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[PMC free article] [PubMed] [CrossRef] [Google Scholar] 13. stimulate capsule-specific antibody responses are effective for other encapsulated bacteria such as type b, and meningitidis (6,C8), there are currently no vaccines available for protection against melioidosis. Therefore, there is a need to identify potential antigens for vaccine development. O-polysaccharide (OPS) and hemolysin coregulated protein 1 (Hcp1) are important virulence factors expressed by and are considered to be promising vaccine candidates (9,C11). OPS is usually a component of lipopolysaccharide (LPS) located on outer membrane of bacteria. Hcp1 is usually a protein component of the cluster 1 type VI secretion system (T6SS) that plays a role in the intracellular way of life of (9, 12,C14). Both OPS and Hcp1 are recognized by the immune systems of melioidosis patients (15). Our previous studies exhibited by ELISAs that melioidosis patients produced high levels of IgG against OPS and Hcp1 antigens (16, 17). OPS induced high levels of IgG1 and IgG2 subclasses while Hcp1 predominantly induced high levels of IgG1 (18). Many studies in animal models have exhibited the association between antibody Bumetanide levels and protection from melioidosis but the mechanisms of protection have not been well investigated (10, 19,C22). A study in human melioidosis showed a lower mortality rate was associated with seropositivity against crude antigen preparations (23). We previously reported an association of survival with high levels of IgG against OPS and Hcp1 in melioidosis patients (18, 24) suggesting a potential functional role for these antibodies in protection against disease (25). In addition, Chaichana et al. exhibited that serum from survivors of melioidosis enhanced bacterial uptake compared to serum from nonsurvivors (25). The same study exhibited that purified IgG against whole-cell antigen promotes antibody-dependent cellular phagocytosis (ADCP). However, this study did not characterize what antigen-specific antibodies in the patient serum samples were associated with the ADCP (26). We hypothesized that specific IgG antibodies against OPS (IgG-OPS) and Hcp1 (IgG-Hcp1) in human melioidosis cases could contribute to enhanced phagocytosis and complement deposition on K96243 via flow cytometry. Results of these studies indicated that in both instances higher activity was associated with IgG-OPS compared to IgG-Hcp1. RESULTS Serum from human melioidosis patients enhances bacterial uptake into THP-1 cells. The opsonophagocytic activity of pooled serum from melioidosis patients and healthy controls was decided using THP-1 cells. We observed that pooled melioidosis serum at dilution of 1 1:10 significantly enhanced bacterial uptake into THP-1 cells compared with pooled serum from healthy donors from endemic and non-areas of endemicity. Results showed that mean concentrations standard deviation (SD) of bacterial uptake for pooled melioidosis serum was 3.83??2.30 103 CFU/mL, pooled healthy donor serum from areas of endemicity was 0.58??0.25 103 CFU/mL and pooled healthy donor serum from non-areas of endemicity was 0.18??0.12 103 CFU/mL (Fig. 1). We examined live/lifeless THP1 cells using an inverted light microscope, but we did not observe the difference in live/lifeless cells among cells incubated with different serum groups Bumetanide and PBS control. Open in a separate Bumetanide windows FIG 1 Antibody-dependent cellular phagocytosis (ADCP) activity from pooled serum from melioidosis patients Tmem24 compared to healthy donors from Northeast Thailand, healthy donors from Bangkok and PBS control in THP-1 cells. The ADCP activity was determined by colony count method to enumerate the live bacteria in Bumetanide THP-1 cells. Three-independent experiments were performed. Purification and analysis of IgG-OPS and IgG-Hcp1 antibodies. IgG-OPS and IgG-Hcp1 samples were obtained from pooled melioidosis patient serum using a two-step approach. The total amount of IgG-OPS and IgG-Hcp1 isolated from 400?mL of the pooled patient serum (290?mL for IgG-OPS and 110?mL for IgG-Hcp1) was 3.92?mg and 6.25?mg, respectively. As expected, SDS-PAGE analysis of the depleted and pooled patient serum samples demonstrated a organic combination of protein. In contrast, the purified total IgG and Ig fractions revealed the current presence of two main rings of ~50?kDa and ~25?kDa (Fig. 2). These observations are in keeping with the molecular weights connected with IgG light and weighty stores within their decreased forms, respectively. Open up in another windowpane FIG 2 Coomassie and SDS-PAGE blue staining of prepurified pooled serum, depleted serum, purified anti-OPS antibodies, purified IgG anti-OPS and purified IgG.