Gene Microarray Analyses of Daboia russelli russelli Daboiatoxin Treatment of THP-1 Human Macrophages Infected with Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis and represents a potential bioterrorism threat. In this study, the transcriptomic responses of B. pseudomallei infection of a human macrophage cell model were investigated using whole-genome microarrays. Gene expression profiles were compared between infected THP-1 human monocytic leukemia cells with or without treatment with Daboia russelli russelli daboiatoxin (DRRDbTx) or ceftazidime (antibiotic control). Microarray analyses of infected and treated cells revealed differential upregulation of various inflammatory genes such as interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α), cyclooxygenase (COX-2), vascular endothelial growth factor (VEGF), chemokine C-X-C motif ligand 4 (CXCL4), transcription factor p65 (NF-kB); and several genes involved in immune and stress responses, cell cycle, and lipid metabolism. Moreover, following DRR-DbTx treatment of infected cells, there was enhanced expression of the tolllike receptor 2 (TLR-2) mediated signaling pathway involved in recognition and initiation of acute inflammatory responses. Importantly, we observed that highly inflammatory cytokine gene responses were similar in infected cells exposed to DRR-DbTx or ceftazidime after 24 h. Additionally, there were increased transcripts associated with cell death by caspase activation that can promote host tissue injury. In summary, the transcriptional responses during B. pseudomallei infection of macrophages highlight a broad range of innate immune mechanisms that are activated within 24 h post-infection. These data provide insights into the transcriptomic kinetics following DRR-DbTx treatment of human macrophages infected with B. pseudomallei.

Acinetobacter calcoaceticus-Acinetobacter baumannii complex species in clinical specimens in Singapore.

This study was performed to determine the prevalence, distribution of specimen sources, and antimicrobial susceptibility of the Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) species complex in Singapore. One hundred and ninety-three non-replicate Acb species complex clinical isolates were collected from six hospitals over a 1-month period in 2006. Of these, 152 (78·7%) were identified as A. baumannii, 18 (9·3%) as 'Acinetobacter pittii' [genomic species (gen. sp.) 3], and 23 (11·9%) as 'Acinetobacter nosocomialis' (gen. sp. 13TU). Carbapenem resistance was highest in A. baumannii (72·4%), followed by A. pittii (38·9%), and A. nosocomialis (34·8%). Most carbapenem-resistant A. baumannii and A. nosocomialis possessed the bla(OXA-23-like) gene whereas carbapenem-resistant A. pittii possessed the bla(OXA-58-like) gene. Two imipenem-resistant strains (A. baumannii and A. pittii) had the bla(IMP-like) gene. Representatives of carbapenem-resistant A. baumannii were related to European clones I and II.

Lack of association of dengue activity with haze.

Dengue activity depends on fluctuations in Aedes populations which in turn are known to be influenced by climate factors including temperature, humidity and rainfall. It has been hypothesized that haze may reduce dengue transmission. Due to its geographical location Singapore suffers almost every year from hazes caused by wildfires from Indonesia. Such hazes have a significant impact on pollution indexes in Singapore. We set out to study the relationship of dengue activity and haze (measured as pollution standard index) in Singapore, using ARIMA models. We ran different univariate models, each encompassing a different lag period for the effects of haze and temperature (from lag 0 to lag 12 weeks). We analysed the data on a natural logarithmic scale to stabilize the variance and improve the estimation. No association between dengue activity and haze was found. Our findings do not lend support to the hypothesis that haze is associated with reduced dengue activity in Singapore.

The use of antibodies in the treatment of infectious diseases.

There is a long history of the use of antibodies in the treatment and prophylaxis of infectious diseases, because these molecules play a critical role in directing the effector mechanisms of the immune system against the pathogens they recognise. However, the widespread application of this therapy has been hampered by allergic reactions, production costs and the availability of alternative drugs such as antibiotics. Some of these obstacles can now be overcome with advances in biotechnology, which has enabled the development of antibody-based drugs for use first in treating cancer, and recently, for treating infectious diseases. The efficacy of such antibodies has been demonstrated in various in vitro studies, animal models and clinical trials for a variety of both viral and bacterial pathogens. Antibodies appear to hold great promise as a new class of drugs against infectious diseases.

The rapid identification of Clostridium perfringens as the possible aetiology of a diarrhoeal outbreak using PCR.

A gastroenteritis outbreak occurred in a military camp where a laboratory and epidemiological investigation was carried out. The early onset of symptoms indicated probable food contamination with Clostridium perfringens. Stool samples collected from affected patients were tested within 4 h via real-time polymerase chain reaction (PCR) for the presence of the C. perfringens plc gene. Ten out of the 12 stool samples were positive. Confirmation of the molecular test results was carried out by enumeration of C. perfringens in stool by culture and shown to be in excess of 106 spores/g stool. The isolates obtained from culture were further analysed by PCR for the presence of the chromosomal enterotoxin (cpe) gene. Based on the clinical symptoms, epidemiological and laboratory investigations, C. perfringens was implicated as the aetiological agent. The ability to conduct real-time PCR analysis greatly shortens the time to diagnosis and allows for preventive and control measures to be effected quickly.

Microarray and real-time RT-PCR analyses of differential human gene expression patterns induced by severe acute respiratory syndrome (SARS) coronavirus infection of Vero cells.

Vero E6 African green monkey kidney cells are highly susceptible to infection with the newly emerging severe acute respiratory syndrome coronavirus (SARS-CoV), and they are permissive for rapid viral replication, with resultant cytopathic effects. We employed cDNA microarray analysis to characterize the cellular transcriptional responses of homologous human genes at 12 h post-infection. Seventy mRNA transcripts belonging to various functional classes exhibited significant alterations in gene expression. There was considerable induction of heat shock proteins that are crucial to the immune response mechanism. Modified levels of several transcripts involved in pro-inflammatory and anti-inflammatory processes exemplified the balance between opposing forces during SARS pathogenesis. Other genes displaying altered transcription included those associated with host translation, cellular metabolism, cell cycle, signal transduction, transcriptional regulation, protein trafficking, protein modulators, and cytoskeletal proteins. Alterations in the levels of several novel transcripts encoding hypothetical proteins and expressed sequence tags were also identified. In addition, transcription of apoptosis-related genes DENN and hIAP1 was upregulated in contrast to FAIM. Elevated Mx1 expression signified a strong host response to mediate antiviral resistance. Also expressed in infected cells was the C-terminal alternative splice variant of the p53 tumor suppressor gene encoding a modified truncated protein that can influence the activity of wild-type p53. We observed the interplay between various mechanisms to favor virus multiplication before full-blown apoptosis and the triggering of several pathways in host cells in an attempt to eliminate the pathogen. Microarray analysis identifies the critical host-pathogen interactions during SARS-CoV infection and provides new insights into the pathophysiology of SARS.

Topographic changes in SARS coronavirus-infected cells at late stages of infection.

Scanning electron and atomic force microscopy was used for the first time to view the maturation of the severe acute respiratory syndrome-associated coronavirus at the cell surface. The surface form of the cells at advanced infection displayed prolific pseudopodia that, in addition to the rest of the plasma membrane, were also active sites of virus release. High magnification of the maturing virus particles showed a rosette appearance with short knoblike spikes under both the scanning electron and atomic force microscopes. The final expulsion step of the maturing virus particles seemed to result in some disruptions to the plasma membrane. The cytoskeletal network along the edge of the infected cells was enhanced and could be involved in transporting and expelling the progeny virus particles. Thickening of the actin filaments at the cell edge provided the bending force to extrude the virus particles.

Proliferative growth of SARS coronavirus in Vero E6 cells.

An isolate of SARS coronavirus (strain 2003VA2774) was obtained from a patient and used to infect Vero E6 cells. The replication cycle of the virus was followed from 1 to 30 h post-infection (p.i.). It was surprising to observe the swift growth of this human virus in Vero cells. Within the first hour of infection, the most obvious ultrastructural change was the proliferation of the Golgi complexes and related vesicles accompanied by swelling of some of the trans-Golgi sacs. Extracellular virus particles were present by 5 h p.i. in about 5 % of the cells and this increased dramatically to about 30 % of the cell population within an hour (6 h p.i.). Swollen Golgi sacs contained virus nucleocapsids at different stages of maturation. These virus precursors were also in large vacuoles and in close association with membrane whorls. The membrane whorls could be the replication complexes, since they appeared rather early in the replication cycle. As infection progressed from 12 to 21 h p.i., the cytoplasm of the infected cells was filled with numerous large, smooth-membraned vacuoles containing a mixture of mature virus and spherical cores. Several of these vacuoles were close to the cell periphery, ready to export out the mature progeny virus particles via exocytosis. By 24 to 30 h p.i., crystalline arrays of the extracellular virus particles were seen commonly at the cell surface.

Localised outbreak of Falciparum malaria in Singapore.

Malaria is primarily an imported disease in Singapore. Local outbreaks are uncommon. We describe a localised outbreak of three patients with Falciparum malaria, which we believe to be locally acquired. There was one fatality due to severe disease and late presentation. Malaria should be considered as a cause of febrile illness as the likelihood of cure depends on early detection and treatment.

Early events of SARS coronavirus infection in vero cells.

An isolate from a patient in the recent severe acute respiratory syndrome (SARS) outbreak in Singapore was used to infect Vero E6 cells. This study concentrated on the first 30 min of infection. It was discovered that the SARS coronavirus attached, entered, and uncoated the nucleocapsids, all within a 30-min period. At 5 min after infection, several virus particles lined the Vero cell plasma membrane. Virus particles were at various stages of fusion at the cell surface, since entry was not a synchronised process. After entry (10 and 15 min), spherical core particles moved into the cytoplasm within large vacuoles. Quite surprising at such early stages of infection (20 min), a virus-induced change in the infected cells was evident. The induction of myelin-like membrane whorls was obvious within the same vacuoles as the core particles. The significance of this virus-induced change is unknown at this stage. By 25-30 min postinfection (p.i.), the spherical core particles appeared to be disassociating and, in their place, doughnut-shaped electron-dense structures were observed. These could be the virus genomes together with the helical nucleocapsids. They were no longer in large vacuoles but packaged into smaller vacuoles in the cytoplasm, and occasionally in small groups.

Development of a simplified assay for the detection of neutralizing antibodies to Japanese encephalitis virus.

Japanese encephalitis virus is a common cause of viral encephalitis in Asia with an estimated 45,000 cases annually. It causes significant morbidity and mortality. It is transmitted primarily by Culex mosquitoes between birds and animals, while man is thought to be an accidental, dead-end host. Since dengue is also prevalent usually in Japanese encephalitis-endemic areas, all Japanese encephalitis positive sera must be confirmed by detecting Japanese encephalitis specific neutralizing antibodies. The plaque reduction neutralization test is the gold standard for detecting and quantifying Japanese encephalitis neutralizing antibodies. This test, however, takes about a week and is carried out in 6 or 24-well plates, which limits its usage for large-scale screening. A simplified assay was developed for the detection and quantification of Japanese encephalitis neutralizing antibodies. The assay, which is carried out in 96-well plates, would be suitable for use in the mass screening of the population's immunity level as well as for use in vaccine efficacy studies.

Dengue seroepidemiology in Singapore.

The resurgence of dengue in Singapore since 1986 had been associated with an adult predominance and a very low incidence in children. No study had been carried out to investigate this finding. Here we report a serological study of 1068 children aged 0 to 15 years. There is a significant rise in seroconversion in children aged 6 years and older coinciding with the start of formal schooling. This suggests that there may be a change in the location where dengue is acquired.

Elevated blood levels of soluble tumor necrosis factor receptors in nasopharyngeal carcinoma: correlation with humoral immune response to lytic replication of Epstein-Barr virus.

Nasopharyngeal carcinoma (NPC) is tightly associated with Epstein-Barr virus (EBV) infection and a heavy infiltration of lymphoid cells in the tumor tissue. Although various lines of evidence have shown that the immune systems of NPC patients have the potential to attack the tumor cells, it is not yet understood how this potential is blocked. In this study we determined the circulatory soluble tumor necrosis factor receptors (sTNFRI and sTNFRII), which are proven to be inhibitory to the anti-tumor effects of tumor necrosis factor-alpha (TNF-alpha), in NPC patients. The serum concentration of both sTNFRI and sTNFRII was determined with an ELISA method, and shown to be significantly higher in 28 NPC patients than in matched healthy controls. This elevation was found to be positively correlated with the serum titers of IgA against EBV early antigens and viral capsid antigens in NPC patients, suggesting that the increased serum concentration of sTNFRI and sTNFRII is possibly due to the EBV infection in NPC tumor cells. This is partly supported by FACS analysis of the circulatory T cells. Phenotypical expression of activation markers such as CD25, CD38, CD69 and CD71 in blood T cells was not significantly different between the NPC and control individuals, indicating the elevation of the sTNFRs is indeed derived from the local immune response in the tumor area. Based on these results, it seems that the increased sTNFRs may act as an inhibitor to decrease the host immune response towards tumor cells in NPC patients.

Association between microsatellites within the human MHC and nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) has been known to be associated with specific HLA haplotypes, in particular HLA A2, B46 and A33, B58 haplotypes. A linkage study based on this observation suggested that HLA antigens are not the cause of NPC but that there exists a gene that lies close to if not within the major histocompatibility complex locus and confers a greatly increased relative risk of NPC. Since then, no further work has elucidated the presence of this gene. One of the difficulties faced by researchers has been the size of the region of chromosome implicated. The MHC locus alone is almost 4 Mb in length, and the number of genes encoded within it is numerous. The purpose of our study was thus to reduce the region of DNA in which the NPC susceptibility gene is likely to be. We report that the NPC susceptibility gene may be within the centromeric end of the class-1 and the telomeric end of the class-III regions of the MHC, near the D6S1624 microsatellite locus, where the presence of allele 4 of the microsatellite conferred a 3 1/2-fold increase in the risk of NPC, the highest reported for a single locus, and the presence of allele 1 of the same microsatellite conferred a highly significant protective effect against NPC.

Neuroendocrine regulation of immunity: the effects of noradrenaline in Xenopus laevis, the South African clawed toad.

A functional association between the peripheral nervous and the immune system in Xenopus laevis, the South African clawed toad, is demonstrated. This association involves the neurotransmitter noradrenaline (NA), produced and released by the sympathetic nerves of the spleen. Chemical sympathectomy prior to immunization reduces splenic NA, and decreases thymus-dependent (TD), but increases thymus-independent (TI), antibody responses. Immune challenge with representatives of the three antigen classes affects splenic NA levels differentially. Thus, the modulatory effect of NA on immunity will depend on the immunogen used. Carrier-priming of helper function in TD responses stimulates a transitory NA release in the spleen, while subsequent immunization activates a more prolonged release. The two types of challenge differ in the antigenic dose given. The effects of NA also depend on the time when it is applied. If used early in the in vivo TD response, antibody production is increased, but if given later, suppressor function is stimulated, thus decreasing antibody production. NA increases both amplifying and suppressing T cell functions in TD responses through stimulation of the alpha 2 adrenoceptor. Alpha 2 adrenoceptor stimulation decreases, and beta adrenoceptor stimulation increases, anti-TNP reactivity. Since an alpha 2 receptor agonist does not affect lectin-stimulated T cell mitogenesis, while a beta receptor agonist depresses it, NA appears to up-regulate T cell functions by affecting their maturation, rather than their clonal expansion.