Comparative analysis of cysteine proteases reveals gene family evolution of the group 1 allergens in astigmatic mites.

BACKGROUND: Astigmatic mites contain potent allergens that can trigger IgE-mediated immune responses, leading to allergic diseases such as asthma, allergic rhinitis and atopic dermatitis. In house dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae, group 1 allergens (Der p 1 and Der f 1), characterized as papain-like cysteine proteases, have been defined as the major allergens that have high prevalence and potency. Previous studies of mite group 1 allergens mainly focused on identification, comparison of sequence and structure, as well as the investigation of cross-reactivity. To achieve a comprehensive view of mite group 1 allergens, we performed a comparative genomic analysis of all the cysteine proteases in six astigmatic mite species to elucidate the evolutionary relationships of group 1 allergens. METHODS: Based on the high-quality and annotated genomes, all the cysteine proteases in six astigmatic mite species were identified by sequence homology search. The phylogenetic relationships, gene synteny and expression levels were revealed by bioinformatic tools. The allergenicity of recombinant cysteine proteases was evaluated by enzyme-linked immunosorbent assay. RESULTS: Tandem duplication was revealed as the major feature of cysteine protease gene evolution in astigmatic mites. The high IgE-binding capacity and the significant expression level of the cysteine protease DP_007902.01 suggested its potential as a novel group 1 allergen of D. pteronyssinus. In addition, gene decay events were identified in the skin-burrowing parasitic mite Sarcoptes scabiei. CONCLUSION: This comprehensive analysis provided insights into the evolution of cysteine proteases, as well as the component-resolved diagnosis of mite allergies.

Genome assembly and annotation of Periplaneta americana reveal a comprehensive cockroach allergen profile.

BACKGROUND: One of the most common cockroach types in urban areas, the American cockroach (Periplaneta americana), has been reported to impose an increased risk of allergies and asthma. Limited groups of allergens (Per a 1-13) have been identified in this species due to the lack of genome-related information. METHODS: To expand the allergen profile of P. americana, genomic, transcriptomic, and proteomic approaches were applied. With the support of a high-quality genome assembled using nanopore, Illumina, and Hi-C sequencing techniques, potential allergens were identified based on protein homology. Then, using enzyme-linked immunosorbent assay, selected allergens were tested in Thai patients allergic to P. americana. RESULTS: A chromosomal-level genome of P. americana (3.06 Gb) has been assembled with 94.6% BUSCO completeness, and its contiguity has been significantly improved (N50 = 151 Mb). A comprehensive allergen profile has been characterized, with seven novel groups of allergens, including enolase (Per a 14), cytochrome C (Per a 15), cofilin (Per a 16), alpha-tubulin (Per a 17), cyclophilin (Per a 18), porin3 (Per a 19), and peroxiredoxin-6 (Per a 20), showing IgE sensitivity in enzyme-linked immunosorbent assay. A new isoallergen of tropomyosin (Per a 7.02) and multiple potential isoallergens of Per a 5 were revealed using bioinformatics and proteomic approaches. Additionally, comparative analysis of P. americana with the closely related Blattodea species revealed the possibility of cross-reaction. CONCLUSION: The high-quality genome and proteome of P. americana are beneficial in studying cockroach allergens at the molecular level. Seven novel allergen groups and one isoallergen in Per a 7 were identified.

A Component-Resolved Therapeutic Vaccine for Cockroach Allergy Made of Per a 9 and Transforming Growth Factor-ß Homologue, an Immunosuppressive Protein of Brugia malayi.

Allergen-specific-immunotherapy (ASIT) can cause long-term resolution of allergic diseases, reduces drug use and chances of new allergen sensitization. Nevertheless, therapeutic vaccine and data on ASIT efficacy for cockroach (CR) allergy are relatively scarce. In this study, efficacy and mechanism of a novel intranasal vaccine consisting of liposome (L)-entrapped mixture of American CR (Periplaneta americana) major allergen (Per a 9) and immunosuppressive protein of Brugia malayi nematode named transforming growth factor-beta homologue (TGH) in treatment of CR allergy were investigated along with two other vaccines (L-Per a 9 alone and L-TGH alone). All three vaccines could reduce pathogenic type 2 response and lung immunopathology in the vaccines-treated CR-allergic mice, but by different mechanisms. L-Per a 9 caused a deviation of the pathogenic type 2 to type 1 response (IFN-γ-upregulation), whereas the L-(TGH + Per a 9) and L-TGH generated regulatory immune responses including up-expression of immunosuppressive cytokine genes and increment of serum adenosine and lung indoleamine-2,3-dioxygenase-1 which are signatures of regulatory T cells (Tregs) and tolerogenic dendritic cells, respectively. The L-(TGH + Per a 9) should be further evaluated towards clinical application, as this vaccine has a propensity to induce broadly effective therapeutic effects for inhalant allergies.

Cockroaches: Allergens, Component-Resolved Diagnosis (CRD) and Component-Resolved Immunotherapy.

Allergic diseases are assuming increasing trend of prevalence worldwide. The diseases confer increasing demand on medical and healthcare facilities. Patients with allergies have poor quality of life and impaired cognition. Adult patients have subpar working efficiency while afflicted children are less effective at school, often have school absenteeism and need more attention of their caregivers. All of them lead to negative socio-economic impact. This narrative review focuses on cockroach allergy including currently recognized cockroach allergens, pathogenic mechanisms of allergy, componentresolved diagnosis and allergen-specific immunotherapy, particularly the component-resolved immunotherapy and the molecular mechanisms that bring about resolution of the chronic airway inflammation.

Immunochromatographic test for rapid serological diagnosis of human angiostrongyliasis.

OBJECTIVES: The serological diagnosis of human infection with Angiostrongylus cantonensis remains problematic because there are no commercially available validated tests. Most laboratories use domestically prepared tests such as the enzyme-linked immunosorbent assay (ELISA) or immunoblotting. Since laboratory facilities are not always available in endemic areas, we developed and assessed a rapid lateral flow immunochromatographic assay (AcQuickDx Test) to detect anti-A. cantonensis antibodies in human serum. METHODS: The test device was assembled with purified 31-kDa glycoprotein as diagnostic antigen and with gold-labelled anti-human immunoglublin-G as the detector reagent. A total of 97 serum samples were tested - 19 samples from clinically diagnosed patients with detectable A. cantonensis-specific antibody in immunoblotting; 43 samples from patients with other parasitic diseases, i.e. gnathostomiasis (n=13), toxocariasis (n=2), trichinellosis (n=2), hookworm infection (n=4), filariasis (n=5), cysticercosis (n=9), paragonimiasis (n=2), opisthorchiasis (n=3), and malaria (n=3); and 35 samples from normal healthy subjects. RESULTS: The sensitivity, specificity, positive predictive value and negative predictive value of AcQuickDx Test to detect anti-A. cantonensis specific antibodies in serologically confirmed angiostrongyliasis cases, were 100%, 98.72%, 95% and 100%, respectively. Positive AcQuickDx was observed in 1 of 4 cases with hookworm infections. No positive AcQuickDx was observed in cases with other parasitic diseases, and the individual healthy subjects. CONCLUSIONS: AcQuickDx Test is rapid, highly sensitive and specific, and easy to perform without additional equipment or ancillary supplies. It yields results that are interpreted visually, and possesses a long shelf-life at room temperature. Thus, it can be applied as an additional test for clinical diagnostic support of angiostrongyliasis either in conventional laboratories or for remote areas where laboratory infrastructure is not available.

Allergenicity of native and recombinant major allergen groups 1 and 2 of Dermatophagoides mites in mite sensitive Thai patients.

BACKGROUND AND OBJECTIVE: Natural allergenic extracts using for diagnosis and immunotherapy may have batch-to-batch variations and contaminations with unrefined allergens or non-allergenic components. Thus, recombinant allergen is believed to overcome these shortcomings. In this study, native and recombinant allergens of group 1 and 2 of Dermatophagoides mites were produced and their allergenicities were compared. METHODS: Native allergens were prepared by MAb affinity chromatography. All recombinant allergens were produced in E. coli expression system. IgE reactivities of these allergens were determined by IgE-ELISA. RESULTS: The native and recombinant Der p 1, Der p 2, Der f 1, Der f 2 had molecular weights of approximately 25, 15, 25 and 15 kDa, respectively. IgE reactivities of nDer p 1, nDer f 1, rDer p 1 and rDer f 1 were 96.67%, 90%, 43.33% and 46.67%, respectively. Allergenicities of nDer p 2, nDer f 2, rDer p 2 and rDer f 2 were 86.67%, 96.43%, 76.67% and 89.29%, respectively. The findings indicated that recombinant group-1 products were minor allergens which revealed no correlation with their native forms. In contrast, recombinant group-2 allergens were major allergens and showed a significant correlation to their native allergens. CONCLUSION: We successfully produced native and recombinant group-1 and group-2 allergens. According to their allergenicities, recombinant Der p 2 and rDer f 2 have potential to replace native allergen in diagnostic and therapeutic extracts. Moreover, they can employ as a standard reagent to measure the amount of group 2 allergen in the environment by sandwich-ELISA and utilise this as an immunogen for MAb production.

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

BACKGROUND: The first documented case of oral mite anaphylaxis has recently been reported in Thailand, with mites possibly originating from cooking flour. OBJECTIVE: Our 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. METHODS: In 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. RESULTS: Levels 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). CONCLUSIONS: In 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.

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, Dfl-1 and Dfl-2, were established. Their secreted MAbs (MAbDfl-1 and MAbDfl-2, respectively) bound to the homologous antigen as well as native Der f 1 and a crude extract of D. farinae. Epitopes of MAbDfI-1 and MAbDfl-2 were located at amino acid residues 206NSQHYGISNYCQ217 and 283DYW---NSWD-WGDSG298 of Der f 1. MAbDf-1 had higher affinity to Der f 1 than the MAbDfl-2. A sandwich ELISA (MAbDfl-1-allergen-PAb) and commercialized reagents (MAbl-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 MAbDfI-1-PAb and MAbl-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.

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 tetanospasmin 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 beta-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.

Ganoderma lucidum: a cause of pseudoparasitosis.

We report a pseudoparasitosis case due to Ganoderma lucidum, (lingzhi or reishi mushroom); we believe this to be a first reported case in Thailand. A 49-year-old male patient with non-Hodgkins lymphoma presented with chronic watery diarrhea. He had a history of consumption of powdered lingzhi extract as a dietary supplement and herbal medicine. Stool examination demonstrated many spores of G. lucidum, which must be differentiated from intestinal helminth ova and coccidia. After discontinuation of mushroom spores ingestion, the diarrheal symptoms improved and fecal examination subsequently showed no Ganoderma spores. Many artifacts in the stool may be confused with parasites. Differentiation of parasites from artifacts depends on characterization of the size, shape, structure, and reactivity with common stains.

The levels of cockroach allergen in relation to cockroach species and allergic diseases in Thai patients.

UNLABELLED: Recently, cockroaches have been established as the second most Important allergen, producing allergic diseases, especially in low socioeconomic populations. In Thailand, about 44-61% of atopic patients were positive to cockroach extract by a skin-prick test. This study examined cockroach allergen levels in relation to cockroach species and allergic diseases in the houses of cockroach-sensitive patients. Sixty households of allergic patients in the Bangkok metropolitan area were surveyed using open- and closed-ended questionnaires. Cockroaches were collected using commercial cockroach traps, while dust samples were obtained from the bedrooms, kitchens and living rooms of the houses using a vacuum cleaner. The cockroaches were counted and their species Identified. The levels of cockroach allergens were determined by specific monoclonal antibodies using a monoclonal antibody-polyclonal antibody based sandwich ELISA kit. Six cockroach species were Identified: Periplaneta americana (American cockroach, 72.15%), Supella longlpalpa (2.75%, found in only one house), Periplaneta brunnea (0.78%), Periplaneta australaslae (0.78%), Neostylopyga rhombifolla (0.78%), Blattella germanica (German cockroach, 0.39%) and nymphs (22.35%). Allergens of the predominant species, P. americana, were detectable in all homes studied, with the highest levels in the kitchen areas. The range of allergen levels in house dust varied from 0.40-162.00 microg per g of dust. The median and mean allergen levels in kitchen dust were 59.16 microg and 62.80 microg per g of dust, respectively, while the median allergen level in bedroom dust was only 15.90 microg per g of dust. The German cockroach allergen (Bla g 2) was undetectable in any of the houses. IN CONCLUSION: P. americana was the most common cockroach and may be the species causing allergic diseases, especially asthma, in Thailand, which differs from the USA and Europe

Blastocystis hominis infection in irritable bowel syndrome patients.

Irritable bowel syndrome (IBS) is a functional bowel disorder in which abdominal pain is associated with a defect or a change in bowel habits. Subtle inflammation, especially after infectious enteritis, has been sometimes suspected as one mechanism of pathogenesis. This research was performed (1) to evaluate the prevalence of parasitic infections and (2) the possible association of IBS and parasitic infections. Fifty-nine IBS patients were recruited using symptom-based criteria (Rome Criteria II) with an absence of intestinal parasitic infection by direct smear method. Stool samples of individual patients were examined using 7 methods, ie examination for stool occult blood, simple saline smear method, formalin-ether technique, culture for Blastocystis hominis, modified trichrome stain, modified Ziehl-Neelsen method, and trichrome stain for parasitic and bacterial infections. Of the 59 patients, stool samples of 13 patients (22.1%) were positive for parasites. These were B. hominis (13.6%), Strongyloides stercoralis larvae (1.7%), Giardia lamblia cysts (1.7%), and non-pathogenic protozoa, ie Endolimax nana cysts (5.1%). The prevalence rate of parasitic infections in the control group (20%) was not statistically different from the patients. There was no statistical difference between B. hominis infection in IBS patients and control was found in this study (p = 0.87). In the IBS group, B. hominis infection predominated (13.6%), while other parasitic infections were found in 8.5%. The culture method for B. hominis is more sensitive than the direct (simple) stool smear method, which is the routine diagnostic method in most laboratories. These results were also found in control group.

Relationship of tobacco smoking with serum vitamin B12, folic acid and haematological indices in healthy adults.

OBJECTIVES: To investigate the effects of tobacco smoking on serum vitamin B12, folic acid and haematological parameters in healthy Thai smokers and non-smokers. DESIGN: Cross-sectional study of smokers and non-smokers in a military unit in Bangkok, Thailand. SETTING: A military unit in Thailand. SUBJECTS: One hundred and twenty-three male smokers from a military unit in Bangkok, who participated voluntarily in the study, were investigated. Sixty-six male non-smokers from the same unit were selected as controls. Fasting blood samples were collected for investigation of vitamin B12, folic acid and haematological variables. RESULTS: The serum folic acid concentration of smokers was lower than that of non-smokers, but was not statistically significantly different. Haemoglobin was lower in smokers than in non-smokers; 16.3% of smokers were anaemic compared with only 3.0% of non-smokers. Anaemia was not related to folate deficiency. The white blood cell count was found to be higher in smokers than in non-smokers. CONCLUSION: The results of this study suggest that there were low serum folic acid concentrations in smokers compared with non-smokers, which might contribute to the development of vascular and cardiovascular diseases. The higher white blood cell count might be indicative alterations in the immune functions of smokers.

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.