• In vitro production of human antibodies reactive with periodontal pathogens, using Epstein-Barr virus immortalization of B lymphocytes derived from peripheral blood of periodontal patients, with an emphasis on the production of a human monoclonal antibody reactive with the heat-modifiable outer membrane protein of Actinobacillus actinomycetemcomitans

      Raulin, Leslie A.; Falkler, William A., Ph.D. (1995)
      The purpose of this research was to develop cell line(s) that produce a human monoclonal antibody (huMAb) specific to an oral pathogenic microorganism using Epstein-Barr virus (EBV) transformation of B cells isolated from the gingiva and the peripheral blood of periodontal patients with active disease. The resulting huMAB was then characterized. Excised gingival tissue was obtained from 6 patients and peripheral blood was obtained from 3 of the same patients. To release cells, the gingival tissue was digested with collagenase. Gingival mononuclear cells (GMC) and peripheral blood mononuclear cells (PBMC) were then separated from the gingival extracts, patient peripheral blood, and leukophoresed blood from an anonymous donor by Ficoll centrifugation. The resulting cells were infected with EBV, cultured in 96-well plates, and observed for foci of transformation. Using a dot-immunobinding assay (DIB), the cell culture supernatant fluids (CCS) of transformation-positive wells were tested for Ab reactivity to a panel of 11 microorganisms. Continuously positive cultures were expanded and cloned by limiting dilution. Using an ELISA, clone CCS was tested for Ab reactivity to the microorganism to which the originating culture had exhibited reactivity (target organism). Positive responders were expanded and retested with the ELISA. Continuously producing clones were expanded further, CCS was collected, and excess cells were cryopreserved. The class of Ab in each CCS was determined with a DIB, IgG subclasses and concentration were determined with a commercial ELISA, and the light chain type was determined with a fixed cell indirect immunofluorescence assay (IFA). Using representative strains of the target microorganism, Western blots were performed (separation of bacterial proteins by SDS-PAGE, electroblotting onto nitrocellulose, and probing with concentrated CCS) to determine the MW of the band(s) to which the human antibodies (huAbs) bound. To exclude the possibility of non-specific Ig binding by the target organism, a Western blot was performed with a huMAb of the same IgG subclass and light chain type but with specificity toward a viral protein.;GMC did not transform. Of 517 wells receiving PBMC, 503 wells (97%) had foci of transformation. CCS from 71 of the 517 (14%) wells initially tested positive for Ab activity, most commonly to Eikenella corrodens, Actinobacillus actinomycetemcomitans (Aa), Bacteroides fragilis, Peptostreptococcus micros, and Campylobacter rectus, in order of decreasing frequency. Subsequent DIB during expansion of the cells demonstrated 28, 12, 7, 3, and finally 1 culture with Ab activity (II-24P, reactive with Aa ATCC 43718).;A similar (pilot) experiment was performed with PBMC from 2 healthy control volunteers and from leukophoresed blood. Although initial Ab-producing cultures were obtained, none of the clones produced significant amounts of Ab. (Abstract shortened by UMI).
    • Multispecies oral biofilms studied at the single community level as a model system for spatiotemporal development of biofilms and interspecies interactions

      Chalmers, Natalia I.; Kolenbrander, Paul E.; Bavoil, Patrik M. (2008)
      Oral biofilms are multispecies communities that are important in the development of the two most prevalent oral diseases---dental caries and periodontal disease. The primary initial colonizers of human enamel are streptococci, veillonellae and actinomyces. Bacteria of these genera coaggregate (cell-cell interactions between genetically distinct bacteria). The streptococci can be classified into two groups: those with receptor polysaccharides (RPS) and those with adhesins that recognize RPS. RPS mediates streptococcal intrageneric coaggregation. Besides coaggregation, veillonellae interact with streptococci metabolically by using lactic acid produced by streptococci. The hypothesis tested in this study is that coaggregation mediated interactions between streptococci and veillonellae are important for the development of oral biofilms in vivo. To translate naturally occurring interactions from undisturbed dental plaque to multispecies in vitro communities, a simple community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by immunofluorescence with quantum-dot-conjugated antibodies and micromanipulated from the tooth surface. Characterization of the application of quantum-dot primary immunofluorescence was accomplished and was essential for the success of this approach. An antibody-unreactive streptococcus invisible during micromanipulation was also obtained. The streptococci were identified as Streptococcus oralis and Streptococcus gordonii. The veillonellae could not be cultured; however, immunoreactive veillonellae cells were present in the original mixture and a Veillonella 16S-rRNA gene sequence was amplified. This sequence was similar to Veillonella sp. PK1910. The two streptococci coaggregated by an RPS-dependent mechanism, and both coaggregated with Veillonella sp. PK1910. Veillonella sp. PK1910 was used as a surrogate in in vitro community reconstruction. In vitro the two streptococcal isolates and Veillonella sp. PK1910 grew on saliva as the sole nutritional source and formed interdigitated multispecies clusters. Veillonella sp. PK1910 grew only in biofilms where streptococci were present. To map the spatial relationship between these species in vivo we used a novel approach integrating immunofluorescence and fluorescence in situ hybridization. Reconstruction in vitro of a community composed of Veillonella sp. PK1910, S. oralis, and S. gordonii demonstrates the vital role of RPS-mediated coaggregation in initial colonization of multispecies communities. The integrated in vitro - in vivo approach serves as a new paradigm for the study of oral biofilms and their development.