Generation of dendritic cells from human monocytes to study immune response against Burkholria pseudomallei

Burkholderia 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.

Utility of polyclonal antibodies binding to specific epitopes of Burkholderia pseudomallei

Burkholderia 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.