0.91) in measuring known amounts of recombinant and native Der f 1. The results indicate that the reagents produced in the present study can be used for measuring the environmental levels of HDM Der f 1. The assay can also be used for standardization of the HDM extract for monitoring patient's allergenic status or for immunotherapeutic purpose.

Prevalence of Listeria monocytogenes in Raw Meats Marketed in Bangkok and Characterization of the Isolates by Phenotypic and Molecular Methods.

Listeria monocytogenes causes listeriosis characterized by septicaemia, encephalitis, and abortion or stillbirth. Regular monitoring of its prevalence in food and characterization of its phenotypes and genotypes are necessary for disease surveillance and tracing the epidemic outbreaks. In this study, the prevalence of L. monocytogenes in raw meats marketed in Bangkok was 15.4%. The bacteria isolated from meat were serotyped and genotyped using enterobacterial repetitive intergenic consensus–polymerase chain reaction (ERIC-PCR). Their virulence-associated genes, antimicrobial susceptibility, and ability to invade intestinal epithelial cells were studied. All 22 L. monocytogenes strains isolated from 104 raw meat samples carried virulence-associated genes, such as actA, flaA, hlyA, iap, inlA, inlB, and prfA. These were serotype 4b, suggesting their pathogenic and epidemic potential. These isolates could be classified into six ERIC-PCR groups: A-F. The majority (59.1%) of the isolates belonged to Group A, and three isolates were Group D which was closely related to the Group A. Two isolates each were Group C and E, and one isolate each was group B and F. Although the isolates belonged to the same serotype and genotype and were all equipped with the virulence-associated genes, they showed a different cell invasion capability and antibiotic susceptibility. All the isolates were susceptible to ampicillin, amikacin, chloramphenicol, gentamicin, imipenem, penicillin G, sulphamethoxazole-trimethoprim, and tetracycline. However, one isolate showed only intermediate susceptibility to tetracycline. The data provide the first molecular insight into the L. monocytogenes isolates in Thailand and elucidate a potential risk of people contracting listeriosis.

Human monoclonal ScFv that inhibits cellular entry and metalloprotease activity of tetanus neurotoxin.

Tetanus is a deadly disease of warm blooded animals and humans caused by an exotoxin called te-tanospasmin or tetanus neurotoxin (TeNT) produced by anaerobic bacterium named Clostridium tetani. TeNT is an A-B toxin; each molecule consists of a heavy chain (HC) containing cellular receptor binding domain and a light chain (LC) with zinc metalloprotease activity. TeNT produced in the infected tissue by the bacteria grown under anaerobic condition binds to ganglioside receptors of peripheral nerve, and endocytosed. The A subunit exits from the endosome and undergoes a retrograde transport via the nerve axon to the spinal cord. This highly toxic enzyme specifically cleaves one of the nerve cell SNARE proteins, i.e., synaptobrevin, resulting in inhibition of the release of neurotransmitters (glycine and GABA) from inhibitory interneuron causing spastic paralysis, the characteristic of tetanus. Current treatment mainstay of human tetanus is by passively administering anti-tetanus toxin produced from animals immunized with adjuvanted tetanus toxoid (TT). There are several obstacles in production and use of the animal derived therapeutic antibody especially the allergic reaction and serum sickness induced by the host immune response to the foreign protein. The animal antibody, mainly IgG, blocks nerve cell entry of the TeNT but does not neutralize the TeNT protease activity per se and cannot reverse the tetanus symptoms. In this study, fully human single chain antibody fragments (HuScFv) were produced from a human antibody phage display library. TT was used as antigen in a single round phage bio-panning to select phage clones that display TT bound-HuScFv from the library. HuScFv from 4 selected huscfv-phagemid transformed E. coli clones inhibited binding of the native TeNT to retinoic acid pulsed human neuroblastoma cells when used at the molecular TeNT:HuScFv ratio of 1:100. HuScFv from one of the 4 clones also inhibited the TeNT mediated cleavage of recombinant synaptobrevin. Further investigation is needed for identification of epitope specificity of these HuScFv and HuScFv effector mechanisms towards the TeNT. Cell penetrating version of the HuScFv that inhibited the TeNT zinc metalloprotease activity should be made. The HuScFv produced in this study either singly or in their suitable combination warrant developing further to a real use in humans as a surrogate of the animal antibody for treatment of tetanus.

Giardia intestinalis in Thailand: Identification of Genotypes.

This study was undertaken to determine the genetic diversities of Giardia intestinalis isolated in Thailand. G. intestinalis cysts were collected from stool samples of 61 subjects residing in Bangkok or in rural communities of Thailand with and without gastrointestinal symptoms. All the cyst samples gave positive tpi amplicons (100% sensitivity), either of the 148- or the 81-bp tpi segments. Cyst assemblage identification of the 148- and 81-bp tpi gene segments by polymerase chain reaction showed that 8% of the cysts were assemblage A, 41% assemblage A and B combined, and 51% assemblage B. The prevalence of assemblage A was significantly lower than that of assemblage B and the mixed types. Restriction fragment length polymorphism (RFLP) of the 384-bp ß-giardin gene segment revealed that 12% and 88% of the assemblage A cysts were AI and AII respectively. RFLP, based on the 432-bp gdh gene segment, showed 45.5% of the assemblage B cysts to be BIII and 54.5% to be BIV. The AI sub-assemblage was less prevalent than the others. All subjects with AI and 50% of the subjects with BIII sub-assemblage cysts were symptomatic; 80% of symptomatic Bangkok residents were adults/elderly while 85% of the rural cases were children.

Allergenicity of native/recombinant tropomyosin, per a 7, of American cockroach (CR), periplaneta Americana, among CR allergic Thais.

In this study, native tropomyosin (Per a 7) of American cockroach (CR), Periplaneta americana, caught in Thailand was purified. Also, gene sequence encoding full length tropomyosin of the CR was PCR ampli-fied by using degenerate primers designed from gene sequences coding for P. americana tropomyosin of the data-base (Per a 7.0101 and Per a 7.0102; accession no.Y14854 and AF106961, respectively). Amino acid sequence deduced from the nucleotide sequence encoding P. americana tropomyosin of this study (GenBank accession no. FJ976895) had 98.59% identity with the sequences of Per a 7.0101 and Per a 7.0102 and was 97.18% identical to the Bla g 7 sequence of German cockroach, Blatella germanica (accession no. AF260897). The native and recom-binant tropomyosins (~34 kDa) were used as antigens in sandwich ELISA for detecting specific IgE in serum sam-ples of 14 consented allergic patients who were positive by skin test to crude CR extract in comparison to 5 indi-viduals who were skin test negative. It was found that 8 (57%) and 6 (43%) of the CR allergic patients gave positive IgE binding results to the native and the recombinant proteins, respectively, while none of the non-allergic counter-parts was positive. Results of immunoblotting conformed to the ELISA results. Tropomyosin extracted from the P. americana caught in Thailand has potential as standard P. americana allergen in clinical monitoring of the allergic Thai patients.

Seasonal levels of the major American cockroach allergen per a 9 (arginine kinase) in Bangkok and their rele-vance for disease severity.

Monitoring the levels of cockroach (CR) allergen in the environment has medical relevance as a clear dose response relationship between CR allergen exposure, sensitization and hospitalization has been reported. In this study, a cross-sectional survey of the levels of a major American cockroach (Periplaneta americana) allergen, i.e. Per a 9 (arginine kinase) in dust samples collected in various seasons throughout the year 2007 from 76 houses of CR allergic Thai patients in the Bangkok metropolitan area were determined. A monoclonal antibody-polyclonal antibody (MAb-PAb) based-sandwich ELISA was used. The MAb was specific to Per a 9 and the PAb was raised in a rabbit against the crude extract of P. americana. The detection limit of the assay was 122 pg of the allergen or 0.024 μg per gram of fine dust powder. The concentrations of Per a 9 were found to be highest during the winter months and lowest in summer. The levels of this CR allergen had a direct correlation with disease exac-erbation; i.e. the majority of the CR allergic patients had their most severe clinical manifestations during winter. Moreover, the CR allergen levels were found to be higher in wood based-houses than in concrete houses.

A murine model of allergy caused by American cockroach (CR), Periplaneta americana.

An animal model resembling the human immuno-pathological features of CR allergy is needed for CR allergy research, e.g., measuring allergenicity of novel allergens, testing immunotherapeutic efficacies of drugs and vaccines. In this study we develop a murine model of American CR, P. americana allergy. BALB/c mice, 6 weeks old, were individually intraperitoneally injected with three doses (days 0, 7 and 14) of alum adjuvanted-crude extract of P. americana. On days 21 and 23, they were given crude CR extract in PBS intranasally (10 microl) and aerosolically (10 ml) via an air-pressure nebulizer, respectively. Mice received alum alone and PBS instead of the CR extract served as non-allergenic controls. All mice were bled twenty four hours after the nebulization and sacrificed. Their serum samples, broncho-alveolar lavage fluids (BALF), and lung tissues were collected. BALF of all allergen-treated mice had marked cellular infiltration notably neutrophils, eosinophils and lymphocytes. The average total cell count in BALF of the allergenic mice was 1.9 x 10(5) cells/ml which out-numbered those of the non-allergenic controls (8 x 10(4) cells/ml). The eosinophil infiltration was pronounced in lungs of the allergen-treated mice. Specific serum IgE to the CR extract elevated in serum samples of all allergen treated mice and nil in the sera of the controls. None of the mice showed detectable level of IgG2a to the CR extract. RT-PCR revealed that all allergen-treated mice had marked increase of IL-13, IL-4 and TNF-alpha gene expressions, slight increase of IL-5 gene expression, and absence of detectable IFN-gamma gene expression in comparison to the non-allergenic controls. None of the allergen-treated mice and 50% of the non-allergenic controls had IL-12 gene expression as detected by RT- PCR. One allergen treated-mouse (25%) had subpar level of the IL-18 gene expression compared to the controls. Results of the quantitative real-time PCR conformed to those of the RT-PCR. A murine model of P. americana resembling human allergic manifestations was successfully developed.