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.

Previous studies on the "spontaneous antibody response" have included in vitro steps and it is possible that the response is an in vitro artifact. The objective of the present study was to induce a spontaneous antibody response entirely in vivo and determine if the response is localized and if the magnitude of the response is related to the location of persisting antigen. Antigen was injected into the right hind footpads of mice, and lymph nodes on the right side were draining and lymph nodes on the left side were controls. Antibody-forming cells (AFCs) were enumerated in both draining and nondraining nodes 2 weeks, 2 months, and 1 year after secondary immunization. Four days prior to determining AFC number, the mice were severely bled to stimulate AFC production. Thousands of AFCs were found in the draining lymph nodes and the numbers were dramatic in nodes closest to the injection site that retain the most antigen. In contrast, the vast majority of nondraining nodes lacked any AFCs. One year after immunization, the response was almost exclusively in the popliteal node, draining the foot where antigen was administered a year earlier. These results are consistent with previous data on the spontaneous response and support the hypothesis that antigen retained on FDCs is essential in the maintenance of serum antibody levels.

This study examines several periodontitis-associated bacterial isolates for the presence of mitogenic activity, as indicated by their capacity to stimulate unsensitized lymphocytes to undergo blastogenesis. Germfree mouse spleen cells responded vigorously to all of the bacterial sonic extracts tested. The kinetics and dose responses to these activators in germfree mouse spleen cell cultures paralleled those seen with the standard murine B-cell mitogen, Escherichia coli lipopolysaccharide. In contrast, Streptokinase-Streptodornase (Varidase; Lederle Laboratories) antigen elicited no response. Human cord blood lymphocytes also responded upon stimulation with these same bacterial isolates but failed to respond to Streptokinase-Streptodornase. The frequency, magnitude, and kinetics of these cord blood lymphocyte responses were remarkably similar to those seen with adult peripheral blood lymphocytes. However, in this and previous studies, individuals with unresponsive peripheral blood lymphocytes have been observed. Studies were initiated to determine whether these unresponsive leukocyte preparations truly lacked the capacity to respond to these bacteria or whether unresponsiveness reflected the presence of a regulatory cell population in these cultures. After the removal of the adherent cells from unresponsive peripheral blood lymphocyte cultures, the nonadherent cells were found to be responsive. Therefore, peripheral blood lymphocyte responsiveness appears to be regulated via an adherent cell population. The removal of adherent cells from unresponsive cord blood lymphocyte preparations resulted in a less consistent alteration to responsiveness. However, cord blood lymphocyte preparations unresponsive at a standard cell density were shown to be responsive at altered cell densities.

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.

A role for activated b‐lymphocytes in mediating the initiation and/or progression of periodontal diseases has been proposed in previous work. The present study was conducted to: (1) assess the proportion of total lymphocyte blastogenic response to selected oral bacteria which is composed of T‐cell and B‐cell activation, respectively, and (2) to determine whether different kinetic patterns exist for the T‐cell vs. the B‐cell responses to these bacteria. Using lymphocyte rosetting and culturing techniques, rosette‐positive and rosette‐negative lymphocyte preparations were examined for blastogenic responsiveness following stimulation with a variety of both Gram‐positive and Gram‐negative periodontitis‐associated bacteria. Results of these studies indicated that both peripheral blood lymphocytes (PBL) from healthy adults and cord blood lymphocytes (CBL) from placental afterbirths responded to these bacteria with similar kinetic patterns. The net PBL blastogenic response appeared to consist of an early B‐cell response, which peaked at Days 2 or 3 of culture, followed by a later T‐cell response, which peaked at Days 5 to 6 of culture. The B‐cell response appeared to be T‐cell‐dependent in that B‐cells cultured alone showed minimal thymidine uptake over the entire 6‐day period, but the addition of irradiated T‐cells to these B‐cell cultures resulted in a greatly enhanced B‐cell response.