Pam2CSK4 and Pam3CSK4 induce iNOS expression via TBK1 and MyD88 molecules in mouse macrophage cell line RAW264.7.

OBJECTIVE: The aim of this study was to investigate the involvement of TLR adaptor molecules, such as TRIF, MyD88, and TBK1 in the induction of iNOS and nitric oxide (NO) production in Pam2CSK4 and Pam3CSK4-treated mouse macrophages. METHOD: Mouse macrophage cell line (RAW264.7) was transfected with trif, myd88, and tbk1 siRNAs before stimulated with Pam2CSK4 and Pam3CSK4. The iNOS gene and protein expression were determined by RT-PCR and immunoblotting, respectively. The NO production was determined by Griess reaction assay. RESULTS: The results showed that the induction of iNOS expression and NO production by Pam2CSK4 and Pam3CSK4 were diminished in tbk1 and myd88-depleted mouse macrophages but not trif-depleted cells. CONCLUSION: These results suggested that the TBK1 and MyD88 molecules were essential for the induction of iNOS expression and NO production by both Pam2CSK4 and Pam3CSK4 via TLR2 signaling.

Regulation of sterile α- and armadillo motif (SARM) containing protein expression in Pam2CSK4- and Pam3CSK4-activated mouse macrophage cell line (RAW264.7) requires TLR9.

INTRODUCTION: We aimed to investigate the involvement of surface TLRs and endosomal TLRs in the regulation of SARM expression by TLR2 ligands (Pam2CSK4 and Pam3CSK4). MATERIALS AND METHODS: Mouse macrophage cell line (RAW264.7) was treated with either Pam2CSK4 or Pam3CSK4 (TLR2 ligands) at a concentration of 100 ng/ml. At indicated time points, the treated cells were lysed. The gene and protein expression of SARM were determined by RT-PCR and immunoblotting, respectively. For silencing of TLR9 function, the cells were transfected with TLR9 siRNAs before stimulation by these two TLR2 ligands RESULTS: The SARM expression was upregulated at both transcriptional and translational levels in time-dependent manner during activation of Pam2CSK4 and Pam3CSK4 in mouse macrophages. Blocking of ligand internalization by cytochalasin D showed interference effect with SARM expression. Moreover, our results also demonstrated that endosomal acidification and TLR9 were required for SARM expression suggesting the essential role of endosomal compartment acidification and TLR9 in regulating SARM expression. CONCLUSION: Our findings suggested the collaboration of TLR2-TLR9 at least in the regulation of SARM expression. However, the underlying mechanism that participated in these two TLRs cooperation is underinvestigated.

Burkholderia pseudomallei induces IL-23 production in primary human monocytes.

Burkholderia pseudomallei, a gram-negative intracellular bacterium, is a causative agent of melioidosis. The bacterium has been shown to induce the innate immune response, particularly pro-inflammatory cytokine production in several of both mouse and human cell types. In the present study, we investigate host immune response in B. pseudomallei-infected primary human monocytes. We discover that wild-type B. pseudomallei is able to survive and multiply inside the primary human monocytes. In contrast, B. pseudomallei LPS mutant, a less virulent strain, is susceptible to host killing during bacterial infection. Moreover, microarray result showed that wild-type B. pseudomallei but not B. pseudomallei LPS mutant is able to activate gene expression of IL-23 as demonstrated by the up-regulation of p19 and p40 subunit expression. Consistent with gene expression analysis, the secretion of IL-23 analyzed by ELISA also showed that wild-type B. pseudomallei induces a significantly higher level of IL-23 secretion than that of B. pseudomallei LPS mutant. These results implied that IL-23 may be an important cytokine for the innate immune response during B. pseudomallei infection. The regulation of IL-23 production may drive the different host innate immune responses between patients and may relate to the severity of melioidosis.

PCR-RFLP and antimicrobial susceptibility profiles of Helicobacter pylori isolated from antrum and corpus of dyspeptic patients in Thailand.

The study determined the genetic heterogeneity of Helicobacter pylori isolates from antrum and corpus of the same dyspeptic patients in a Thai population and determined the relationship between the antimicrobial susceptibility (AS) profile (antibiogram) and PCR-restriction fragment length polymorphism (PCR-RFLP) pattern. One hundred and nineteen H. pylori isolates comprising 7 single and 56 paired antrum and corpus isolates obtained by gastric biopsy from 160 dyspeptic patients were analyzed. For PCR-RFLP, the 820 bp amplicon of ureC was digested with Sau3AI and HhaI, which revealed 16 (A-Q) and 19 (a- s) different PCR-RFLP patterns after Sau3AI and HhaI digestion, respectively. Combination of the restriction enzyme digestion patterns resulted in 35 distinct RFLP types. Among the 56 paired isolates, 47 were infected with H. pylori having the same AS and PCR-RFLP profiles, 7 with different AS profiles but the same PCR-RFLP profiles and 2 with different PCR-RFLP profiles but the same AS profiles. No patient was infected with H. pylori different in both PCR-RFLP and AS profiles. The results indicate that the majority of the paired H. pylori isolates displayed identical AS profile and PCR-RFLP patterns suggesting that most patients were infected with a single strain. Some patients could have been infected with single strains that were different in the AS profiles.

Comparison of media and antibiotic supplements for isolation of Helicobacter pylori from gastric biopsies.

Our objective was to improve the media and the antibiotic supplements in order to increase the detection rate of Helicobacter pylori from gastric biopsy specimens. For the primary isolation of H. pylori taken from gastric biopsies, we compared the efficacy of two media: Columbia blood agar (CBA, Difco); brain heart infusion agar (BHIA, Difco); and two antibiotic supplement sets--a commercial antibiotic supplement (SR147, Oxoid) and an in-house antibiotic supplement (IHS). Gastric biopsies obtained from 210 patients were diagnosed by culture, rapid urease test (RUT) and histology. The true positive criteria were defined as a culture or both urease and histology tests being positive. The H. pylori infection rate was 44.3% (93/ 210). To compare the two media, a total of 106 gastric biopsies were plated on CBA or BHIA with 7% human blood, containing the antibiotic supplement SR147 and incubated under microaerophilic conditions. Of the 106 samples, 48 (45.3%) case of H. pylori infection, compared to the true positive criteria. The isolation rate using a combination of the two media was 83% (40/48). Of the 40 samples, 36 (90%) and 35 (87.5%) were positive on CBA and BHIA, respectively. To compare the two antibiotic supplement sets, a total of 104 gastric biopsies were plated on CBA, containing the commercial antibiotic supplement SR147 (5 mg/l trimethoprim, 10 mg/l vancomycin, 5 mg/l amphotericin B and 5 mg/l cefsulodin) or containing IHS (5 mg/l trimethoprim, 10 mg/l vancomycin, 2 mg/l amphotericin B and 2,500 U/l polymyxin B). Of the 104 samples, 45 (43.2%) case of H. pylori infection were found compared to the true positive criteria. The isolation rate using a combination of the two selective supplement sets was 82% (37/45). Of the 37 samples, 35 (95%) and 34 (92%) were positive with SR147 and IHS, respectively. Our study indicates that the combination of the two media and two antibiotic supplements is useful for maximum recovery of H. pylori isolated from gastric biopsies. CBA, and the commercial antibiotic supplement SR147 provided higher detection rates for H. pylori than BHIA, and IHS but the differences were not statistically significant.