Narisara Anuntagool

Recent developments in laboratory diagnosis of melioidosis.

a Laboratory of Immunology, Chulabhorn Research Institute, Bangkok 10210, Thailand b Department of Microbiology, Faculty of Science, Mahidol Uni6ersity, Rama VI Road, Bangkok 10400, Thailand c Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol Uni6ersity, Bangkok, Thailand d Department of Microbiology, Faculty of Medicine, Khon Kaen Uni6ersity, Khon Kaen, Thailand e National Institute of Health, Department of Medical Ser6ices, Ministry of Public Health, Nonthaburi, Thailand f Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol Uni6ersity, Bangkok, Thailand g Department of Internal Medicine, Faculty of Medicine Siriraj Hospital, Mahidol Uni6ersity, Bangkok, Thailand

Induction of iNOS expression and antimicrobial activity by interferon (IFN)-β is distinct from IFN-γ in Burkholderia pseudomallei-infected mouse macrophages

Burkholderia pseudomallei is a causative agent of melioidosis. This Gram‐negative bacterium is able to survive and multiple inside both phagocytic and nonphagocytic cells. We previously reported that exogenous interferons (both type I and type II) enhanced antimicrobial activity of the macrophages infected with B. pseudomallei by up‐regulating inducible nitric oxide synthase (iNOS). This enzyme thus plays an essential role in controlling intracellular growth of bacteria. In the present study we extended our investigation, analysing the mechanism(s) by which the two types of interferons (IFNs) regulate antimicrobial activity in the B. pseudomallei‐infected macrophages. Mouse macrophage cell line (RAW 264·7) that was exposed simultaneously to B. pseudomallei and type I IFN (IFN‐β) expressed high levels of iNOS, leading to enhanced intracellular killing of the bacteria. However, neither enhanced iNOS expression nor intracellular bacterial killing was observed when the macrophages were preactivated with IFN‐β prior to being infected with B. pseudomallei. On the contrary, the timing of exposure was not critical for the type II IFN (IFN‐γ) because when the cells were either prestimulated or co‐stimulated with IFN‐γ, both iNOS expression and intracellular killing capacity were enhanced. The differences by which these two IFNs regulate antimicrobial activity may be related to the fact that IFN‐γ was able to induce more sustained interferon regulatory factor‐1 (IRF‐1) expression compared with the cells activated with IFN‐β.

CpG ODN activates NO and iNOS production in mouse macrophage cell line (RAW 264·7)

Synthetic CpG containing oligodeoxynucleotide (CpG ODN) is recognized for its ability to activate cells to produce several cytokines, such as IL‐12 and TNF‐α. In the present study we have demonstrated that CpG ODN 1826, known for its immunostimulatory activity in the mouse system could, by itself, induce nitric oxide (NO) and inducible nitric oxide synthase (iNOS) production from mouse macrophage cell line (RAW 264·7). Neutralizing antibody against TNF‐α was not able to inhibit NO or iNOS production from the CpG ODN 1826‐activated macrophages, suggesting that although the TNF‐α was also produced by CpG ODN‐activated macrophages, the production of iNOS was not mediated through TNF‐α. Although both CpG ODN 1826 and lipopolysaccharide (LPS) were able to stimulate NO and iNOS production, the exposure time required for maximum production of NO and iNOS for the CpG ODN 1826‐activated macrophages was significantly longer than those activated with LPS. These results were due probably to a delay of NF‐κB translocation, as indicated by the delay of IκBα degradation. Moreover, the fact that chloroquine abolished NO and iNOS production from the cells treated with CpG ODN 1826 but not from those treated with LPS suggested that the induction of NO and iNOS production from the cells stimulated with CpG ODN (1826) also required endosomal maturation/acidification.

Antigenic differences between clinical and environmental isolates of Burkholderia pseudomallei.

Burkholderia pseudomallei is a free‐living organism that causes the potentially lethal tropical infection melioidosis. The disease is endemic in many parts of eastern Asia and northern Australia. The presence of two distinct biotypes in soil can be reliably distinguished by their ability to assimilate l‐arabinose. Whereas some soil isolates could utilize this substrate (Ara+), the remaining soil isolates and all clinical isolates tested so far could not (Ara−). Only the Ara− isolates were virulent in animal models. We have raised a murine monoclonal antibody (MAb) that can readily distinguish Ara− from Ara+ biotypes. The MAb reacted with a high molecular weight component present only on the Ara− biotype. With this MAb, clinical and soil Ara−isolates gave identical positive reactions in agglutination, immunofluorescence, ELISA and immunoblot assays. Using these same assay systems, the soil Ara+ biotype did not react with the MAb. Similar but distinct immunoblot patterns were also noted when these two Ara biotypes were probed with sera from patients with melioidosis or with polyclonal immune rabbit sera. These data showed that the Ara− biotype from both clinical and environmental isolates is antigenically different from its Ara+ environmental counterpart. The SDS‐PAGE protein and lectin‐binding profiles of both groups of Ara− isolates were also found to be different from those of the Ara+ biotype.

Burkholderia pseudomallei stimulates low interleukin-8 production in the human lung epithelial cell line A549.

Melioidosis is a life‐threatening disease caused by Burkholderia pseudomallei. The lung is the most commonly affected organ, resulting in abscess formation in patients with chronic melioidosis. Previous study has shown that B. pseudomallei was able to invade and multiply in epithelial cells. In the present study, we have demonstrated that B. pseudomallei is able to stimulate interleukin 8 (IL‐8) production from the human alveolar lung epithelium cell line A549. However, the level of IL‐8 production was significantly lower than when the cells were infected with other Gram‐negative bacteria such as Salmonella enterica serovar Typhi (S. typhi) which were used for comparison. The degree of IκBα degradation in the B. pseudomallei‐infected cells was lower than that of the S. typhi‐infected cells, suggesting that B. pseudomallei is also a poorer cell activator. Inhibition of B. pseudomallei invasion by cytochalasin D did not interfere with either IL‐8 production or IκBα degradation, indicating that bacterial uptake is not required for the production of this chemokine. Thus, it appears that the signalling initiated by the interaction of B. pseudomallei with the epithelial cell surface is sufficient for epithelial cell activation.

A radical form of nitric oxide suppresses RNA synthesis of rabies virus

Hydrophobia is an incurable disease of the central nervous system. Therefore, every mode of the immune response is important to inhibit and clear infection. Innate immunity such as nitric oxide is significantly upregulated during rabies virus infection in vivo. In this report, the possible role of nitric oxide in inhibition of rabies virus replication was studied. Rabies virus infected neuroblastoma cells were treated with nitric oxide generated from SNP or SNP in the presence of ascorbate. SNP-ascorbate generates mainly NO* in culture medium while NO(+) is the major product of SNP alone. Treatment with SNP-ascorbate resulted in delay and suppression of infectious viral particle production. In contrast, treatment with SNP alone did not interfere with multiplication of this virus. The mechanism of inhibition by NO was at the level of gene expression, which was demonstrated by reduction in the level of N, G and L gene expression. The effect of SNP-ascorbate generated NO on rabies virus protein synthesis was also investigated. Synthesis of N protein in the presence of NO was suppressed which correlated to down regulation of N gene expression. We hypothesize that one of the roles of NO in the central nervous system during rabies virus infection is to limit viral dissemination by down-regulating rabies virus production through transcription inhibition.

Shedding of lipopolysaccharide and 200-kDa surface antigen during the in vitro growth of virulent Ara- and avirulent Ara+Burkholderia pseudomallei.

Non-virulent Ara+ B. pseudomallei environmental isolates differ from virulent Ara- clinical isolates by their ability to assimilate L-arabinose and the absence of a 200 kDa antigen on their surface. The latter, present only on the Ara- isolates from either clinical or environmental origin, was recently demonstrated by its immunoreactivity with monoclonal antibody (MAb) 5F8. We recently demonstrated that lipopolysaccharide (LPS) from both biotypes were indistinguishable from one another with regard to SDS-PAGE profiles and immunoreactivities with immune sera. In this study, the shedding of LPS and 200-kDa antigen into the culture medium during the in vitro growth of Ara- was compared with that of its Ara+ counterpart, using MAb-based sandwich ELISAs. The results showed that the LPS shedding profiles from the two biotypes were similar to one another. This was in contrast to the situation with the 5F8-reactive antigen. The culture fluid of all Ara- isolates and none of the Ara+ isolates were found to react strongly with the MAb 5F8 during the early log phase of growth. However, during the late stationary phase, a trace amount of the 5F8-reactive material could also be detected in the culture fluid of the Ara+ isolates.