[Show abstract][Hide abstract] ABSTRACT: Purpose
Cockroach (CR) is a common source of indoor allergens, and Per a 1 is a major American CR (Periplaneta americana) allergen; however, several attributes of this protein remain unknown. This study identifies a novel specific B cell epitope and anatomical locations of Per a 1.0105.
Recombinant Per a 1.0105 (rPer a 1.0105) was used as BALB/c mouse immunogen for the production of monoclonal antibodies (MAb). The MAb specific B cell epitope was identified by determining phage mimotopic peptides and pair-wise alignment of the peptides with the rPer a 1.0105 amino acid sequence. Locations of the Per a 1.0105 in P. americana were investigated by immunohistochemical staining.
The rPer a 1.0105 (~13 kDa) had 100%, 98% and ≥90% identity to Per a 1.0105, Per a 1.0101, and Cr-PII, respectively. The B-cell epitope of the Per a 1.0105 specific-MAb was located at residues99 QDLLLQLRDKGV110 contained in all 5 Per a 1.01 isoforms and Per a 1.02. The epitope was analogous to the Bla g 1.02 epitope; however, this B-cell epitope was not an IgE inducer. Per a 1.0105 was found in the midgut and intestinal content of American CR but not in the other organs. The amount of the Per a 1 was ~544 ℃g per gram of feces.
The novel Per a 1 B-cell epitope described in this study is a useful target for allergen quantification in samples; however, the specific MAb can be used as an allergen detection reagent. The MAb based-affinity resin can be made for allergen purification, and the so-purified protein can serve as a standard and diagnostic allergen as well as a therapeutic vaccine component. The finding that the Per a 1 is contained in the midgut and feces is useful to increase yield and purity when preparing this allergen.
[Show abstract][Hide abstract] ABSTRACT: Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus
hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5’-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.
[Show abstract][Hide abstract] ABSTRACT: Vespa affinis (Asian wasp, Thai banded tiger wasp or local name: Tor Hua Seua) causes the most frequent incidence of medically important Hymenoptera sting in South and Southeast Asia. However, data on the venom components attributable to the sting derived-clinical manifestations (local reactions, IgE mediated-anaphylaxis and/or systemic envenomation) are lacking. This study provides first set information on V. affinis venom proteome, allergenome and IgE reactivity of individual venom components. From 2DE-gel based-proteomics, the venom revealed 93 protein spots of which proteins in 51 spots could be identified and classified into three groups: typical venom components and structural and housekeeping proteins. Venom proteins in 32 spots reacted with serum IgE of wasp allergic patients. Major allergenic proteins which reacted to IgE of >50% of the wasp allergic patients included PLA1 (100%), arginine kinase (73%), heat shock 70 kDa protein (73.3%), venom allergen-5 (66.7%), enolase (66.7%), PLA(1) magnifin (60%), glyceraldehyde-3-phosphate dehydrogenase (60%), hyaluronidase (53.3%) and fructose-bisphosphate aldolase (53.3%). The venom minor allergens were GB17876 transcript (40%), GB17291 transcript (20%), malic enzyme (13.3%), aconitate hydratase (6.7%) and phosphoglucomutase (6.7%). The information has diagnostic and clinical implications for future improvement of case diagnostic sensitivity and specificity, component-resolve diagnosis (CRD) and design of specific Hymenoptera venom immunotherapy.
Journal of Proteome Research 01/2014; 13(3). DOI:10.1021/pr4009139 · 5.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: NS3 helicase is a pivotal enzyme involved in the early and late phases of hepatitis C virus (HCV) replication. The primary sequence and tertiary structure of this virus enzyme differ from human helicase to a certain extent; thus this virus protein has potential as a novel anti-HCV target. In this study, recombinant C-terminal NS3 protein of HCV genotype 3a with endowed helicase activity was produced and used as antigen by selecting VH/VHH display phage clones from an established humanized-camel single domain antibody library that bound specifically to HCV helicase. The VH/VHH derived from phage transfected E. coli clones were linked molecularly to a cell penetrating peptide, i.e., penetratin (PEN). The cell penetrable VH/VHH (transbodies) could reduce the amounts of the HCV RNA released into the cell culture fluid and inside Huh7 cells infected with pJFH1 replicon with a greater effect on the former compared to the latter. Regions and residues of the helicase bound by the transbodies were determined by phage mimotope searching and multiple alignments as well as homology modeling and molecular docking. The epitope of one transbody (PEN-VHH9) encompassed residues 588RLKPTLHGPTPLLYRLGA605 of the domain 3 necessary for helicase activity while another transbody (PEN-VH59) interacted with the areas covering the phenylalanine loop and arginine clamp of the domain 2 which are important for the proper folding of the enzyme as well as nucleic acid substrate binding. Although the molecular mechanisms of the prototypic transbodies on NS3 helicase need further investigation, these transbodies have high potential as novel, safe and mutation tolerable anti-HCV agents.
[Show abstract][Hide abstract] ABSTRACT: Periodic monitoring of Staphylococcus aureus characteristics in a locality is imperative as their drug-resistant variants cause treatment problem. In this study, antibiograms, prevalence of toxin genes (sea-see, seg-ser, seu, tsst-1, eta, etb, and etd), PFGE types, accessory gene regulator (agr) groups, and ability to form biofilm of 92 S. aureus Thailand clinical isolates were investigated. They were classified into 10 drug groups: groups 1-7 (56 isolates) were methicillin resistant (MRSA) and 8-10 (36 isolates) were methicillin sensitive (MSSA). One isolate did not have any toxin gene, 4 isolates carried one toxin gene (seq), and 87 isolates had two or more toxin genes. No isolate had see, etb, or tsst-1; six isolates had eta or etd. Combined seg-sei-sem-sen-seo of the highly prevalent egc locus was 26.1%. The seb, sec, sel, seu, and eta associated significantly with MSSA; sek was more in MRSA. The sek-seq association was 52.17% while combined sed-sej was not found. Twenty-three PFGE types were revealed, no association of toxin genes with PFGE types. All four agr groups were present; agr group 1 was predominant (58.70%) but agr group 2 strains carried more toxin genes and were more frequent toxin producers. Biofilm formation was found in 72.83% of the isolates but there was no association with antibiograms. This study provides insight information on molecular and phenotypic markers of Thailand S. aureus clinical isolates which should be useful for future active surveillance that aimed to control a spread of existing antimicrobial resistant bacteria and early recognition of a newly emerged variant.
[Show abstract][Hide abstract] ABSTRACT: Currently, there is a need of new anti-influenza agents that target influenza virus proteins other than ion channel M2 and neuraminidase. Non-structural protein-1 (NS1) is a highly conserved multifunctional protein which is indispensable for the virus replication cycle. In this study, fully human single chain antibody fragments (HuScFv) that bound specifically to recombinant and native NS1 were produced from three huscfv-phagemid transformed E. coli clones (nos. 3, 10 and 11) selected from a human ScFv phage display library. Western blot analysis, mimotope searching/epitope identification, homology modeling/molecular docking and phage mimotope ELISA inhibition indicated that HuScFv of clone no. 3 reacted with NS1 R domain important for host innate immunity suppression; HuScFv of clone nos. 10 and 11 bound to E domain sites necessary for NS1 binding to the host eIF4GI and CPSF30, respectively. The HuScFv of all clones could enter the influenza virus infected cells and interfered with the NS1 activities leading to replication inhibition of viruses belonging to various heterologous A subtypes and type B by 2-64 fold as semi-quantified by hemagglutination assay. Influenza virus infected cells treated with representative HuScFv (clone 10) had up-expression of IRF3 and IFN- genes by 14.75 and 4.95 fold, respectively, in comparison with the controls, indicating that the antibodies could restore the host innate immune response. The fully human single chain antibodies have high potential for developing further as a safe (adjunctive) therapeutic agent for mitigating, if not abrogating, severe symptoms of influenza.
Antiviral research 08/2013; 100(1). DOI:10.1016/j.antiviral.2013.07.019 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Cockroach (CR) allergens frequently cause severe asthma in CR-sensitized subjects. Allergen-specific immunotherapy causes a shift of allergic Th2 responses towards Th1 and/or regulatory T cell (Treg) responses which reduce airway inflammation and prevent disease progression. Data are relatively limited on immunotherapy via CR allergy vaccine. Methods: The therapeutic efficacy of an intranasal liposome-adjuvant vaccine made of a refined Periplaneta americana arginine kinase (AK) was compared to the liposome-entrapped P. americana crude extract (CRE) vaccine. Adult BALB/c mice were rendered allergic to CRE. Three allergic mouse groups were immunized intranasally on alternate days with 8 doses of liposome-entrapped CRE (L-CRE), liposome-entrapped AK and placebo, respectively. One week later, all mice received a nebulized CRE provocation. Evaluation of vaccine efficacy was performed 1 day after provocation. Results: Liposome-entrapped native AK attenuated airway inflammation after the CRE provocation and caused a shift of allergic Th2 to Th1 and Treg responses. The L-CRE also induced a shift from the Th2 to the Th1 response but did not induce a Treg response and could not attenuate the airway inflammation upon allergen reexposure. Conclusions: Intranasal liposome-adjuvant CR allergy vaccine containing native AK (Per a 9) is better than L-CRE in attenuating allergic airway inflammation. The findings of this study not only document a more comprehensive and beneficial immune response induced by the refined allergen vaccine but also raise the point that the shift from the Th2 to the Th1 response alone might not correlate with improved airway histopathology, clinical outcome and quality of life.
International Archives of Allergy and Immunology 05/2013; 161(4):351-362. DOI:10.1159/000348314 · 2.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Most allergen extracts/vaccines used today in clinical practice are derived from natural allergen sources. Therefore, their allergic components may vary as these are prone to natural variation. This study aims to compare the allergenic components and biological potency of crude extracts from wild and laboratory reared American cockroaches.
Crude extracts of male and female of wild and laboratory reared American CR, were prepared by the same method. Their allergenic components were evaluated by in vitro assays such as protein contents, protein profiles, quantification of major allergens (Per a 1 and Per a 9) and IgE inhibition ELISA assay.
There was no statistically significant difference between the protein contents and the concentrations of Per a 1 in the crude extracts from both groups. However, the Per a 9 levels in extracts of wild CR were significantly higher than those from the extracts of laboratory reared CR. The protein patterns of the extracts of laboratory reared CR exhibited more consistency in the number of bands with higher intensity than those of wild CR. Pooled extracts of laboratory reared CR could inhibit IgE binding to that of wild CR up to 78%. The endotoxin content of extracts of laboratory reared CR were ten times less than those of the the wild CR. We have successfully determined the allergenic potency of the extracts of laboratory reared CR versus those of the wild CRs by in vitro assays. Further studies should be performed to determine the biological potency of CR extracts by in vivo assays for clinical application.
Our finding indicates that the laboratory reared CR would be the better source of material in vaccine production than the wild CR.
Asian Pacific journal of allergy and immunology / launched by the Allergy and Immunology Society of Thailand 09/2012; 30(3):231-8. · 1.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA(2)). The PLA(2) exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/V(H)H) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/V(H)H phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-V(H)H, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/V(H)H purified from the E. coli homogenates neutralized PLA(2) enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/V(H)H covered the areas around the PLA(2) catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/V(H)H would ameliorate/abrogate the principal toxicity of the venom PLA(2) (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.
[Show abstract][Hide abstract] ABSTRACT: Measuring allergen levels in the environment provides useful information to guide the management of allergic patients. A laboratory-based test kit sandwich ELISA for quantification of Per a 9, the major allergen of Periplaneta americana was recently developed. However, it is not suitable for screening.
To develop a simple, rapid, and economic format for semi-quantification of Per a 9 assay using dot-blot ELISA technique.
The efficacy of direct dot-blot ELISA and sandwich dot-blot ELISA was evaluated. Direct dot-blot ELISA was selected for further modification into 6 protocols. The selected protocol of direct dot-blot was further compared with the laboratory-based test kit, sandwich ELISA.
The lowest detection limits in protocols no. 1-6 were 3.9, 15.6, 15.6, 62.5, 125 and 62.5 microg/ml of native Per a 9 whereas time required for each protocol was 145, 45, 30, 26, 18 and 26 minutes, respectively. The sensitivity of direct dot-ELISA was 3.9 microg/ml of Per a 9. Protocol no. 3 was the most suitable assay because its detection limits were as low as 15.6 microg/ml of CR allergen and the total process took only 30 minutes. In comparison with the 2 days required for laboratory sandwich ELISA, the selected protocol provided a similar yield of allergen detection but it offers significant savings of time. Additionally, this method could be easily interpreted by various groups of people.
This modified direct dot-blot ELISA is the first membrane ELISA which is a semiquantitative test appropriate for screening American cockroach allergen owing to its simplicity, speed and good yield.
Asian Pacific journal of allergy and immunology / launched by the Allergy and Immunology Society of Thailand 06/2012; 30(2):99-106. · 1.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tetrodotoxin (TTX) is a heterocyclic guanidinium alkaloid (C11H17N3O8) with molecular mass of ∼320 Da. The TTX and toxic analogs block sodium ion activity of mammalian nerve cells resulting in failure to conduct nerve impulse which manifested clinically in host as variable degrees of organ paralysis. Human intoxication occurs after consuming food containing the toxins. Current treatment of the poisoning is supportive and symptomatic. There has been no specific drug or antidote for the TTX mediated malady. In this study, phage clones displaying human single chain antibody fragments (HuScFv) were selected from a human ScFv phage display library. HuScFv derived from phagemid transformed Escherichia coli clones (clones s16 and s35) bound to the TTX as tested by indirect ELISA and band shift assay. Homology modeling and molecular docking revealed that VL domain of the s16-HuScFv interacted with the hydroxyl groups of C6, C9, C10 and C11 of the TTX by means of Tyr 223, Ser226 and Tyr228, while the Asp53 and Asp55 of the VH domain of s35-HuScFv interacted with the positions 1 and 2 of the guanidinium group and the hydroxyl groups at C9 and C10 of the TTX. The s16- and s35-HuScFv neutralized the TTX bioactivity in nerve cell based- and mouse bio-assays. Moreover, the HuScFv could rescue the intoxicated mice from the TTX mediated lethality. Thus, the HuScFv derived from the transformed E. coli clones have high potential as a safe, effective and specific therapeutic remedy for TTX intoxication in humans and warrant further trials.
[Show abstract][Hide abstract] ABSTRACT: A total of 155 puffers caught from two of Thailand's seas, the Gulf of Siam and the Andaman seas, during April to July 2010 were included in this study. Among 125 puffers from the Gulf of Siam, 18 were Lagocephalus lunaris and 107 were L. spadiceus which were the same two species found previously in 2000-2001. Thirty puffers were collected from the Andaman seas, 28 Tetraodon nigroviridis and two juvenile Arothron reticularis; the two new species totally replaced the nine species found previously in 1992-1993. Conventional mouse bioassay was used to determine the toxicity in all fish tissue extracts, i.e., liver, reproductive tissue, digestive tissue and muscle. One of each of the species L. lunaris and L. spadiceus (5.56 and 0.93%, respectively) were toxic. All 28 T. nigroviridis and 2 A. reticularis (100%) from the Andaman seas were toxic. The toxicity scores in T. nigroviridis tissues were much higher than in the respective tissues of the other three fish species. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) revealed that the main toxic principle was tetrodotoxin (TTX). This study is the first to report TTX in L. spadiceus. Our findings raised a concern for people, not only Thais but also inhabitants of other countries situated on the Andaman coast; consuming puffers of the Andaman seas is risky due to potential TTX intoxication.
[Show abstract][Hide abstract] ABSTRACT: El Tor Vibrio cholerae O1 carrying ctxB C trait, so-called El Tor variant that causes more severe symptoms than the prototype El Tor strain, first detected in Bangladesh was later shown to have emerged in India in 1992. Subsequently, similar V. cholerae strains were isolated in other countries in Asia and Africa. Thus, it was of interest to investigate the characteristics of V. cholerae O1 strains isolated chronologically (from 1986 to 2009) in Thailand.
A total of 330 V. cholerae O1 Thailand strains from hospitalized patients with cholera isolated during 1986 to 2009 were subjected to conventional biotyping i.e., susceptibility to polymyxin B, chicken erythrocyte agglutination (CCA) and Voges-Proskauer (VP) test. The presence of ctxA, ctxB, zot, ace, toxR, tcpA C , tcpA E, hlyA C and hlyA E were examined by PCR. Mismatch amplification mutation assay (MAMA) - and conventional- PCRs were used for differentiating ctxB and rstR alleles.
All 330 strains carried the El Tor virulence gene signature. Among these, 266 strains were typical El Tor (resistant to 50 units of polymyxin B and positive for CCA and VP test) while 64 had mixed classical and El Tor phenotypes (hybrid biotype). Combined MAMA-PCR and the conventional biotyping methods revealed that 36 strains of 1986-1992 were either typical El Tor, hybrid, El Tor variant or unclassified biotype. The hybrid strains were present during 1986-2004. El Tor variant strains were found in 1992, the same year when the typical El Tor strains disappeared. All 294 strains of 1993-2009 carried ctxBC ; 237 were El Tor variant and 57 were hybrid.
In Thailand, hybrid V. cholerae O1 (mixed biotypes), was found since 1986. Circulating strains, however, are predominantly El Tor variant (El Tor biotype with ctxB C).
The Indian Journal of Medical Research 04/2011; 133(4):387-94. · 1.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
Asian Pacific journal of allergy and immunology / launched by the Allergy and Immunology Society of Thailand 03/2011; 29(1):78-85. · 1.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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, lap, 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-E 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.
Journal of Health Population and Nutrition 02/2011; 29(1):26-38. DOI:10.3329/jhpn.v29i1.7565 · 1.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A non-egg, non-culture based influenza vaccine that intervenes large influenza outbreaks and protects against heterosubtypic infections is needed. Candidates of such vaccine are likely to be conserved influenza virus proteins or their coding DNA. The vaccine must be conveniently produced at reasonable cost, safe, highly immunogenic and should be able to recall rapidly the immunological memory upon the antigenic re-exposure. In this study vaccines made of full length recombinant NP and M2 of the H5N1 influenza A virus were entrapped either alone or together into liposome (L) made of phosphatidylcholine and cholesterol. The vaccines (L-NP, L-M2 or L-NP+M2) and mocks (L or PBS) were safe without causing any adverse reaction in the intramuscularly injected mice. They were readily immunogenic at a single dose and a recalled response could be detected within one day post booster. Cytokine and antibody data indicated that the vaccines induced a Th1 bias immune response. NP containing vaccines stimulated a marked increase of cytotoxic lymphocytes, i.e., CD8(+), intracellular IFNγ(+) cells, while M2 containing vaccines elicited good antibody response which neutralized infectivity of heterologous influenza viruses. Although the three vaccines elicited different immunological defense factors; nevertheless, they similarly and readily abrogated lung histopathology mediated by viruses belonging to different H5N1 clade/subclade and heterosubtypes including swine H1N1 and human H1N1/2009 viruses. They protected the vaccinated mice against lethal challenges with mouse adapted avian H5N1 virus. The liposome adjuvanted vaccines which demonstrated high protective efficacy in mice warrant testing further in a non-rodent model as well as in humans.
[Show abstract][Hide abstract] ABSTRACT: Among cockroaches (CR) that live in people's homes, two species, i.e., German CR (Blattella germanica) and American CR (Periplaneta americana) predominate in temperate and tropical areas, respectively. CR is an important source of inhalant indoor allergens that sensitize atopic subjects to (localized) type I hypersensitivity or atopy including allergic rhinitis and atopic asthma. In Thailand the predominant CR species is P. americana. CR allergens are found throughout CR infested houses; the number found in kitchens correlates with the degree of CR infestation while sensitization and reactivation of the allergic morbidity are likely to occur in the living room and bedroom. Levels of the CR allergens in homes of CR allergic Thais, measured by using locally made quantification test kits, revealed that the highest levels occur in dust samples collected from the wooden houses of urban slums and in the cool and dry season. CR allergens are proteins that may be derived from any anatomical part of the insect at any developmental stage. The allergens may be also from CR secretions, excretions, body washes or frass. The proteins may be the insect structural proteins, enzymes or hormones. They may exist as dimers/multimers and/or in different isoforms. Exposure to CR allergens in infancy leads to allergic morbidity later in life. Clinical symptoms of CR allergy are usually more severe and prolonged than those caused by other indoor allergens. The mechanisms of acute and chronic airway inflammation and airway hyper-responsiveness (AHR) have been addressed including specific IgE- and non-IgE-mediated mechanisms, i.e., role of protease-activated receptor-2 (PAR2). Participation of various allergen activated-CD4+ T cells of different sublineages, i.e., Th2, Th17, Th22, Th9, Th25, Tregs/Th3 as well as invariant NKT cells, in asthma pathogenesis have been mentioned. The diagnosis of CR allergy and the allergy intervention by CR population control are also discussed.
Asian Pacific journal of allergy and immunology / launched by the Allergy and Immunology Society of Thailand 06/2010; 28(2-3):95-106. · 1.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: House dust mite is a major cause of allergic asthma and rhinitis in Thai population. Skin prick test (SPT) is a useful tool for the diagnosis of the IgE-mediated reactions. The imported commercial mite vaccine for SPT is available but it is relatively expensive. Aim of this study is to compare Siriraj Mite Allergen Vaccine (SMAV) with standardized commercial mite allergen vaccine by skin prick testing in normal Thai adults. A double blind, self-controlled study between the SMAV and standardized commercial mite allergen vaccine was performed by SPT in 17 normal Thai adult males and non-pregnant or non-lactating females aged 18-60 years. The study showed that 35.29 % of non atopic adults had positive SPT reaction to Dp and Df of both SMAV and standardized commercial mite allergen vaccine. Mean wheal and flare diameters from SPT of Dp and Df of SMAV showed strong correlation with standardized commercial mite allergen vaccine (r= 0.768 and 0.897 in Dp and Df respectively, p <0.001). The intraclass correlation was also excellent (0.893 and 0.775 in Dp and Df respectively). There was no significant difference in wheal and flare diameter between SMAV and standardized commercial mite allergen vaccine. No systemic or large local reaction was found in any of the study cases.
Asian Pacific journal of allergy and immunology / launched by the Allergy and Immunology Society of Thailand 03/2010; 28(1):41-5. · 1.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Asian Pacific journal of allergy and immunology / launched by the Allergy and Immunology Society of Thailand 03/2010; 28(1):85-93. · 1.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Health Population and Nutrition 02/2010; 28(1):42-52. DOI:10.3329/jhpn.v28i1.4522 · 1.39 Impact Factor