The purpose of this study was to identify and quantify mononuclear cells and invasive bacteria in consecutive histological sections in diseased gingiva. Gingival biopsies were obtained from sites displaying evidences of severe Periodontitis (pocket depth >5 mm, bleeding on probing) in six patients. Specimens were frozen and serially sectioned at 8 fim in a cryostat. Monoclonal antibodies (mAb) directed against membrane markers of mononuclear infiltrate cells and rabbit polyclonal sera against specific bacteria shown to be invasive in association with an immunoperoxidase technique and specific point quantification were used. The mAb panel included Leu 1 (Pan T), Leu 2a (T suppressor/cytotoxic), Leu 3a (T helper/inducer), Leu 6 (Langerhans/dendritic), Leu 7 (NK/K), Leu M3 (monocyte/macrophage), and B7 (B cell). This methodology allows for in situ per cent quantification of mononuclear cell subsets along with identification and quantification of invasive bacteria in the same gingival tissue site. This may be a practical approach to establish the relationship between bacterial invasion and cellular immune response by the host. This technique enabled the characterization of the mononuclear infiltrate in relationship to specifically identified invasive bacteria in diseased gingiva.

Gingival biopsies of the mesial papilla area of the first molar were obtained from each patient at 0‐, 14‐ and 21‐day intervals during plaque formation. The biopsies were fixed, serially sectioned, and Gram‐stained. The incidence and distribution of the bacteria‐like structures were studied by microscopy. In all the specimens the bacterial nature of Gramstained material was substantiated. In the epithelium the highest number of bacteria was found at the outer layer of marginal oral epithelium, sulcular epithelium and apical oral epithelium along with a decreasing pattern of penetration progressing deeper into the layers of tissue. For junctional epithelium the situation was just the opposite. Each subject had significantly higher counts at Day 21 than at Day 14 for both epithelium and connective tissue. Also significantly higher counts were found in connective tissue compared with the epithelium. The higher bacterial density of intragingival bacteria was associated with the higher gingival and plaque indices. This study suggests that early stages of gingival inflammation may be mediated by invasion of bacteria.

Previous investigations have shown that, in biopsies taken from untreated sites of periodontitis, bacteria were present between the epithelial cells and within the connective tissue. In the present study we have examined Gram-stained sections of diseased gingival sites where the disease had recurred after surgical periodontal treatment. The six subjects chosen for the study were patients who had undergone surgical therapy for the treatment of periodontitis and who, upon subsequent recall visits, showed evidence of at least one site recurrent after treatment as detected by bleeding on probing and increased pocket depth of 5 mm or more. A normal control site from the same patient was chosen, preferably contralaterally, showing positive response to treatment with no signs of disease. Sections were stained with either hematoxylin and eosin for tissue survey or Gram for assessment of bacteria and examined by light microscopy. In many specimens, the bacterial nature of Gram-stained material was substantiated. Preliminary results showed a significantly increased number of bacteria in the refractory sites when compared with control sites which responded positively to treatment. The results of this investigation provided further evidence that bacterial presence inside the periodontal tissue may be an important pathogenic factor in periodontal disease.