Serum antibodies and cytokines in C4-deficient mice and their responses to exercise.

Psychological stress is believed to be one of the predisposing factors for systemic lupus erythemato-sus (SLE), whereas physical stress such as exercise has never been reported to be related. We measured the cir-culating levels of antibodies (IgM, IgG, anti-dsDNA IgG), Th1 (IFN-γ), Th2 (IL-4, IL-6), and of pro-inflammatory (TNF-α, IL-1β) and anti-inflammatory (TGF-β) cytokines of C4-/- female mice at rest, after acute exercise and after exercise training, using an antibody-capture ELISA. Prior to the exercise, the C4-/- mice had higher levels of IgG and anti-dsDNA IgG but lower levels of IFN-γ, IL-1β, IL-6 and IL-4 than wild-type C57BL/6 (B6) mice. A single bout of exercise to exhaustion increased serum IgG, TNF-α, IL-1β and TGF-β in the B6 mice but only TGF-β in the C4-/- mice was increased. We conclude that exhaustive or moderate exercise has no effect on the levels of serum anti-bodies and cytokines and is thus unlikely to promote the onset of SLE.

Analysis of Foxp3, CD25, and CD127 expressed on regulatory T cells in Thai subjects.

Foxp3+ natural regulatory T cells (nTreg) play a distinct role in maintaining self tolerance at the pe-riphery. CD25hi and CD127lo were proposed for the identification and purification of nTreg but they have not been confirmed in non-Caucasian populations. This study examined the sensitivity and purity of Foxp3 nTreg identified by CD25 and CD127 in the peripheral blood of Thai subjects (13 males, 15 females with age range of 20-42 years old). The proportions of nTreg/CD4+ as identified by the different markers were as follows: Foxp3+, 18.3 ± 6.4%; CD25hi, 6.4 ± 3.2%; and CD127lo, 54.3 ± 14.2%. Sensitivity tests showed the following results: CD25hi, 23.1%; CD127lo, 40.6%; CD25hiCD127lo, 7.4%. Purity tests concluded as follows: CD25hi, 63.6%; CD25int, 24.9%; CD25lo, 8.7%, CD127lo, 26.5%; CD127hi, 14.9%, and CD25hiCD127lo, 52.0%. In conclusion, the proportions of nTreg in Thai subjects are similar to Caucasian populations. CD25hi is superior to CD127lo for separating Foxp3+ nTreg. Com-bining CD25hi and CD127lo does not improve the nTreg purity Abbreviations: CD25hi, CD25 highly expressed cells; CD25int, CD25 intermediately expressed cells; CD25lo, CD25 low expressed cells; CD127hi, CD127 high expressed cells; CD127lo, CD127 low expressed cells.

Differential gene expression profiles of lung epithelial cells exposed to burkholderia pseudomallei and burkholderia thailandensis during the initial phase of infection.

Burkholderia pseudomallei is the causative agent of melioidosis, and its infection usually affects pa-tients’ lungs. The organism is a facultative intracellular Gram-negative bacillus commonly found in soil and water in endemic tropical regions. Another closely related Burkholderia species found in soil and water is B. thailandensis. This bacterium is a non-pathogenic environmental saprophyte. B. pseudomallei is considerably more efficient than B. thailandensis in host cell invasion and adherence. A previous study by our group demonstrated that after suc-cessfully invading cells, there was no difference in the ability to survive and to replicate between both Burkholderia species in cultured A549 human lung epithelial cells. In this study, Human Affymetrix GeneChips were used to identify the difference in gene expression profiles of A549 cells after a 2-h exposure to B. pseudomallei and B. thai-landensis. A total of 280 of 22,283 genes were expressed at higher levels in the B. pseudomallei-infected cells than in the B. thailandensis-infected cells, while 280 genes were expressed at lower levels in the B. pseudomallei-infected cells. Approximately 9% of these genes were involved in immune response and apoptosis. Those genes were further selected for gene expression analysis using reverse transcription PCR and/or real-time RT-PCR. The results of RT-PCR and real-time RT-PCR are in accordance with data from the microarray data in that bcl2 gene expression in the B. pseudomallei-infected cells was 2-fold higher than the level in the B. thailandensis-infected cells even though no apoptosis was seen in the infected cells. The levels of E-selectin, ICAM-1, IL-11, IRF-1, IL-6, IL-1 and LIF genes expression in the B. pseudomallei-infected cells were 1.5-5 times lower than in the B. thailan-densis-infected cells. However, both species stimulated the same level of IL-8 production from the tested epithelial cell line, and no difference in the ratio of adherent polymorphonuclear cells (PMNs) to infected A549 cells of both species was observed. Taken together, our results suggest that B. pseudomallei manipulates host response in fa-vor of its survival in the host cell, which may explain the more virulent characteristics of B. pseudomallei when compared with B. thailandensis.

Atorvastatin attenuates TLR4-mediated NF-κB activation in a MyD88-dependent pathway.

Antigen presenting cells such as dendritic cells and macrophages have recently been detected in atherosclerotic plaques. Toll-like receptors expressed on the surface of these cells, have been implicated in ongo-ing inflammatory responses in the plaques. In this study, we investigated the anti-inflammatory effect of atorvas-tatin, a lipid lowering drug, via Toll-like receptor 4 (TLR4) in vitro, employing murine pro-B cell lines transfected with hTLR4/MD2 and MyD88/hTLR4/MD2 systems. The results showed that atorvastatin at 10 μM significantly attenu-ated NF-κB activation within 24 hours while at lower doses of 0.1 and 1 μM, treatment time had to be prolonged up to 48 hours for a significant inhibition to occur. The inhibition of NF-κB was also observed in a cell line co-transfected with MyD88 and TLR4 suggesting that the attenuation of NF-κB by atorvastatin occurred in a MyD88 dependent fashion.

Differential gene expression profiles of human monocyte-derived antigen presenting cells in response to Penicillium marneffei: roles of DC-SIGN (CD209) in fungal cell uptake.

DNA microarray technology was used to determine the gene expression profile of human monocyte-derived macrophages (hMDMs) after stimulation by Penicillium marneffei yeast. The expression levels of 175 macrophage genes were found to be altered by a minimum of two-fold in magnitude following 4 hours of P. marneffei exposure. Among those, 41 genes were upregulated in activated hMDMs while 134 genes were downregulated. Real-time PCR and RT-PCR were performed to further examine gene expression associated with the inflammatory response. Increased levels of TNF-alpha and IL-1 beta gene expression in both hMDMs and human monocyte-derived dendritic cells (hMoDCs) were observed after stimulation by P. marneffei yeast. Furthermore, the genes encoding T-bet, IL-6 and ICAM-1 were also upregulated in hMDMs. Functional analysis of the adhesion of P. marneffei to dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN, CD209) was performed in hMoDCs since the microarray data revealed an increased expression of DC-SIGN in activated hMDMs. We found that DC-SIGN-Fc bound preferentially to P. marneffei yeast rather than to conidia. Moreover, an anti-DC-SIGN monoclonal antibody inhibited the binding of P. marneffei yeast to hMoDCs, but did not inhibit endocytosis of P. marneffei yeast. The mannose receptor, on the other hand, was important in both adhesion and phagocytosis. These results suggest that P. marneffei may exploit DC-SIGN as a receptor to facilitate the systemic spread of infection. Taken together, our study demonstrates the usefulness of microarray technology in generating valuable expression data to permit conventional immunologic investigations of host-fungal interactions.

Molecular typing of Vibrio cholerae O1 isolates from Thailand by pulsed-field gel electrophoresis.

The aim of the present study was to genotypically characterize Vibrio cholerae strains isolated from cholera patients in various provinces of Thailand. Two hundred and forty V. cholerae O1 strains, isolated from patients with cholera during two outbreaks, i.e. March 1999-April 2000 and December 2001-February 2002, in Thailand, were genotypically characterized by NotI digestion and pulsed-field gel electrophoresis (PFGE). In total, 17 PFGE banding patterns were found and grouped into four Dice-coefficient clusters (PF-I to PF-IV). The patterns of V. cholerae O1, El Tor reference strains from Australia, Peru, Romania, and the United States were different from the patterns of reference isolates from Asian countries, such as Bangladesh, India, and Thailand, indicating a close genetic relationship or clonal origin of the isolates in the same geographical region. The Asian reference strains, regardless of their biotypes and serogroups (classical O1, El Tor O1, O139, or O151), showed a genetic resemblance, but had different patterns from the strains collected during the two outbreaks in Thailand. Of 200 Ogawa strains collected during the first outbreak in Thailand, two patterns (clones)--PF-I and PF-II--predominated, while other isolates caused sporadic cases and were grouped together as pattern PF-III. PF-II also predominated during the second outbreak, but none of the 40 isolates (39 Inaba and 1 Ogawa) of the second outbreak had the pattern PF-I; a minority showed a new pattern--PF-IV, and others caused single cases, but were not groupable. In summary, this study documented the sustained appearance of the pathogenic V. cholerae O1 clone PF-II, the disappearance of clones PF-I and PF-III, and the emergence of new pathogenic clones during the two outbreaks of cholera. Data of the study on molecular characteristics of indigenous V. cholerae clinical isolates have public-health implications, not only for epidemic tracing of existing strains but also for the recognition of strains with new genotypes that may emerge in the future.

Distinct immunologic properties of Penicillium marneffei yeasts obtained from different in vitro growth conditions.

A dimorphic fungus Penicillium marneffei is a causative agent of penicilliosis, a life-threatening disseminated disease in immunocompromised hosts predominantly found in southeast Asia and southern China. P. marneffei is the only known Penicillium that possesses a dimorphic characteristic. Since it is difficult to produce large amount of P. marneffei yeasts in vivo for experimentation purpose, yeast cells were produced in different in vitro conditions as alternatives. We interested in investigating the immunologic properties of yeast cells from different culture preparations. It was found that yeast cells obtained from brain heart infusion broth and Sabouraud dextrose broth did not resemble those resided in clinical specimens. A solution of 1% peptone, on the other hand, could induce a direct conidial transition into fission yeasts. Ability of yeast cells in each preparation to activate macrophages was determined by analyzing surface expression of CD40 and CD86 co-stimulatory molecules after two days of co-cultivation. Every P. marneffei yeast cell preparation demonstrated such ability. However, the ones from Sabouraud dextrose broth seemed to induce less phagocytosis. Additionally, although distinct antigenic profiles and lack of conformity in antigenic expression were observed among yeast cells from different culture conditions, most major immunogenic bands were present when Western analysis was performed using polyclonal antisera from penicilliosis patients. The results of the study raise attention on immunological and biochemical characteristics of P. marneffei yeasts if such preparations are to be used in future laboratory investigations.