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310 hornbills. Subsequently, we increase breeding success and conservation awareness through nest cavities improvements, ceased poaching of hornbill chicks and getting community involvement in hornbill conservation. Currently supported by BIOTEC, we are investigating the characteristics of Phylogenetic of Thai hornbills and genetic variations of hornbills in fragmented forest landscapes and determining their population and habitat status throughout Thailand. (Oral Presentation (Special Topic) at Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, 4 August 2005. Supported by Thailand Hornbill Project, Hornbill Research Foundation and The National Center for Genetic Engineering and Biotechnology (BIOTEC) STUDY ON THE POLYMORPHISM OF MULTIPLE LOCI OF VARIABLE NUMBER OF TANDEM REPEATS IN MYCOBACTERIUM TUBERCULOSIS (NO. 818) 1 Nat Smittipat; 1 Pamaree Billamas; 2 Manee Palittapongarnpim; 2 Arunee Thong-On; 2 Mansuet M. Temu; 3 Prathep Thanakijcharoen; 3 Opart Karnkawinpong; 2 Prasit Palittapongarnpim 1 National Center for Genetic Engineering and Biotechnology, Phatumthani, 2 Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, and 3 Amnat Charoen Hospital, Amnat Charoen, Thailand. E-mail: Prasit@biotech.or.th Key words : VNTR, polymorphisms, Mycobacterium Tuberculosis Genotyping based on variable number of tandem repeats (VNTR) is currently a very promising tool for studying the molecular epidemiology and phylogeny of M. tuberculosis. Here, we investigated the polymorphisms of 48 loci of direct or tandem repeats in M. tuberculosis previously identified by our group. Thirty-nine loci including nine novel ones are polymorphic. Ten VNTR loci had high allelic diversity (Nei’s diversity values ≥0.6) and subsequently were used as the representative VNTR-typing set for comparison to IS6110-based restriction fragment length polymorphism (RFLP) typing. The 10-loci VNTR set, potentially providing >2×10 9 allele combinations, obviously showed the discriminating capacity over IS6110-RFLP method for M. tuberculosis isolates with less than 6 IS6110-hybridized bands whereas it had equivalent resolution to the IS6110-RFLP for the isolates having >5 IS6110 bands. Allelic diversity of many VNTR loci varied in each IS6110 type. Genetic relationships inferred from the 10-VNTR set supported the notion that M. tuberculosis may be evolved from two different lineages (high and low-IS6110 copy). In addition, we found that many VNTR loci had significant relationships to each other. These relationships could cause a restriction of the VNTR-typing discriminating capability to some extent. Our results suggested that VNTR-PCR typing is practically useful for application to molecular epidemiological and phylogenetic studies of M. tuberculosis. The discriminating power of VNTRtyping system could still be enhanced by the supplementation of an extra number of (new) VNTR loci. (Journal of Clinical Microbiology 43 (10), (2005): (Accepted). Faculty of Science RESTRICTION FRAGMENT LENGTH POLY- MORPHISM STUDY OF NATION-WIDE SAMPLES OF MYCOBACTERIUM TUBERCULOSIS IN THAILAND, 1997-1998 (NO. 819) 1 Dhanida Rienthong; 2 Pravech Ajawatanawong; 1 Somsak Rienthong; 3 Angkana Chaiprasert ; 1 Saijai Smithtikarn; A Pasakorn; 4 Prasit Palittapongarnpim. 1 Tuberculosis Division, Department of Disease Control, Bangkok, 2 National Center for Genetic Engineering and Biotechnology, Phatumthani, 3 Department of Microbiology, Faculty of Medicine, Siriraj Hospital and 4 Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand. Email: Prasit@biotech.or.th Key words: RFLP, recent transmission, Mycobacterium tuberculosis Setting : During 1997-1998, a national anti-tuberculosis drug resistance surveillance was conducted in Thailand as a part of a global project. Objective : To evaluate the IS6110-hybridization patterns and the level of clustering, which was expected to be low due to the short duration of the sample collection. Design : 828 bacterial isolates were available for fingerprinting by standard IS6110 hybridization. Results : The RFLP patterns varied with geographic locations, ages of the patients, and resistance to rifampin and streptomycin. The Beijing strains were more common among younger patients and their prevalence appeared to decrease with the distance from Bangkok, while the opposite was true for the singlebanded isolates. Excluding isolates containing 5 or less copies of IS6110, 26.4 % were clustered. The clustering was more common among female. The clustered isolates were sometimes from different provinces and, if resistant to drugs, usually possessed different resistance profiles. Conclusions : The results raised a question of the validity of inferring the recent transmission from the clustering of IS 6110hybridization patterns in some settings in Thailand. Evaluating the level of recent transmission in a nation-wide study in a country with high incidence of tuberculosis should be done cautiously. (International Journal of Tuberculosis Lung Disease 9(5), (2005): 576-581.) BURKHOLDERIA PSEUDOMALLEI INVASION AND ACTIVATION OF EPITHELIAL CELLS REQUIRES ACTIVATION OF P38 MITOGEN- ACTIVATED PROTEIN KINASE (NO. 820) P. Utaisincharoen 1 , S. Arjcharoen 1 , I. Lengwehasatit 2 , K. Limposuwan 1 , S. Sirisinha 1 1 Department of Microbiology, Faculty of Science, Mahidol University; 2 Department of Biotechnology, Faculty of Science, Mahidol University. E-mail: scput@mahidol.ac.th PDF created with FinePrint pdfFactory Pro trial version http://www.pdffactory.com
Mahidol University Annual Research Abstracts, Vol. 33 311 Key words: Burkholderia pseudomallei, melioidosis, p38 MAP kinase, human lung epithelial cell line, IκBα Burkholderia pseudomallei is a causative agent of melioidosis. This gram-negative bacterium is able to survive inside the macrophages and also able to invade non-phagocytic cells including epithelial cells. Interaction of pathogenic bacteria to the host cells is frequently associated with activation of mitogenactivated protein (MAP) kinases signaling activity. In this study, we demonstrated that B. pseudomallei stimulated p38 MAP kinase of human alveolar lung epithelial cell line (A549). Phosphorylation of p38 was observed after 15 min, attained a maximal level at 60 min after the infection. A specific inhibitor of p38 phosphorylation, SB 203580, was able to inhibit invasion of this bacterium into the cells suggesting that invasion of B. pseudomallei required activation of p38. In contrast, wortmannin which is a specific inhibitor of phosphoinositide 3-kinase (PI3-kinase) failed to inhibit the invasion. Moreover, SB 203580 can also interfere with IkBa degradation and IL-8 mRNA expression, indicating that the phosphorylation of p38 occurred upstream of NF-kB activation. Cytochalasin D, an inhibitor of actin polymerization needed for internalisation of bacteria, did not have any effect on the phosphorylation of p38. These results indicate that B. pseudomallei stimulate phosphorylation of p38 making by initial contact with the cell surface components and do not require internalisation and interaction with intracellular cytoplasmic components of the cells. (Microb Pathogenesis 38, (2005); 107-112.) DENGUE VIRUS-ENHANCING ANTIBODY POSSIBLY SUPPRESSES NITRIC OXIDE PRODUCTION VIA IL-10 MEDIATED SUPPRESSIVE PATHWAY (NO. 821) Takol Chareonsirisuthigul 1 , Siriphen Kalayanarooj 2 , Sukathida Ubol 1* 1 Department of Microbiology, Faculty of Science Mahidol University, Bangkok, Thailand; 2 WHO Collaborating Centre Case Management of Dengue/DHF/DSS, Queen Sirikit National Institute of Child Health, Bangkok Thailand. Key words : Dengue virus, Enhancing antibody, Nitric oxide Background: Dengue virus (DV) causes a spectrum of clinical manifestation ranging from asymptomatic infection, classical dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Recently, we demonstrated that patients who developed DHF during secondary infection generate low level of nitric oxide (NO) but high level of viral load and vice versa for DF patients. Thus, NO suppression might be one of the factors which determine high viremia stage in secondary DHF patients. Since this phenomenon occurred in secondary DHF patients but not in primary DHF or DF patients, therefore, we hypothesized that enhancing antibody may play a role on NO production suppression. In the presence study, we performed in vitro experiments to test the suppressive effect of enhancing antibody on NO production in human monocyte cell line infected with DV. The suppressive mechanism was partially investigated. Methods: Human monocyte cells, THP-1, were infected with DV in the presence or absence of enhancing antibodies. Viral production in supernatant was quantitated by real-time RT-PCR. Amount of NO in supernatant was measured by Griess reaction. Level of IL-10, IL-12 and IFN-ã productions were determined by ELISA. Results: In the presence of antibodies from DHF patients, enhancing viral replication was found, 3.77±1.81x10 5 versus 3.64±2.56x10 4 copies/ml for the presence and absence of DHF antibody, respectively. The synthesis of NO was suppressed in the presence of antibody (4.43±2.62 versus 8.36±2.83 µM). IL-10 production was 98.87±4.51 versus 6.28±0.32 pg/ml for the presence and absence of enhancing antibody, respectively. Level of IL-12 and IFN-γ productions were undetectable in our studies by ELISA. Conclusions: Our study demonstrated that sera from DHF patients enhanced replication of DV by down regulation of NO production. The possible mechanism of antibody mediated NO suppression is via IL-10 activity. Our hypothesis is supported by studies on Ross River virus (RRV) which IL-10 diminished NO production via suppression of transcription factor (STAT-1 and NFκB) complexes. IMMUNOHISTOLOGICAL CHARACTERIZATION OF MACROPHAGE MIGRATION INHIBITORY FACTOR EXPRESSION IN PLASMODIUM FALCIPARUM-INFECTED PLACENTAS. (NO. 822) Sujittra Chaisavaneeyakorn 1,5,6 , Naomi Lucchi 2 , Carlos Abramowsky 3 , Caroline Othoro 4 , Sansanee C. Chaiyaroj 6 , Ya Ping Shi 1,4 , Bernard L. Nahlen 1,7 , David S. Peterson 2 , Julie M. Moore 2 , and Venkatachalam Udhayakumar 1 Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Geolgia 30333 1 ; Center for Tropical and Emerging Global Diseases and Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602 3 , Department of Pathology, Egleston Children’s Hospital, Emory University School of Medicine, Atlanta, Georgia 30322 3 , Vector Biology and Control Research Center, Kenya Medical Research Institute, Kisumu, Kenya 4 ; Atlanta Research and Education of Foundation, Atlanta, Georgia 30033 5 , Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand 6 ; and Roll Back Malaria, World Health Organization, Geneva, Switzerland 7 Key words: macrophage migration inhibitory factor (MIF), intervillous blood (IVB), Plasmodium falciparum Previously, we have shown that macrophage migration inhibitory factor (MIF) was highly elevated in the placental intervillous blood (IVB) of Plasmodium falciparum-infected women. Here, we compared the expression of MIF in placental tissues obtained from P. falciparum-infected and -uninfected women. Immunoperoxidase staining showed a consistent pattern of MIF expression in syncytiotrophoblasts, extravillous trophoblasts, IVB mononuclear cells, and amniotic epithelial cells, irrespective of their malaria infection status. Cytotrophoblast, villous stroma, and Hofbauer cells showed focal staining. Only amniotic epithelial and IVB mononuclear cells from P. falciparum-infected placentas exhibited significantly higher level of MIF expression than uninfected placentas. Stimulation of syncytilized human trophoblast BeWo cells with P. falciparum-infected erythrocytes that were selected to bind these cells resulted in significant increases in MIF PDF created with FinePrint pdfFactory Pro trial version http://www.pdffactory.com
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