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Several observed clinical and immunological similarities between lipopolysaccharide (LPS), an integral component of the outer cell membranes of gram-negative bacteria, and peptidoglycan (PGN), a component of the cell walls of gram-positive bacteria, suggest that the two bacterial components initiate analogous signaling pathways after being recognized by monocytes/macrophages (8,12,15,33,49). These signaling pathways are thought to be cascades for NF-B activation. Activated NF-B controls the transcription of numerous genes including those involved in cell proliferation, transformation, inflammation, and survival (2, 3, 32). Besides activating NF-B, LPS and PGN similarly activate other transcription factors, such as CREB/ATF and AP-1, in a membrane CD14 (mCD14)-dependent or -independent manner (13, 14, 18). The real dissimilarity between their signal pathways was revealed recently, and their initial discrimination by different Drosophila melanogaster Toll proteins, evolutionarily conserved from fruit flies to humans, is under way (4, 21). To date, six human Toll-like receptors (TLRs) have been cloned (40,44). Of these, TLR2 and TLR4 have been extensively investigated and are thought to be signal transducers of PGN and LPS, respectively (7,24,32,35,50).TLR2, predominantly distributed in monocytes/macrophages and polymorphonuclear cells (PMNs) (34), had been thought to be a signal transducer of LPS. Conclusive evidence mainly came from the following data. First, the overexpression of TLR2 in human embryonic 293 cells conferred LPS responsiveness on the cells, resulting in NF-B activation and interleukin-8 (IL-8) mRNA expression. Second, the cotransfection of TLR2 with mCD14 synergistically enhanced LPS signaling (24, 50). Third, the putative complex of mCD14-TLR2 after LPS exposure to the cotransfected cells was found to initiate an IL-1 receptor-like NF-B signaling cascade (51). Fourth, the LPS-elevated expression of the TLR2 gene, as measured by reverse transcription-PCR (RT-PCR), was observed in human monocytes and in mouse peritoneal macrophages (31,50). Like the transcriptional expression of TLR2, the transcriptional expression of mCD14 in human monocytes is upregulated by LPS (30). The elevated expression of the TLR2 and mCD14 genes undoubtedly contributes to the resensitization of macrophages to invasive pathogens. However, the finding that TLR4 but not TLR2 senses LPS has challenged the above conclusions.TLR4 is predominantly present in monocytes/macrophages, PMNs, dermal microvessel and umbilical vein endothelial cells, and intestinal epithelial cells (6,9,34). Several studies have shown that a TLR4 transfectant initiates a series of events for NF-B activation, as does TLR2 (7,32). Moreover, the macrophages and B cells from mice (C3H/HeJ) that have a TLR4 mutation in the intracellular domain consisting of the replacement of a conserved proline residue by histone are hyporesponsive to LPS in vitro and in vivo (22,36,38). Further evidence from TLR2 and TLR4 knockout mice has confirmed

Oral viridans group streptococci are the major commensal bacteria of the supragingival oral biofilm and have been detected in human atheromatous plaque. Atherosclerosis involves an ongoing inflammatory response, reportedly involving chronic infection caused by multiple pathogens. The aim of this study was to examine the invasion of human aortic endothelial cells (HAECs) by oral viridans group streptococci and the subsequent cytokine production by viable invaded HAECs. The invasion of HAECs by bacteria was examined using antibiotic protection assays and was visualized by confocal scanning laser microscopy. The inhibitory effects of catalase and cytochalasin D on the invasion of HAECs were also examined. The production of cytokines by invaded or infected HAECs was determined using enzyme-linked immunosorbent assays, and a real-time polymerase chain reaction method was used to evaluate the expression of cytokine messenger RNA. The oral streptococci tested were capable of invading HAECs. The number of invasive bacteria increased with the length of the co-culture period. After a certain co-culture period, some organisms were cytotoxic to the HAECs. Catalase and cytochalasin D inhibited the invasion of HAECs by the organism. HAECs invaded by Streptococcus mutans Xc, Streptococcus gordonii DL1 (Challis), Streptococcus gordonii ATCC 10558 and Streptococcus salivarius ATCC 13419 produced more cytokine(s) (interleukin-6, interleukin-8, monocyte chemoattractant protein-1) than non-invaded HAECs. The HAECs invaded by S. mutans Xc produced the largest amounts of cytokines, and the messenger RNA expression of cytokines by invaded HAECs increased markedly compared with that by non-invaded HAECs. These results suggest that oral streptococci may participate in the pathogenesis of atherosclerosis.

doc-1 is a putative tumor suppressor gene isolated and identified from the hamster oral cancer model. Here, we report the molecular cloning and the functional characterization of the human ortholog of the hamster doc-1 gene. Human doc-1 cDNA is 1.6 kilobase pairs in length and encodes for a 115-amino acid polypeptide (12.4 kDa, pI 9.53). Sequence analysis showed 98% identity between human and hamster doc-1 protein sequences. DOC-1 is expressed in all normal human tissues examined. In oral keratinocytes, expression of DOC-1 is restricted to normal oral keratinocytes. By immunostaining of normal human mucosa, DOC-1 is detected in both the cytoplasm and nuclei of basal oral keratinocytes; while in suprabasilar cells, it is primarily found in the nuclei. Human oral cancers in vivo did not exhibit immunostaining for DOC-1. Like murine DOC-1, human DOC-1 associates with DNA polymerase ␣/primase and mediates the phosphorylation of the large p180 catalytic subunit, suggesting it may be a potential regulator of DNA replication in the S phase of the cell cycle. Using a human doc-1 cosmid as a probe, human doc-1 is mapped to chromosome 12q24. We identified four exons in the entire human doc-1 gene and determined the intron-exon boundaries. By polymerase chain reaction and direct sequencing, we examined premalignant oral lesion and oral cancer cell lines and found no intragenic mutations. Squamous cell carcinoma (SCC)1 of the oral cavity is newly diagnosed in 38,000 Americans each year and in 350,000 people worldwide (1, 2). Approximately half of the patients afflicted die within 5 years of diagnosis, while surviving patients may be left with severe cosmetic and/or functional compromise (1-3). Survival curves of oral cancer patients have plateaued over the past 2 decades and remained among the worst of all cancer sites.The hamster oral cancer model is an excellent model to study the molecular event during oral carcinogenesis (4 -7). doc-1 is a putative tumor suppressor gene identified and isolated from the carcinogen-induced hamster oral cancer model (8). DOC-1 is predicted to be a 114-amino acid peptide with a molecular mass of 12.4 kDa. Transfection of doc-1 into malignant oral keratinocytes led to the reversion of transformation phenotypes including anchorage independence, doubling time, and morphology. The genetic sequence of doc-1 matched to a tumor necrosis factor-␣-induced early-response murine transcript, TU-166 (9), suggesting that doc-1 may be a downstream event in the tumor necrosis factor-␣ signaling pathway. We have recently cloned the full-length mouse doc-1 cDNA (GenBank TM number AF011644); its DNA sequence in the open reading frame is 94% identical to that of the hamster. The predicted amino acid peptides encoded by the mouse and hamster doc-1 open reading frames are identical. Each open reading frame encodes for a 114-amino acid peptide that has a predicted molecular mass of 12.4 kDa and a pI of 9.53.The highly conserved nature of the rodent doc-1 genes prompted us to clone and examine the role of doc-1 in...

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