Studies of Proliferative Responses by Long-term-cryopreserved Peripheral Blood Mononuclear Cells to Bacterial Components Associated with Periodontitis

Freezing techniques provide a means for repeating and extending immunological assays with frozen aliquots of an individual's peripheral blood mononuclear cell fraction. Lymphocytes which are stored frozen for a limited time retain their ability to respond to polyclonal B-cell activators, mitogens, and antigens of dental interest. Our studies extend these previous findings by determining lymphocyte functional activity following frozen storage for up to 100 weeks. In addition, the autologous immune response was measured by spontaneous lymphocyte proliferation following 0, 1, 40, and 60 weeks of frozen storage. Peak responses for all individuals occurred at day 7 of incubation. The lymphocyte proliferative response to the superantigens toxic shock syndrome toxin-1 (TSST-1) and Staphylococcus enterotoxin A (SEA) were not changed after 100 weeks of frozen storage. Maximum responses varied among the individuals but occurred at equivalent stimulator concentrations. However, slopes generated from data obtained following 0, 4, 13, 20, 30, 50, 88, and 100 weeks of frozen storage showed no significant deviation from zero (P > 0.05) for all individuals tested. After 100 weeks of storage, the total changes in proliferative activity (counts per minute per week) were -2.1% +/- 16.8% and -5.5% +/- 17.0% for TSST-1 and SEA, respectively. The lymphocyte proliferative responses to pokeweed mitogen, concanavalin A, and sonicates of two periodontal pathogens (Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans) following frozen storage were similar to those with TSST-1 and SEA. These results indicate that peripheral blood mononuclear cells stored frozen may serve as appropriate controls to monitor changes in the disease state long-term periodontal treatment.