Blastogenic responsiveness in periodontally healthy subjects. Evidence for mitogenic activity in oral bacteria.
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Biomedical subjects
Publications and source records attributed to R R Ranney.
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From replicate trials of experimental gingivitis in four periodontally healthy subjects, 166 bacterial species and subspecies were detected among 3,034 randomly selected isolates from 96 samples. Of these bacteria, Actinomyces naeslundii (serotype III and phenotypically similar strains that were unreactive with available antisera), Actinomyces odontolyticus (serotype I and phenotypically similar strains that were unreactive with available antisera), Fusobacterium nucleatum, Lactobacillus species D-2, Streptococcus anginosus, Veillonella parvula, and Treponema species A appeared to be the most likely etiological agents of gingivitis. Statistical interpretations indicated that the greatest source of microbiological variation of the total flora observed was person-to-person differences in the floras. The next greatest source of variation was the inflammatory status of the sample sites. Person-to-person differences were smallest at experimental day 4. The floras became more diverse with time and as gingivitis developed and progressed. Analyses indicated that sequential colonization by certain species was repeatable and therefore probably predictable. Variation was relatively small between replicate trials, between two sites on the same teeth sampled on the same day, and between the same sites sampled at the same relative time in a replicate trial.
A total of 78 bacteriological samples were taken from the supragingival tooth surface after superficial cleaning with toothpicks or from the periodontal sulci of 42 affected sites in 21 adolescents or young adults with severe generalized periodontitis. Of 190 bacterial species, subspecies, or serotypes detected among 2,723 isolates, 11 species exceeded 1% of the subgingival flora and were most closely associated with the diseased sulci. Eleven others were also sufficiently frequent to be suspect agents of tissue destruction. Many of these species are known pathogens of other body sites. In addition, 10 species of Treponema were isolated. One of these and the "large treponeme" were also more closely associated with severe periodontitis than they were with healthy sites or gingivitis. There were highly significant differences between the composition of the flora of the affected sulci and the flora of (i) the adjacent supragingival tooth surface, (ii) the gingival crevice of periodontally healthy people, and (iii) sites with a gingival index score of 0 or 2 in experimental gingivitis studies. The floras of different individuals were also significantly different. There was no statistically detectable effect of sampling per se upon the composition of the flora of subsequent samples from the same sites. The composition of the supragingival flora of the patients with severe generalized periodontitis that had serum antibody to Actinobacillus actinomycetemcomitans was significantly different from the supragingival flora of patients without this serum antibody. However, there was no statistically significant difference in the composition of their subgingival floras.
The purpose of this study was to determine if adolescents and young adults with severe periodontal disease had precipitating serum antibody reactive with Actinobacillus actinomycetemcomitans, and, if so, to correlate presence or absence of the precipitin with clinical patterns of periodontal destruction. Double diffusion in gel was used to detect precipitins. Test and known positive standard sera were run in different wells against a common well containing a minute of sonicates from two strains of A. actinomycetemcomitans (Y4 and N27). Periodontal loss of attachment was measured with a probe on four surfaces per tooth; missing and unerupted teeth were noted with the aid of radiographs. Precipitating antibody was seen in 77% of sera from 22 subjects with localized juvenile periodontitis, 40% of 35 young subjects with more generalized severe disease, and 2% of 44 periodontally healthy individuals. About 80% of the positive reactions were with strain Y4. Within the diseased groups, there were significantly fewer involved teeth in subjects with precipitating antibody (Ab+) than among those in whose sera precipitins were not seen (Ab-). Comparisons of the distribution and severity of involved teeth between Ab+ and Ab- subjects were consistent with an hypothesis that high levels of antibody specific for A. actinomycetemcomitans have a protective effect against periodontal destruction.
Studies of blastogenic responsiveness induced in peripheral blood lymphocytes (PBL) from periodontally healthy subjects by periodontal bacteria have conflicted. This study was undertaken to examine the blastogenic response of periodontally healthy subjects under experimental conditions which provide improved control of laboratory variables. By using cryopreserved PBL the responses of all subjects in an experiment were determined on the same day under identical conditions. The periodontally healthy populations consisted of subjects of the same age range and sex matched to three distinct groups of periodontally diseased subjects (i.e., juvenile (JP), severe (SP), and moderate (MP) periodontitis). The bacterial stimulants tested were chosen on the basis of known association with and isolation from persons fitting the disease classifications. If PBL response differences between periodontally healthy and diseased subjects exist they should be most obvious in response to predominant organisms associated with the disease states. PBL cultures were harvested after a 4 hour pulse with 3H-thymidine on days 4 and 6 of culture. Three separate experiments were conducted comparing one healthy group and one diseased group. In all three experiments subjects in the healthy group responded as frequently as did those in the diseased group, the dose-response distribution was indistinguishable, and the magnitude of the responses was not substantially different between groups. These results suggest nonspecific activation as the major determinant in the blastogenic response, rather than specific sensitization occurring during initiation or progression of periodontitis.
The objective of this study was to identify and test a convenient means for long-term storage of lymphocytes taken from clinically characterized patients without losing B- or T-cell function. Accordingly, peripheral blood lymphocytes were frozen and stored, and portions of each sample were subsequently assayed for T-cell blastogenic response and B-cell Jerne plaquing at various time intervals after freezing. A comparison of the cell counts of fresh and frozen cultures indicated that cell were recovered after freezing. Furthermore, these cells showed no significant differences in (i) cell viability; (ii) blastogenic response to antigens of Actinomyces maeslandii, Bacteroides melaninogenicus, Fusobacterium nucleatum, and tetanus toxoid; (iii) blastogenic response to phytohemagglutinin and pokeweed mitogen; and (iv) polyclonal B-cell response to pokeweed mitogen as measured by the direct Jerne plaque assay. The retained blastogenic and plaquing responses seen in frozen cultures indicated the maintenance of both T-cell and B-cell function, respectively. This is the first reported demonstration of Jerne plaquing of normal human lymphocytes after freezing. It appears that freezing techniques provide a means for repeating and extending both T- and B-cell assays using frozen stored portions of the same cell samples.
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The objective of this research was to determine whether gram-negative bacteria frequently isolated from periodontally diseased sites contained polyclonal B-cell activators. Polyclonal B-cell activation, which results in nonspecific activation of multiple B-cell clones was analyzed by a hemolysis-in-gel assay designed to detect a broad range of antibody specificities. Extracts from numerous bacterial strains, including Bacteroides gingivalis, Bacteroides melaninogenicus subsp. melaninogenicus, B. melaninogenicus subsp. intermedius, Fusobacterium nucleatum, Selenomonas sputigena, Capnocytophaga ochracea, and Actinobacillus actinomycetemcomitans, were tested. Extracts of the above organisms were found to stimulate polyclonal antibody responses in cultures of normal human peripheral blood lymphocytes, although the magnitude of stimulation varied among the extracts. Optimal antibody-forming cell responses were found at stimulator doses between 5 and 1,000 micrograms/ml. We conclude that the resident gram-negative subgingival flora associated with periodontal lesions possesses potent polyclonal B-cell activators. These activators may contribute to disease pathogenesis by inducing B lymphocytes to produce antibody, osteolytic factors, or both and possibly other mediators of inflammation.
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