Application of flow cytometry to determine the cytotoxicity of urethane dimethacrylate in human cells.

The effects of an oligomer, urethane dimethacrylate (UDMA), on two human cell lines were studied using flow cytometry (FCM). Untreated and treated cultures of propidium iodine-stained KB (epidermal oral carcinoma cells) and human foreskin fibroblast (HFF) cells were analyzed for cellular DNA content. Concentrations of 10 and 25 microM of UDMA slightly perturbed the KB cell cycle progression at 24 and 48 h of incubation. However, the effect of 50 microM was more pronounced at the latter incubation time period. In cell growth experiments, the sublethal concentrations (10 and 25 microM) produced inhibition of KB cell growth rate at a moderate level, which resulted in the prolongation of cell population doubling time. Significant inhibition of cell growth occurred when 50 microM (lethal concentration) was used. Data obtained from the cell cycle perturbation analysis, evidenced by FCM, correlated with the extent of inhibition in KB cell growth rates. The effects of sublethal concentrations were reversible during a 24 h period of oligomer withdrawal from culture medium. In contrast, the effects of 50 microM were not reversible. In HFF cells the depletion of S phase in the cell cycle was the major effect of 50 microM of UDMA. It was concluded that FCM technology is an ideal and practical approach for studying the cytotoxicity of components of dental composites.

Immune response in human melanoma after transfer of an allogeneic class I major histocompatibility complex gene with DNA-liposome complexes.

Analysis of the antitumor immune response after gene transfer of a foreign major histocompatibility complex class I protein, HLA-B7, was performed. Ten HLA-B7-negative patients with stage IV melanoma were treated in an effort to stimulate local tumor immunity. Plasmid DNA was detected within treated tumor nodules, and RNA encoding recombinant HLA-B7 or HLA-B7 protein was demonstrated in 9 of 10 patients. T cell migration into treated lesions was observed and tumor-infiltrating lymphocyte reactivity was enhanced in six of seven and two of two patients analyzed, respectively. In contrast, the frequency of cytotoxic T lymphocyte against autologous tumor in circulating peripheral blood lymphocytes was not altered significantly, suggesting that peripheral blood lymphocyte reactivity is not indicative of local tumor responsiveness. Local inhibition of tumor growth was detected after gene transfer in two patients, one of whom showed a partial remission. This patient subsequently received treatment with tumor-infiltrating lymphocytes derived from gene-modified tumor, with a complete regression of residual disease. Thus, gene transfer with DNA-liposome complexes encoding an allogeneic major histocompatibility complex protein stimulated local antitumor immune responses that facilitated the generation of effector cells for immunotherapy of cancer.

Generation of therapeutic T-lymphocytes after in vivo tumor transfection with an allogeneic class I major histocompatibility complex gene.

In an effort to enhance the generation of tumor-reactive T-lymphocytes for adoptive immunotherapy, we examined the effects of in vivo transfection of an allogeneic major histocompatibility complex (MHC) class I gene (H-2Ks) of the poorly immunogenic B16BL6 (BL6) melanoma of H-2b origin. Cells from lymph nodes (LNs) draining these tumors after transfection were assessed in adoptive immunotherapy experiments for tumor reactivity after sequential activation with anti-CD3 monoclonal antibody (mAb) followed by culture in interleukin (IL)-2. H-2Ks lipofection of progressively growing BL6 subcutaneous tumors did not reduce tumorigenicity. However, in vivo lipofection of BL6 by intratumor inoculation or admixture of H-2Ks cDNA/liposome complexes and tumor cells prior to inoculation resulted in enhanced development of sensitized T-lymphocytes in the draining LN, which mediated the reduction of the numbers of established 3-day parental lung metastases in six of six experiments. In subsequent studies, in vivo transfection of BL6 with naked H-2Ks cDNA was found to be more effective than lipofection in eliciting sensitized T-cells in the draining LN. Admixture of liposomes alone or control plasmid DNA did not have an adjuvant effect similar to H-2Ks cDNA. Relative tumor transfection efficiency was assessed by an indirect assay with the chloramphenicol acetyltransferase (CAT) reporter gene. BL6 tumors were more efficiently transfected by intratumor inoculation with naked cDNA compared with lipofection. In summary, in vivo allogenization of the poorly immunogenic BL6 tumor resulted in enhanced generation of therapeutic T-cells effective in the treatment of parental tumor.