Shihoko Tajika

Infective endocarditis caused by Granulicatella elegans originating in the oral cavity

ABSTRACT We studied the pheno- and genotypes of an oral Granulicatella elegans strain in comparison with those of a blood-derived isolate which caused infective endocarditis. The two isolates exhibited identical biochemical characteristics and had the same drug MICs. Their genotypes were indistinguishable, indicating that these were from the same clone. The transmission of G. elegans from the oral cavity thus should be noted as a possible cause of infective endocarditis.

Antigenic characterisation of a novel Streptococcus anginosus antigen that induces nitric oxide synthesis by murine peritoneal exudate cells.

A novel antigen that induces nitric oxide (NO) synthesis by murine peritoneal exudate cells (PEC) was prepared from a culture supernate of Streptococcus anginosus NCTC 10713 in dialysed medium by column chromatography with DEAE-Sephacel followed by size-exclusion high performance liquid chromatography (HPLC). A chemical analysis of the S. anginosus antigen (SAA) revealed that it mainly consisted of carbohydrates (rhamnose, N-acetylglucosamine, glucose and galactose), smaller quantities of protein and a trace amount of phosphorus. The SAA stimulated PEC from C57BL/6N mice to produce NO and accumulate induced NO synthetase (iNOS) mRNA in a dose-dependent manner, reaching a plateau with 10-30 microg/ml. Furthermore, a reverse transcription-PCR assay revealed that SAA 10 microg/ml could induce mRNA accumulation of tumour necrosis factor-alpha, interleukin (IL)-1beta and IL-6 as well as iNOS. In contrast, Rantz-Randall antigen (RRA), a carbohydrate antigen prepared from the organisms, could not induce NO synthesis or cause the accumulation of iNOS mRNA, although cytokine production was observed after stimulation. The SAA-induced NO synthesis, but not the cytokine production, was sensitive to heat. Furthermore, an immunoblot analysis of SAA indicated that the 43-kDa protein band reacted with anti-SAA but not anti-RRA antibodies. In immunodiffusion, SAA reacted with both anti-SAA and anti-RRA antibodies, and the precipitin bands formed crossing lines, suggesting that SAA could possess two different antigenic components--one that reacts specificially with anti-SAA antibodies and another that has an identity similar to that of RRA. Taken together, SAA, a novel antigen of S. anginosus, was found to induce NO synthesis as well as produce inflammatory cytokines in murine PEC. It is suggested that the protein molecule of SAA may exclusively induce NO synthesis, and its carbohydrate component(s) could have a relationship to cytokine production.

The production of secretory leukocyte protease inhibitor from gingival epithelial cells in response to Porphyromonas gingivalis lipopolysaccharides

Secretory leukocyte protease inhibitor (SLPI) has been recognized as not only a protease inhibitor but also an important defense component in mucosal secretory fluids. To elucidate the functional role in innate immunity in gingival crevices, the SLPI production from a gingival epithelial cell line, GE1, with and without a stimulation of Porphyromonas gingivalis lipopolysaccharides (Pg-LPS), and the inhibitory effect on P. gingivalis proteases were investigated. The unstimulated GE1 cells showed low, but significant, levels of SLPI mRNA expression, which increased after stimulation with Pg-LPS. The upregulation of SLPI mRNA expression in GE1 cells was accompanied by the inductions of IL-6, TNF-α, and IL-1β mRNA expressions. Further experiments using rSLPI indicated that SLPI showed a direct inhibitory effect on the P. gingivalis protease of Lys-gingipain. Thus, it was suggested that gingival epithelial cells could be a substantial producer of SLPI that functions inhibitory to the pathogenic P. gingivalis protease in gingival crevices.

Proinflammatory cytokine production and leukocyte adhesion molecule expression of endothelial cells in response to Abiotrophia defectiva infection

Abiotrophia defectiva, one of the oral streptococci, possessed a relatively higher adhesive ability to the cultured human umbilical vein endothelial cells (HUVEC) as well as fibronectin and vitronectin than other oral streptococci tested. Further, A. defectiva induced HUVEC to produce IL-8 and TNF-α, and subsequently to express E-selectin, ICAM-1 and VCAM-1. Thus, A. defectiva entering into blood streams could adhere to endothelial cells and induce proinflammatory responses through cytokine productions and leukocyte adhesion molecule expressions.

Rapid identification of HACEK group bacteria using 16S rRNA gene PCR-RFLP

HACEK bacteria (Haemophilus spp., Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens and Kingella spp.), classified as fastidious and slow-growing Gram-negative coccobacilli, inhabit human oral cavity, and can cause infective endocarditis. The identification of the HACEK group of bacteria in blood samples from the infective endocarditis patients is known to be rather difficult and occasionally inconclusive by conventional culture methods, because the biochemical characteristics resemble each other. In this study, we developed a rapid and highly sensitive identification method for the HACEK group bacteria by means of 16S rRNA gene PCR amplification followed by restriction fragment length polymorphism analysis (PCR-RFLP) with HinfI and MspI. The 16S rRNA genes were successfully amplified from all the five representative strains of HACEK bacterial species, and typical restriction patterns of the five bacteria were obtained. The restriction patterns were readily distinguishable from each other and are also different from those of other 15 causative pathogens of infective endocarditis.

CD14-dependent and independent B-cell activations by stimulation with lipopolysaccharide from Porphyromonas gingivalis

The functional role of CD14 and Toll-like receptor (TLR) in the Porphyromonas gingivalis lipopolysaccharide (Pg-LPS)-induced activation of B cells was assessed, using CD14-, TLR2- and TLR4-overexpressed murine B cells (CH12.LX). After stimulation with Pg-LPS, CD14- and TLR4-transfected, but not TLR2-transfected, CH12.LX showed higher induction of nuclear factor (NF)-κB activation. Although Pg-LPS induced control CH12.LX to proliferate accompanying by up-regulations of TGF-β and IL-6 mRNA, CD14-transfected cells showed the up-regulation of TGF-β mRNA, but not IL-6 mRNA nor proliferative responses to Pg-LPS. Thus, Pg-LPS could induce B cell activation both in CD14-dependent and independent pathways: In a CD14-dependent pathway, TGF-β production could be induced through TLR4 and NF-κB activation. In contrast, Pg-LPS induced proliferation and IL-6 production in the CD14-independent pathway.