Browsing by Author "Ganjana Lertmemongkolchai"
Now showing 1 - 4 of 4
Results Per Page
Sort Options
Item Generation of dendritic cells from human monocytes to study immune response against Burkholria pseudomallei(Khon Kaen University, 2009-12-24) Maneerat Pinsiri; Darawan Rinchai; Ganjana LertmemongkolchaiBurkholderia pseudomallei (B. pseudomallei) is a major causative agent of melioidosis in southeast Asia andnorthern Australia. Clinical manifestations range from asymptomatic, sub-acute and chronic infections to acutesepsis. However, the mechanism of immune response to B. pseudomallei is known very little. This study aimed togenerate monocyte derived dendritic cells (MoDcs) from human peripheral blood samples as a model to studyimmune response to B. pseudomallei. By using immune-magnetic bead sorting, monocytes (CD14+ cells) wereisolated from pheripheral blood and the purity of obtained monocytes was approximately 95%. Moreover, themonocytes differentiated into ็immature้ DC by GM-CSF and IL-4 activation as evidenced by their surfacephenotypes assayed by flow cytometry eg. HLA-DR and CD11c upregulation whilst CD14 downregulation. WhenMoDCs were collected and stimulated with heat killed- B. pseudomallei or lipopolysaccharide from E. coli for24 or 48 hours, the results indicated the highest levels of IL-6 induced by B. pseudomallei and it was comparablebetween 24 and 48 hours. In conclusion, we demonstrated the generation of MoDCs which could be used for furtherstudy of immune responses to B. pseudomallei and other pathogens.Item Optimization of Enzyme-linked Immunospot Assay for quantitative analysis of interferon gamma producing cells(Khon Kaen University, 2010-12-21) Jirawan Mahawantung; Duangchan Suwannasaen; Wipawee Saenwongsa; Ganjana LertmemongkolchaiEnzyme-Linked Immunospot (ELISPOT) is the most sensitive assay for the enumeration of IFN-g producing cells at single cell level. This study aimed to optimize the ELISPOT assay to study the IFN-g production in response to B. pseudomallei and dengue virus in healthy individuals. The optimal peripheral blood mononuclear cells (PBMCs) concentrations, incubation time and concentration of each stimulator to maximize IFN-g production were examined. The results showed that using PBMCs at 2.5 x 104 cells and stimulating with a positive control stimulator, phytohemagglutinin (PHA), at 3 µg/ml was the optimal condition for IFN-g responses. The number of IFN-g-Spot Forming Cell (IFN-g-SFC) was appropriate for enumeration by Stereomicroscope. In contrast, incubation of 5 x 105 PBMCs with 3 x 106 cfu/ml heat-killed B. pseudomallei or dengue virus at a dilution of 1:2700 was the optimal condition. These optimal conditions were then used to study the IFN-g production in 8 healthy volunteers. The results clearly showed that B. pseudomallei and dengue virus could stimulate all healthy PBMCs to produce IFN-g at higher levels than those of medium control. However, the degree of IFN-g responses was different for each individual. Moreover, linear regression analysis showed that the number of IFN-g-SFC obtained from B. pseudomallei stimulation was correlated with the dengue virus stimulation. In conclusion, in this study, the ELISPOT for IFN-g production using B. pseudomallei and dengue virus stimulations have been optimized. Our results of IFN-g production in response to B. pseudomallei and dengue virus provides basic information for further study on the ability of B. pseudomallei or Dengue’s peptides in IFN-g induction in screening for vaccine candidate.Item A Study on Immune Response to Cryptococcus Neoformans Antigens in Rabbits(Faculty of Medicine, Khon Kaen University, 2010-05-13) Srivilai Varopastrakul; Ganjana Lertmemongkolchai; Nareas Waropastrakul; Arunrat RomphrukA study on immune response to Cryptococcus neoformans antigens was done in rabbits. Three different doses of encapsulated C. neoformans antigens, 1.5x107, 1.5x108 and 1.5x109 cells/milliliter respectively, were injected intravenously via ear vein. Results showed that the concentration of 1.5x108 cells/milliliter stimulated high agglutination titers of antibody i.e., 1024 in one rabbit and 32768 in the other. For comparison, the titer of antiserum obtained from rabbits immunized with 1.5x107 cells/milliliter did not exceed 64, while the dose of 1.5x109 cells/milliliter killed rabbits. Results suggested that a high quantity of anti-C. neoformans antiserum could be prepared in rabbits. This antiserum would be an important reagent in the development of the serological test kit for Cryptococcal diagnosis.KEY WORD : Cryptococcus neoformans, Antibody response.Item Utility of polyclonal antibodies binding to specific epitopes of Burkholderia pseudomallei(Khon Kaen University, 2009-12-25) Darawan Rinchai; Aroonlug Lulitanond; Ganjana LertmemongkolchaiBurkholderia pseudomallei (B. pseudomallei) is a major causative agent of melioidosis in endemic areas such as Southeast Asia and Northern Australia. Rapid diagnosis is required for appropriate treatments in septicemic melioidosis. In this study, we aimed to evaluate the specific polyclonal antibody (pAb) to B. pseudomallei for potentially use in the development of diagnostic assays and pathogenesis studies. In determination the cross reaction of the pAb to other bacteria by indirect ELISA, the pAb to B. pseudomallei has no cross-reactivity with other bacteria. In contrast, the pAb reacted with 24 clinical isolates of B. pseudomallei-extracted antigens. By ELISA, the pAb to B. pseudomallei could be used to detect secreted antigens in 3 hour culture supernatants of 1.5 x 104 cfu/ml B. pseudomallei. In addition, opsonization of the bacteria with the pAbs could enhance bacterial internalization by U937-derived macrophages when compared with bacterial culture alone. The mechanism of these observations, however, needs to be further investigated. In conclusion, we suggest that the pAbs to B. pseudomallei can be potentially used for development of diagnosis method and pathogenesis study.