Frequency analysis of B lymphocytes specific for Rh antigens in naturally immunized Rh-negative women.

BACKGROUND AND OBJECTIVES: Despite a successful outcome of the anti-D prophylaxis programme, alloimmunization still occurs. The aim of this study was to estimate the frequency of Rh-specific B lymphocytes in the peripheral blood of nine Rh-alloimmunized individuals at different time intervals after parturition. MATERIALS AND METHODS: The donors' B lymphocytes were transformed with Epstein-Barr virus (EBV) and cultured at different cell densities over a feeder of human fetal fibroblasts. Culture supernatants were screened for human immunoglobulin by enzyme-linked immunosorbent assay (ELISA) and for anti-Rh antibody by using a direct haemagglutination technique. The percentage of CD19+ B lymphocytes in peripheral blood was determined by flow cytometry, and the frequency of Rh-specific B lymphocytes was estimated by limiting-dilution assay (LDA). RESULTS: The frequency of Rh-specific B lymphocytes varied from 1 : 150 to 1 : 27,850 in different donors. There was a decrease in this frequency and level of anti-Rh antibody with increase in time interval between bleeding and last exposure to the antigen. Furthermore, a positive correlation was observed between the titre of Rh-specific antibody and frequency of Rh-specific B cells in each of three subjects bled at multiple time-points postdelivery. CONCLUSIONS: The magnitude of the specific antibody response to Rh antigens varies greatly in Rh-alloimmunized women, which partly reflects the difference in frequency of specific B cells in these individuals.

In vitro cytotoxicity of a new epoxy resin root canal sealer.

The cytotoxic effects of a new epoxy resin-based root canal sealer (AH-plus), together with those of two other commonly used endodontic sealers (AH26 and zinc oxide-eugenol), have been studied in vitro on a culture of human gingival fibroblasts. Cytotoxicity was assessed by direct incubation of sealers' extracts with the cultured fibroblasts at different time intervals. Morphological and cytotoxic effects of the sealers were evaluated microscopically and spectrophotometrically using the neutral red cytotoxicity assay. Our results demonstrated that the cytotoxic effects induced by zinc oxide-eugenol were detectable as early as 1 hr after mixing and remained at a high level until completion of the experiment (5 wk). AH26, however, induced early cytotoxic effects that lasted for 1 wk, followed by a substantial reduction in cytotoxicity. Cytotoxicity of the AH-plus was confined to the early period of experiment and was no longer detectable after 4 hr of mixing. Comparison between the results obtained for each sealer revealed significant differences at particular time intervals. Our findings suggest the potential advantage of this sealer over the other two sealers.