Jean-San Chia

A 60-kilodalton immunodominant glycoprotein is essential for cell wall integrity and the maintenance of cell shape in Streptococcus mutans.

ABSTRACT We have demonstrated previously by Western blotting that in naturally sensitized humans, the serum or salivary antibody response toStreptococcus mutans was directed predominantly to a protein antigen with a size of approximately 60-kDa. To identify this immunodominant antigen, specific serum antibodies were eluted from immunoblots and five positive clones with inserts ranging in length from 3 to 8 kb from identical chromosomal loci were obtained by screening a genomic expression library of Streptococcus mutans GS-5. Amino acid sequencing established the identity of this immunodominant antigen, a 60-kDa immunodominant glycoprotein (IDG-60), to be a cell wall-associated general stress protein GSP-781, which was originally predicted to have a molecular mass of approximately 45 kDa based on the derived nucleotide sequence. Discrepancy in the molecular mass was also observed in recombinant his-tagged IDG-60 (rIDG-60) expressed from Escherichia coli. Glycosylation, consisting of sialic acid, mannose galactose, and N-acetylgalactosamine, was detected by lectin binding to IDG-60 in cell wall extracts from S. mutans and rIDG-60 expressed in vivo or translated in vitro. Despite the presence of multiple Asn or Ser or Thr glycosylation sites, IDG-60 was resistant to the effect of N-glycosidase F and multiple O-glycosidase molecules but not to β-galactosidase. Insertional inactivation of the gene encoding IDG-60, sagA, resulted in a retarded growth rate, destabilization of the cell wall, and pleiomorphic cell shape with multifold ingrowth of cell wall. In addition, distinct from the parental GS-5 strain, the isogenic mutant GS-51 was unable to survive the challenge of low pH and high osmotic pressure or high temperature. Expression of the wild-type gene in trans within GS-51 from plasmid pDL277 complemented the growth defect and restored normal cell shape. These results suggested that IDG-60 is essential for maintaining the integrity of the cell wall and the uniformity of cell shape, both of which are indispensable for bacteria survival under stress conditions.

Identification of Stress-Responsive Genes in Streptococcus mutans by Differential Display Reverse Transcription-PCR

ABSTRACT Streptococcus mutans, which causes dental caries in the human oral cavity and occasionally causes infective endocarditis in the heart, withstands adverse environmental stress through diverse alterations in protein synthesis. Differential gene expression in response to environmental stress was analyzed by RNA fingerprinting using arbitrarily primed PCR with a panel of 11mer primers designed for differential display in Enterobacteriaceae. Dot and Northern blot hybridization confirmed that the transcription of several genes was up- or down-regulated following exposure to acid shock from pH 7.5 to 5.5. RNA of a gene designated AP-185 (acid-stress protein) was induced specifically by acid treatment, while RNA of GSP-781 (general-stress protein) was up-regulated significantly when bacteria were exposed to high osmolarity and temperature, as well as low pH. The deduced amino acid sequence of AP-185 shares homology (78% identity) with branched-chain amino acid aminotransferase. Cloning and sequence analysis of GSP-781 revealed a potential secreted protein of a molecular mass of about 43 kDa and with a pI predicted to be 5.5. Transcriptional levels of another gene, designated AR-186 (acid-repressed protein), which encodes putative aconitase, were repressed by acid treatment but were enhanced by plasma or serum components. Analogous results were identified in icd andcitZ genes, and repression of these genes, along with AR-186, was also observed when they were exposed to high osmolarity and temperature. These results indicate that differential regulation of specific genes at the transcriptional level is triggered by different stress and that genes responsible for glutamate biosynthesis in the citrate pathway are coordinately regulated during the stress response of S. mutans.

Inhibition of metastasis, angiogenesis, and tumor growth by Chinese herbal cocktail Tien-Hsien Liquid

BackgroundAdvanced cancer is a multifactorial disease that demands treatments targeting multiple cellular pathways. Chinese herbal cocktail which contains various phytochemicals may target multiple dys-regulated pathways in cancer cells and thus may provide an alternative/complementary way to treat cancers. Previously we reported that the Chinese herbal cocktail Tien-Hsien Liguid (THL) can specifically induce apoptosis in various cancer cells and have immuno-modulating activity. In this study, we further evaluated the anti-metastatic, anti-angiogenic and anti-tumor activities of THL with a series of in vitro and in vivo experiments.MethodsThe migration and invasion of cancer cells and endothelial cells was determined by Boyden chamber transwell assays. The effect of THL on pulmonary metastasis was done by injecting CT-26 colon cancer cells intravenously to syngenic mice. The in vitro and in vivo microvessel formation was determined by the tube formation assay and the Matrigel plug assay, respectively. The in vivo anti-tumor effect of THL was determined by a human MDA-MB-231 breast cancer xenograft model. The expression of metalloproteinase (MMP)-2, MMP-9, and urokinase plasminogen activator (uPA) was measured by gelatin zymography. The expression of HIF-1α and the phosphorylation of ERK1/2 were determined by Western blot.ResultsTHL inhibited the migration and invasion ability of various cancer cells in vitro, decreased the secretion of MMP-2, MMP-9, and uPA and the activity of ERK1/2 in cancer cells, and suppressed pulmonary metastasis of CT-26 cancer cells in syngenic mice. Moreover, THL inhibited the migration, invasion, and tube formation of endothelial cells in vitro, decreased the secretion of MMP-2 and uPA in endothelial cells, and suppressed neovascularization in Matrigel plugs in mice. Besides its inhibitory effect on endothelial cells, THL inhibited hypoxia-induced HIF-1α and vascular endothelial growth factor-A expression in cancer cells. Finally, our results show that THL inhibited the growth of human MDA-MB-231 breast cancer xenografts in NOD-SCID mice. This suppression of tumor growth was associated with decreased microvessel formation and increased apoptosis caused by THL.ConclusionOur data demonstrate that THL had broad-spectra anti-cancer activities and merits further evaluation for its use in cancer therapy.

Platelets Enhance Biofilm Formation and Resistance of Endocarditis-Inducing Streptococci on the Injured Heart Valve

Infective endocarditis is a typical biofilm-associated infectious disease frequently caused by commensal streptococci, but the contribution of host factors in biofilm formation is unclear. We found that platelets are essential for in vitro biofilm formation by Streptococcus mutans or Streptococcus gordonii grown in human plasma. The biofilms were composed of bacterial floes embedded with platelet aggregates in layers, and a similar architecture was also detected in situ on the injured valves of a rat model of experimental endocarditis. Similar to planktonic cells, the streptococci in biofilms were also able to induce platelet aggregation, which facilitates multilayer biofilm formation. Entrapping of platelets directly enhances the resistance of streptococcal biofilms to clindamycin. Prophylactic antibiotics or aspirin can reduce but not prevent or abolish biofilm formation on injured heart valves. Therefore, the platelet is a host factor for commensal streptococci in the circulation to consolidate biofilm formation and protect bacteria against antibiotics.

Glucosyltransferases of Viridans Streptococci Are Modulins of Interleukin-6 Induction in Infective Endocarditis

ABSTRACT The glucosyltransferases (GTFs) of viridans streptococci, common pathogens of infective endocarditis, are extracellular proteins that convert sucrose into exopolysaccharides and glucans. GTFs B, C, and D of Streptococcus mutans are modulins that induce, in vitro and in vivo, the production of cytokines, in particular interleukin-6 (IL-6), from monocytes. The roles of S. mutans GTFs in infectivity and inflammation in situ were tested in a rat experimental model of endocarditis. No significant differences in infectivity, in terms of 95% infective dose and densities of bacteria inside vegetations, were observed between laboratory strain GS-5 and two clinical isolates or isogenic mutant NHS1DD, defective in the expression of GTFs. In aortic valves and surrounding tissues, IL-6 was detected by Western blots and immunostaining 24 h after GS-5 infection, was maintained over 72 h, and was followed by production of tumor necrosis factor alpha but not IL-1β. Animals infected with NHS1DD showed markedly lower levels of IL-6 (less than 5% of that of parental GS-5-infected rats), while tumor necrosis factor alpha was unaffected. In contrast, animals infected with NHR1DD, another isogenic mutant expressing only GtfB, showed a much smaller reduction (down to 56%). These results suggest that GTFs are specific modulins that act during acute inflammation, inducing IL-6 from endothelial cells surrounding the infected valves without affecting bacterial colonization in vegetations, and that IL-6 might persist in chronic inflammation in endocarditis.

Platelet Aggregation Induced by Serotype Polysaccharides from Streptococcus mutans

ABSTRACT Platelet aggregation plays an important role in the pathogenesis of infective endocarditis induced by viridans streptococci or staphylococci. Aggregation induced in vitro involves direct binding of bacteria to platelets through multiple surface components. Using platelet aggregometry, we demonstrated in this study that two Streptococcus mutans laboratory strains, GS-5 and Xc, and two clinical isolates could aggregate platelets in an irreversible manner in rabbit platelet-rich plasma preparations. The aggregation was partially inhibited by prostaglandin I2 (PGI2) in a dose-dependent manner. Whole bacteria and heated bacterial cell wall extracts were able to induce aggregation. Cell wall polysaccharides extracted from the wild-type Xc strain, containing serotype-specific polysaccharides which are composed of rhamnose-glucose polymers (RGPs), could induce platelet aggregation in the presence of plasma. Aggregation induced by the serotype-specific RGP-deficient mutant Xc24R was reduced by 50% compared to the wild-type strain Xc. In addition, cell wall polysaccharides extracted from Xc24R failed to induce platelet aggregation. The Xc strain, but not the Xc24R mutant, could induce platelet aggregation when preincubated with plasma. Both Xc and Xc24R failed to induce platelets to aggregate in plasma depleted of immunoglobulin G (IgG), but aggregation was restored by replenishment of anti-serotype c IgG. Analysis by flow cytometry showed that S. mutans RGPs could bind directly to rabbit and human platelets. Furthermore, cell wall polysaccharides extracted from the Xc, but not the Xc24R, strain could induce pseudopod formation of both rabbit and human platelets in the absence of plasma. Distinct from the aggregation of rabbit platelets, bacterium-triggered aggregation of human platelets required a prolonged lag phase and could be blocked completely by PGI2. RGPs also trigger aggregation of human platelets in a donor-dependent manner, either as a transient and reversible or a complete and irreversible response. These results indicated that serotype-specific RGPs, a soluble product of S. mutans, could directly bind to and activate platelets from both rabbit and human. In the presence of plasma containing IgG specific to RGPs, RGPs could trigger aggregation of both human and rabbit platelets, but the degree of aggregation in human platelets depends on the donors.

Do all the patients with gastric parietal cell antibodies have pernicious anemia

OBJECTIVE This study evaluated whether all the patients with serum gastric parietal cell antibody (GPCA) positivity had pernicious anemia (PA). MATERIALS AND METHODS The blood hemoglobin (Hb), iron, and vitamin B12 concentrations, and mean corpuscular volume (MCV) in 124 GPCA-positive patients were measured and compared with the corresponding data in 124 age- and sex-matched healthy controls. PA was defined by World Health Organization (WHO) as having an Hb concentration < 13 g dl(-1) for men and < 12 g dl(-1) for women, an MCV ≥ 100 fl, and a serum vitamin B12 level < 200 pg ml(-1) . RESULTS We found that 20, 25, and 20 GPCA-positive patients had deficiencies of Hb (men < 13 g dl(-1) , women < 12 g dl(-1) ), iron (<60 μg dl(-1) ), and vitamin B12 (<200 pg ml(-1) ), respectively. Moreover, 16 GPCA-positive patients had abnormally high MCV (≥ 100 fl). GPCA-positive patients had a significantly higher frequency of Hb, iron, or vitamin B12 deficiency and of abnormally high MCV (all P-values < 0.001) than healthy controls. However, only 12.9% of 124 GPCA-positive patients were diagnosed as having PA by the WHO definition. CONCLUSION Only 12.9% of GPCA-positive patients are discovered to have PA by the WHO definition.

Increased prevalence of interleukin-17-producing CD4(+) tumor infiltrating lymphocytes in human oral squamous cell carcinoma.

T helper 17 (Th17) and regulatory T cells share plasticity in the expression of interleukin (IL)‐17 and forkhead box P3 (FOXP3), but their mutual presence in human diseases is unclear.

Identification of a Fibronectin Binding Protein from Streptococcus mutans

ABSTRACT The interaction of viridans streptococci with components of the extracellular matrix (ECM) plays an important role in the pathogenesis of infective endocarditis. We have identified a surface protein ofStreptococcus mutans which binds the ECM constituent fibronectin (Fn). Initially, we found that S. mutans could adsorb soluble Fn in plasma, but with lower efficiency thanStreptococcus pyogenes. In addition, S. mutanscould bind immobilized Fn in a dose-dependent manner when tested using an enzyme-linked immunosorbent assay. Crude extracts of cell wall-associated proteins or extracellular proteins from S. mutans MT8148 specifically bound Fn through a protein with the molecular mass of ca. 130 kDa, as detected by far-Western immunoblotting. The candidate Fn binding protein (FBP-130) was purified to near homogeneity by using Fn coupled Sepharose 4B affinity column chromatography. A rabbit polyclonal antibody against FBP-130 reacted specifically with a protein of molecular mass of ca. 130 kDa in both cell wall and extracellular fractions, and the abundance of FBP was higher in the former than in the latter fractions. The purified FBP bound specifically to immobilized Fn, whereas the binding of soluble Fn to coated FBP could only be detected in the presence of high concentrations of Fn. The purified FBP, as well as anti-FBP immunoglobulin G, inhibited the adherence of S. mutans to immobilized Fn and endothelial cells (ECV304) in a dose-dependent manner. These results demonstrated that FBP-130 mediated the adherence of S. mutans specifically to Fn and endothelial cells in vitro. The characteristics of S. mutans and FBP-130 in binding Fn confirmed that viridans streptococci adopt different strategies in their interaction with ECM.

Serum interleukin-8 level is a more sensitive marker than serum interleukin-6 level in monitoring the disease activity of oral lichen planus

Background  Oral lichen planus (OLP) is a T‐cell‐mediated inflammatory disease. Interleukin (IL)‐8 is a pro‐inflammatory cytokine of host response to injury and inflammation.