Surface of hydroxyapatite disks at 2000 (A) and 5000 (B) magnification (HA disk was a standard hexagonal structure). The colonies and depth of the biofilm in the co-culture group were 27.4% and 22.81% less than the control group. The assembly of and S-IgA was observed under CLSM after co-cultivation. In the mature-stage biofilm, no differences were observed between the different groups. Conclusion: These results demonstrate that the anti-caries DNA vaccine induces the production of specific S-IgA antibodies that may prevent dental caries by inhibiting the initial adherence of onto tooth surfaces, thereby reducing the accumulation of on the acquired pellicles. (onto the GNE-495 tooth surface is the initial step and a major factor in the formation of dental plaque4,5. Because is the primary bacterial agent of dental caries, the virulent factors by which it adheres to tooth surfaces are important potential targets for anticariogenic intervention. There are two major virulent factors related to adherence: the glucosyltransferase (GTF) enzyme and the surface protein antigen AgI/II (PAc)6,7,8,9. The GTF enzyme synthesizes glucans from sucrose and mediates the sucrose-dependent accumulation of on the tooth surface10. One region of the GTF, the glucan-binding domain (GLU) is responsible for the composition of the glucans. Glucans mediate the sucrose-dependent adherence of and help tightly adhere to the tooth surface, forming compact dental plaque11. The PAc protein possesses two important functional and immunogenic regions: an N-terminal region that is alanine-enriched and the middle P-region that is proline-enriched, both of which initialize adherence to the acquired pellicle on GNE-495 the tooth surface12. These two virulent factors mediate the adherence of PAC genes. After intranasally immunization of an experimental animal model, this vaccine elicited a specific S-IgA response in saliva that produced a significant anti-caries effect, as determined by the altered caries scores1,13,14. In our previous studies, the targeted fusion DNA vaccine pGJA-P/VAX was administered intranasally in a rat model and demonstrated a significant inverse correlation between the levels of anti-PAc IgA in saliva and the total caries score. In response to pGJA-P/VAX immunization, the secretory IgA inhibits colonization of from adhering to the tooth’s surface and forming plaque biofilms, which are the hallmark of the initiation and formation of dental caries. The aim of this study is to investigate the impact of S-IgAs on adherence and to identify how S-IgAs are induced by the anti-caries DNA vaccine and how S-IgAs interfere with the pathogenesis of infections strains were used: MT 8148 and UA140:: (mutAp-mrfp1) (provided by Kreth, UCLA School of Dentistry); the monomeric red fluorescent protein (mrfp1) exhibits a red fluorescence when analyzed using fluorescence microscopy18. Both strains of were routinely cultured in brain heart infusion (BHI) agar or Modified Scholtens’ Broth (MSB) agar at 37 C in an atmosphere with 85% N2, 5% H2, and 10% CO2; spectinomycin (800 g/mL) was added to the medium to select for the transformants carrying the reporter plasmid in the UA140:: (mutAp-mrfp1) strain. A standard curve of concentrations was obtained and used to determine the bacterial concentration levels of the test samples. Plasmid preparation The anti-caries DNA vaccine pGJA-P was constructed as previously described14. Briefly, pGJA-P encodes the following: the A-P fragment of the gene from gene, the hinge and Fc regions IEGF of the human gene, and the GLU domain of the gene from MT8148 to S-HA beads was examined as previously described20. Briefly, 5 mg of HA beads were equilibrated in buffered KCl at room temperature overnight. The experimental pellicles were prepared by treating the beads with 125 L of normal rat saliva at room temperature for 1 h while continuously inverting the mixture at 6 r/min. After washing twice with buffered KCl, the HA beads were treated for 30 min with buffered KCl, which contained BSA (5 mg/mL) to GNE-495 block any uncoated regions of the HA. After two washes with buffered KCl, the treated beads were incubated for 1 h with 3H]thymidine-labeled cells (2.0109 cells) suspended in 100 L of buffered KCl containing BSA (5 mg/mL) and 100 L of 4-week, 10-week, and 16-week saliva, and 100 L of the serial diluted saliva, for the 10-week immunized mice (1:2; 1:4; 1:8; 1:16; 1:32). The saliva from the unimmunized mice and PBS was used as the control. The beads were washed three times with buffered KCl, and the bound radioactivity was measured using a liquid scintillation spectrometer (TRICARB-2000A; Packard Instruments, Downers Grove, IL, USA). The number of streptococcal cells attached to the beads was determined from the calculated specific radioactivity of the bacteria. The preparation of biofilms biofilms [UA140:: (mutAp-mrfp1)] were formed on saliva-coated hydroxyapatite discs; to grow the biofilms, a technique.
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