Optimization of Enzyme-linked Immunospot Assay for quantitative analysis of interferon gamma producing cells

Enzyme-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.

A Study on Immune Response to Cryptococcus Neoformans Antigens in Rabbits

 A 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.

Detection of Burkholria pseudomallei DNA in whole blood samples of bacterial septicemia patients using conventional PCR and real-time PCR

Burkholderia pseudomallei is a bacterium that causes a disease known as melioidosis. Infections of B. pseudomallei appear in several organs including acute septicemia which showed high mortality rate. The rapid and high efficient diagnostic test may reduce the mortality rate of melioidosis patients. We performed conventional PCR using LPS primers and real-time PCR using 16S rDNA primers for detection of B. pseudomallei in clinical blood specimens. Bacterial culture was used as a gold standard. Blood specimens from 32 suspected bacterial septicemia patients were obtained from admitted patients in Khon Kaen Hospital and other hospitals in the region. These consist of the patients with 19 B. pseudomallei and 13 other bacterial infections including 1 Burkholderia cepacia, 1 Escherichia coli, 4 Klebsiella pneumoniae, 1 Enterobacter species, 3 Staphylococcus aureus, 1 Streptococcus pneumoniae and 2 group A Streptococcus. The sensitivity, specificity, positive and negative predictive values were determined. Conventional PCR showed low sensitivity of 37 % (95% CI:15-59) and high specificity of 92 % (95% CI:78-100) with 88 % (95% CI: 65-100) and 50 % (95% CI:30-70) of positive predictive value (PPV) and negative predictive value (NPV), respectively. In contrast, a real-time PCR and using of combination test showed higher sensitivity (63 %, 95% CI: 41-85) and lower specificity (69 %, 95% CI: 44-94) with 75 % (95% CI:54-96)  and 56 % (95% CI:32-81) of PPV and NPV, respectively. In 5 melioidosis patients who have died, both PCR methods showed rising of sensitivity [80 % (95 % CI: 45-100) and 100 % (95% CI: 100-100)], respectively. The lower detection limit of B. pseudomallei by conventional PCR was 103 cfu/ml. The conventional PCR and real-time PCR could detect B. pseudomallei DNA of 10 pg and 50 fg per PCR reaction, respectively. The higher sensitivity of real-time PCR may be useful for early screening test, whereas the conventional PCR with higher specificity and PPV may be used as a confirmatory test for diagnosis of B. pseudomallei. Therefore, the two assays may be used in combination as rapid molecular diagnostic for melioidosis which might lead to a reduction in the mortality rate of melioidosis patients. 

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.