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Information on co-adherence of different oral bacterial species is important for understanding interspecies interactions within oral microbial community. Current knowledge on this topic is heavily based on pariwise coaggregation of known, cultivable species. In this study, we employed a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to systematically analyze the co-adherence profiles of oral bacterial species, and achieved a more profound knowledge beyond pairwise coaggregation. Two oral bacterial species were selected to serve as "bait": Fusobacterium nucleatum (F. nucleatum) whose ability to adhere to a multitude of oral bacterial species has been extensively studied for pairwise interactions and Streptococcus mutans (S. mutans) whose interacting partners are largely unknown. To enable screening of interacting partner species within bacterial mixtures, cells of the "bait" oral bacterium were immobilized on nitrocellulose membranes which were washed and blocked to prevent unspecific binding. The "prey" bacterial mixtures (including known species or natural saliva samples) were added, unbound cells were washed off after the incubation period and the remaining cells were eluted using 0.2 mol x L(-1) glycine. Genomic DNA was extracted, subjected to 16S rRNA PCR amplification and separation of the resulting PCR products by DGGE. Selected bands were recovered from the gel, sequenced and identified via Nucleotide BLAST searches against different databases. While few bacterial species bound to S. mutans, consistent with previous findings F. nucleatum adhered to a variety of bacterial species including uncultivable and uncharacterized ones. This new approach can more effectively analyze the co-adherence profiles of oral bacteria, and could facilitate the systematic study of interbacterial binding of oral microbial species.
Adult ; Animals ; Bacterial Adhesion ; DNA, Bacterial ; analysis ; Denaturing Gradient Gel Electrophoresis ; Fusobacterium nucleatum ; physiology ; Humans ; Membranes, Artificial ; Mice ; Microbial Interactions ; physiology ; Polymerase Chain Reaction ; Protein Binding ; Saliva ; microbiology ; Streptococcus mutans ; physiology

7.Involvement of Toll-like receptor in silica-induced tumor necrosis factor alpha release from human macrophage cell line.

Zhen YAN ; Qiao ZHANG ; Lei XU ; Wei-dong WU ; Wen-jie REN ; Lin-hong LIU ; Wu YAO ; Yi-ming WU

Chinese Journal of Industrial Hygiene and Occupational Diseases 2010;28(6):427-429

OBJECTIVETo characterize the role of Toll-like receptor 4 (TLR4) in silica-induced production of tumor necrosis factor alpha (TNFalpha) from macrophage cell line.

METHODSThe human macrophage cell line THP-1 was incubated with silica suspension. Cell media were collected and TNFalpha levels in the supernatants measured with ELISA. To examine the involvement of TLR4 in silica-induced TNFalpha release, the neutralizing antibody (HTA125) against human TLR4 receptor was employed to pretreat THP-1 cells prior to silica treatment. Moreover, murine macrophages expressing wild type or mutated TLR4 were also treated with silica to verify the effect of TLR4 in silica-induced TNFalpha release.

RESULTSCompared with the control group [(3.18 +/- 0.41) pg/ml], the TNFalpha release in cells exposed to 100 microg/ml silica for 4 h and 8 h [(4.71 +/- 0.84), (6.22 +/- 0.58) pg/ml, respectively] increased 1.48 and 1.96 fold, respectively. Pretreatment of THP-1 cells with 20 microg/ml HTA125 antibody significantly blocked silica-induced TNFalpha release by 27%. Furthermore, the TNFalpha content released from cells expressing mutated TLR4 reduced by 30% in compared with that from the cells expressing wild type TLR4 after silica stimulation.

CONCLUSIONTLR4 mediates silica-induced TNFalpha release from macrophages.

Antibodies ; pharmacology ; Cell Line ; Humans ; Macrophages ; drug effects ; metabolism ; Silicon Dioxide ; toxicity ; Toll-Like Receptor 4 ; immunology ; Tumor Necrosis Factor-alpha ; metabolism