Nat Malainual

Genetic polymorphisms of major house dust mite allergens

Background Polymorphic sequence substitutions in the major mite allergens can markedly affect immunoglobulin E binding and T cell responses, but there are few studies on environmental isolates from Dermatophagoides pteronyssinus and none for D. farinae.

Laboratory evaluation of aromatic essential oils from thirteen plant species as candidate repellents against Leptotrombidium chiggers (Acari: Trombiculidae), the vector of scrub typhus

Scrub typhus, a rickettsial disease transmitted by several species of Leptotrombidium chiggers (larvae), is endemic in many areas of Asia. The disease is best prevented by the use of personal protective measures, including repellents. In this study commercially produced aromatic, essential oils of 13 plant species and ethanol (control) were tested in the laboratory for repellency against host-seeking chiggers of Leptotrombidium imphalum Vercammen-Grandjean and Langston (Acari: Trombiculidae). A rapid, simple and economic in vitro test method was used by exposing the chigger for up to 5xa0min. Repellency was based on relative percentages of chiggers attracted to test and control substances. Four of the 13 essential oils showed promise as effective repellent against L. imphalum chiggers. Syzygium aromaticum (clove) oil exhibited 100% repellency at 5% concentration (dilution with absolute ethanol), whereas Melaleuca alternifolia (tea tree) oil exhibited 100% repellency at 40% concentration. Undiluted oils of Zingiber cassamunar (plai) and Eucalyptus globules (blue gum) exhibited 100% repellency. Of the remaining nine essential oils, only 100% Pelargonium graveolens (geranium) exhibited >50% repellency (viz. 57%). Styrax torkinensis (benzoin) oil did not exhibit any repellency. These findings show that several aromatic, essential oils of plants may be useful as chigger repellent for the prevention of scrub typhus. Syzygium aromaticum oil may be safer and more economical to prevent chigger attacks than commercially available synthetic chemicals, such as DEET that may have harmful side effects.

Structural and IgE Binding Analyses of Recombinant Der p 2 Expressed from the Hosts Escherichia coli and Pichia pastoris

Background: The house dust mite allergen Der p 2 is one of the most important indoor allergens associated with allergic disease. Recombinant Der (rDer) p 2 with high IgE binding activity can be readily produced in Escherichia coli and Pichia pastoris, but the structure and IgE binding of the different methods of preparation have not been compared. Methods: Secondary structure was assessed by circular dichroism (CD). Intrinsic fluorescence and hydrophobic probe (1-anilinonaphthalene 8-sulphonic acid, ANS) were used to study the Der p 2 hydrophobic cavity. IgE binding was assessed by ELISA inhibition. Results: CD analysis showed the expected secondary structure for both nDer p 2 and refolded Der p 2 prepared from E. coli inclusion bodies but primarily random structure for Der p 2 secreted from P. pastoris. The secreted product, however, had disulphide bonding and could be refolded to a similar structure to natural Der (nDer) p 2 after precipitation with trichloro-acetic or ammonium sulphate. ANS binding and intrinsic Trp92 fluorescence showed that all recombinant proteins were different to nDer p 2 and that the allergen secreted from P. pastoris did not form a hydrophobic cavity. Despite the marked structural changes, all preparations of Der p 2 had similar IgE binding to nDer p 2. Conclusion: Despite almost identical IgE binding, rDer p 2 prepared from both E. coli and P. pastoris showed structural differences to nDer p 2. Der p 2 secreted from P. pastoris lacked most of the natural structure, but refolding could induce the natural structure.

Partial mattress encasing significantly reduces house dust mite antigen on bed sheet surface: a controlled trial.

BACKGROUNDnThe most effective measure in house dust mite antigen reduction is mattress encasing with an impermeable membrane. A reduction in encasing costs will help increase patients compliance in mite antigen avoidance.nnnOBJECTIVEnTo investigate the effectiveness of partial mattress encasing with a nylon sheet produced in Thailand on the reduction of group I mite antigens from beddings.nnnMETHODSnSixty regularly-used beds from the house officers dormitory of the Siriraj Hospital Mahidol University, Thailand, were randomly matched into two groups according to mite antigen levels. The control group (CG) used only regular cotton bed sheets whereas the partial encasing group (PG) used mattresses partially covered with a locally produced nylon sheet underneath the regular cotton bed sheets. Dust collection from the beddings was performed at baseline, 2, 4 and 6 months after application of the nylon sheet. Mite antigen levels were detected by a two step monoclonal antibody ELISA.nnnRESULTSnMite antigen levels in both groups were not different at the beginning of the study. The PG had significantly lower group I antigen levels on regular bed sheet surfaces than the CG (P < .004) at the 2, 4 and 6 month timepoints. However, antigen levels on the mattress surface of the PG was significantly higher than the CG at the end of the study (P < .004). The barrier efficacy of the nylon sheet in preventing migration of group I mite antigens from the mattress to the surface of the regular cotton bed sheet was 94% whereas that of the regular cotton bed sheet was 66% (P = .007).nnnCONCLUSIONnPartial mattress encasing with a locally made nylon sheet can reduce mite antigens on the regular cotton bed sheet surfaces for up to 6 months.

Cytopathogenesis of Naegleria fowleri Thai strains for cultured human neuroblastoma cells.

The aim of this study is to evaluate cellular interaction between free-living amoebae Naegleria fowleri strains and mammalian target cells in vitro. Two Thai strains of N. fowleri; Khon Kaen strain from the environment and Siriraj strain from the patient’s cerebrospinal fluid and the Center of Disease Control VO 3081 strain from Atlanta (US) were studied. Human neuroblastoma (SK-N-MC) and African Green monkey Kidney (Vero) cells were used as target cells. Each cell line was inoculated with each strain of N. fowleri at a ratio of 1:1 and observed for 7xa0days. The uninoculated target cells and each strain of N. fowleri were used as control. The numbers of the challenged and unchallenged cells as well as the free-living amoebae were counted three times by trypan blue exclusion method. The inoculation began when the amoebae attached to the cell membrane and ingested the target cells. In this study, extensive cytopathogenesis with many floating inoculated cells and abundant number of amoebae were observed. The destruction pattern of both inoculated SK-N-MC and Vero target cells were similar. Interestingly, SK-N-MC was more susceptible to N. fowleri strains than the Vero cell. In addition, N. fowleri Siriraj strain showed the highest destruction pattern for each target cell. Our findings suggest that the SK-N-MC should be used as a base model for studying the neuropathogenesis in primary amoebic meningoencephalitis patients.

Scanning electron microscopic study of human neuroblastoma cells affected with Naegleria fowleri Thai strains

In order to understand the pathogenesis of Naegleria fowleri in primary amoebic meningoencephalitis, the human neuroblastoma (SK-N-MC) and African green monkey kidney (Vero) cells were studied in vitro. Amoeba suspension in cell-culture medium was added to the confluent monolayer of SK-N-MC and Vero cells. The cytopathic activity of N. fowleri trophozoites in co-culture system was elucidated by scanning electron microscope at 3, 6, 9, 12, and 24xa0h. Two strains of N. fowleri displayed well-organized vigorous pseudopods in Nelson’s medium at 37°C. In co-culture, the target monolayer cells were damaged by two mechanisms, phagocytosis by vigorous pseudopods and engulfment by sucker-like apparatus. N. fowleri trophozoites produced amoebostomes only in co-culture with SK-N-MC cells. In contrast, we could not find such apparatus in the co-culture with Vero cells. The complete destruction time (100%) at 1:1 amoeba/cells ratio of SK-N-MC cells (1xa0day) was shorter than the Vero cells (12xa0days). In conclusion, SK-N-MC cells were confirmed to be a target model for studying neuropathogenesis of primary amoebic meningoencephalitis.

Complete mattress encasing is not superior to partial encasing in the reduction of mite allergen

Background: Partial mattress encasing was found to be effective in reducing exposure to mite allergen in our previous investigation. We aimed to compare the short‐term efficacy of partial and complete mattress encasing and to study mite‐allergen levels within these mattresses.

Dust mite infestation in cooking flour: experimental observations and practical recommendations

BACKGROUNDnThe first documented case of oral mite anaphylaxis has recently been reported in Thailand, with mites possibly originating from cooking flour.nnnOBJECTIVEnOur study was designed to assess the effects of cooking flours enhancement and storage conditions on mite proliferation and to provide practical recommendations to prevent mite anaphylaxis.nnnMETHODSnIn a factorial experiment, six commercial brands of cooking flours were selected and either inoculated or set free of mites and stored in one of the four containers chosen for the study: original package, plastic bag, plastic box and glass bottle. The resulting experimental units where then stored at either room temperature or in a refrigerator (+4C). In order to determine levels of Der f 1 allergen, 0.1 gram of flour was sampled from each experimental unit and tested by ELISA. Sampling was carried out immediately after inoculation and subsequently at week 2, 4, 6, 8, 10, 12, 16 and 20.nnnRESULTSnLevels of Der f 1 allergen in the inoculated samples increased significantly in all conditions 6 weeks after inoculation (p <0.001) and reached the highest levels at week 8. While experimental units left at room temperature showed higher levels of mite growth (p <0.001), no statistical differences were found among types of containers. The highest amount of Der f 1 was observed for Gogi, followed by Gold Label, tempura flour, corn flour, wheat flour and tapioca starch, respectively (p <0.01).nnnCONCLUSIONSnIn the context of our experiment, mites preferably grew in cooking flours containing high amounts of wheat at room temperature, particularly after 8 week of storage. According to our results, we thus advise to keep household cooking flour refrigerated and while the type of container does not matter, storage should not exceed 20 weeks.

Genetic Polymorphisms and Allergenicity of Blo t 5 in a House Dust Mite Allergic Filipino Population

BACKGROUNDnBlo t 5, a major allergen from the house dust mite Blomia tropicalis had been identified as an important trigger of allergic diseases. Effects of sequence variations to the IgE binding activity of Blo t 5 remain unclear.nnnOBJECTIVEnThe study determined gene polymorphisms of Blo t 5, expressed the recombinant Blo t 5 allergen in E. coli system and determined its IgE reactivity in house dust mite allergic Filipino patients.nnnMETHODnGene polymorphism was determined using Polymerase Chain Reaction and DNA sequencing. Recombinant Blo t 5 was expressed as a GST-fusion protein in E. coli and purified by affinity chromatography. IgE-binding activity of 230 age-and sex-matched allergic and non-allergic subjects was subjected to Enzyme-linked Immunosorbent Assay and Western blot analysis.nnnRESULTSnFive polymorphic variants of Blo t 5 were identified with 26 DNA base and 12 amino acid substitutions thus implying a high degree of sequence diversity. Of the 115 cases, 68.70% and 59.13% showed reactivity to Blo t extract and rBlo t 5 respectively. Total and specific IgE levels of allergic and non-allergic subjects differ significantly (p< 0.0001).nnnCONCLUSIONSnBlo t 5 gene exhibits polymorphic variants with predicted amino acid sequences resulting to changes in its IgE epitopes. These polymorphisms may suggest variability of allergenic properties of Blo t 5. The incorporation of the Blo t 5 variants in the panel of diagnostic allergens and immunotherapy is highly recommended.

The Blomia tropicalis allergen Blo t 7 stimulates innate immune signalling pathways through TLR2

Although the house dust mite species Blomia tropicalis is a leading cause of allergic diseases in tropical and subtropical regions, the identification and characterization of the allergenic proteins remain incomplete.