Sansanee C. Chaiyaroj

P. marneffei is a thermal dimorphic fungus which causes penicilliosis, an opportunistic infection in immunocompromised patients in South and Southeast Asia. Little is known about the innate immune response to P. marneffei infection. Therefore, the initial response of macrophages to P. marneffei conidia was evaluated by us. Adhesion between monocytes from healthy humans and fungal conidia was examined and found to be specifically inhibited by MAbs against PRR, such as MR, (TLR)1, TLR2, TLR4, TLR6, CD14, CD11a, CD11b, and CD18. To study the consequences of these interactions, cytokines were also examined by ELISA. Binding of P. marneffei conidia to monocytes was significantly inhibited, in a dose‐dependent manner, by MAbs against MR, TLR1, TLR2, TLR4, TLR6, CD14, CD11b and CD18. When monocytes were co‐cultured with the conidia, there was an increase in the amount of surface CD40 and CD86 expression, together with TNF‐α and IL‐1β production, compared to unstimulated controls. In assays containing anti‐TLR4 or anti‐CD14 antibody, reduction in the amount of TNF‐α released by monocytes stimulated with P. marneffei conidia was detected. In addition, it was found that production of TNF‐α and IL‐1β from adherent peripheral blood monocytes was partially impaired when heat‐inactivated autologous serum, in place of untreated autologous serum, was added to the assay. These results demonstrate that various PRR on human monocytes participate in the initial recognition of P. marneffei conidia, and the engagement of PRR could partly initiate proinflammatory cytokine production.

Sap2 is the most abundant virulence factor expressed during Candida infection, and the principal protein known to induce antibody response during Candida infection in humans. Its role in T‐cell activation however, has not yet been determined. Sequence analysis revealed that Sap2 contains two variable regions: Var1 and Var2. Computational predictions by the Hotspot Hunter program identified that Var1 contains three candidate T‐cell epitopes, whereas Var2 contains four. Thirty‐nine overlapping peptides of Sap2 were then synthesized, and tested for their ability to induce proliferation of PBMC from 12 donors. Peptides P11, P17 and P31 exhibited significantly higher proliferative indices when compared with those of other peptides or controls. P17 and P31 are located in the areas of prediction, while P11 is not. There were other peptides outside the prediction areas that could stimulate PBMC proliferation at low levels. Nevertheless, the proliferative noise caused by such peptides was ruled out by IL‐2 ELISpot analysis. Only P17 and P31 were shown to induce clonal proliferation of IFN‐γ producing lymphocytes, suggesting that these two peptides contain T cell epitopes. P11, which stimulated IL‐2 producing clones, contains a known B‐cell epitope. Interestingly, P17 and P31 elicited both Th1 and Th2 cell responses with significant numbers of IL‐13 secreting clones in response to stimulation. Taken together, the computer‐based T cell epitope prediction method could identify the immunogenic T cell epitopes of C. albicans Sap2 that promiscuously bind to the HLA‐DRB1 supertype.

Chondroitin sulfate A (CSA) is an important receptor for Plasmodium falciparum-infected erythrocytes in the placenta. To study the molecular interaction between parasitized erythrocytes (PE) to CSA, we performed in vitro cytoadherence inhibition assays of PE infected with wild and laboratory isolates of P. falciparum to CSA using various glycosaminoglycans (GAGs). Marked decrease in PE adhesion to immobilized CSA and CSA-expressed cells was achieved with soluble chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE) at low concentrations. The effect was dose dependent with the degree of inhibition exceeded that of soluble CSA in certain clinical isolates. The results suggested the influence of oversulfation of CS variant chains on PE adherence to CSA. Interestingly, PE of the tested wild isolates could adhere to immobilized CSD and CSE at different levels while PE of CSA-selected laboratory lines could not. Partial inhibitory activity was observed when chondroitin sulfate C (CSC), chondroitin sulfate B (CSB), and polyolpolysulfate were used even at high concentrations. Keratan sulfate, colominic acid, and Suramine were unable to inhibit PE adherence. Taken together, the results confirm that the 4-sulfate amino sugar moiety, as well as the basic disaccharide structure of N-acetylgalactosamine linked to glucuronic acid, may influence the degree of this molecular interaction. However, other sulfation patterns that could influence the interaction could not be overlooked, as in the case of CSD which contains 2-O-sulfation at glucuronic acid. Studies using pentosan polysulfate, an oversulfated molecule with a xylan backbone, as an inhibitor also showed a reduction of PE adherence of most isolates tested. Thus, only the sulfate content and pattern of this molecule could affect the adhesive interactions. In addition, difference in capacity of low molecular weight heparins to inhibit CSA-mediated PE cytoadherence of clinical isolates was also observed, thereby providing evidence on the heterogeneity in cytoadherence characteristics of maternal parasite isolates as well as their therapeutic potentials.

Two recently produced monoclonal antibodies were used to develop an antigen capture enzyme-linked immunosorbent assay (ELISA) for rapid diagnosis ofPenicillium marneffei. The method was evaluated with 53 patients with culture-confirmed penicilliosis and 240 controls. The diagnostic sensitivity, specificity, and accuracy of the ELISA were 92.45, 97.5, and 96.59%, respectively.

We reported previously two biochemically and antigenically distinct biotypes of Burkholderia pseudomallei. These two distinct biotypes could be distinguished by their ability to assimilate l‐arabinose. Some B. pseudomallei isolated from soil samples could utilize this substrate (Ara⁺), whereas the other soil isolates and all clinical isolates could not (Ara). Only the Ara⁻ isolates were virulent in animals and reacted with monoclonal antibody directed at the surface envelope, most likely the exopolysaccharide component. In the present study, pulsed‐field gel electrophoresis was employed for karyotyping of these previously identified B. pseudomallei strains. We demonstrate here that the DNA macrorestriction pattern allows the differentiation between B. pseudomallei, which can assimilate l‐arabinose, and the proposed B. thailandensis, which cannot do so. Bacterial strains from 80 melioidosis patients and 33 soil samples were examined by genomic DNA digestion with NcoI. Two major reproducible restriction patterns were observed. All clinical (Ara) isolates and 9 Ara soil isolates exhibited macrorestriction pattern I (MPI), while 24 soil isolates (Ara⁺) from central and northeastern Thailand displayed macrorestriction pattern II (MPII). The study here demonstrated pulsed‐field gel electrophoresis to be a useful tool in epidemiological investigation possibly distinguishing virulent B. pseudomallei from avirulent B. thailandensis or even identifying closely related species of Burkholderia.