Effects of platelet growth factors on human mesenchymal stem cells and human endothelial cells in vitro.

The aim of the present in vitro study has been to investigate the effects of a enriched platelet derived growth factors on proliferation and migration of human endothelial and mesenchymal stem cells and on osteogenic differentiation of stem cells. Platelet rich plasma has been produced, yielding a four time higher number of thrombocytes than normal plasma. Degranulation of platelets has been performed by means of calcium and thrombin. Plasma has served as a control, whereas plasma in combination with calcium and thrombin was used to distinguish the difference between calcium and/or thrombin mediated effects and growth factor induced effects on the cells. The observed enhanced proliferation and migration of endothelial cells towards the platelet derived growth factors was driven by the plasma component of these preparations. However PDGF solely stimulated the migration and proliferation of mesenchymal stem cells. The increased osteogenic differentiation of growth factor treated mesenchymal stem cells was mostly driven by the high level of calcium used for the platelets degranulation. In summery, the different components of platelet derived growth factors work together to influence human endothelial and mesenchymal stem cells. This is of special clinically interest regarding the stimulation of bone healing in orthopaedic and traumatic surgery.

Radiation enhancement of gemcitabine in two human squamous cell carcinoma cell lines.

BACKGROUND: Gemcitabine (dFdC) is a new nucleoside analogue with promising activity in different solid tumors. We investigated whether dFdC enhances the effect of irradiation in human squamous carcinoma cells of the oropharynx (#4197) and of the uterine cervix (HeLa) with special regard to the time-dose-relationship concerning dFdC and the dependence upon the timing of irradiation. MATERIALS AND METHODS: Under standardized conditions monolayers of cells were exposed to various dFdC concentrations (0.003-10 mumol/l) for different times (4-24 h). Irradiation (0-6 Gy) followed immediately or 12 h after dFdC exposure (0.003-0.03 mumol/l; 4-24 h). RESULTS: The cytotoxic effect of dFdC depends on its concentration and the exposure duration. Exposed to non and/or slightly cytotoxic concentrations (> or = 0.003-0.03 mumol/l) for 4, 8, 16 and 24 h and followed by immediate irradiation the radiation enhancement ratio (RER) is 1.03-1.67 in #4197 cells and 1.04-2.47 in HeLa cells, respectively. Irradiated 12 h after 24 h exposure (dFdC 0.01-0.03 mumol/l) the RER is reduced to 1.10-1.17 (#4197) and 1.18-1.72 (HeLa). CONCLUSIONS: Depending on the drug concentration, exposure duration, and timing of irradiation, dFdC enhances the irradiation effect on human squamous cell carcinoma cell lines (#4197, HeLa).

[The intensification of the radiotherapeutic effect on HeLa cells by gemcitabine]. / Verstärkung der radiotherapeutischen Wirkung auf HeLa-Zellen durch Gemcitabine.

BACKGROUND: Gemcitabine (2'.2'-difluorodeoxycytidine; dFdC) is a new nucleoside analog with promising activity in different solid tumors in vivo and in vitro. As published up to now, combined with irradiation dFdC demonstrates a radiosensitizing effect on pancreas and colon carcinoma cell lines. We investigated the influence of dFdC on the radiosensitization of human squamous carcinoma cells of the cervix (HeLa-cells, ATCC CCL-2). MATERIAL AND METHODS: Under standardized conditions monolayer cultures of HeLa-cells were incubated in medium with dFdC for different times (4 to 24 hours) and exposed to different concentrations (0.003, 0.01 and 0.03 mumol/l). Irradiation (2 to 6 Gy, electron beam) followed immediately or 12 hours after dFdC-exposure. Cell survival was determined by colony forming assay. Using the linear-quadratic model cell survival curves were fit after correction for drug-induced cytotoxicity and the mean inactivation dose (MID) was calculated. Radiation enhancement was defined as the ratio MIDRT(= Control)/MIDRT + dFdC > 1. RESULTS: Exposed to gemcitabine for 4 and 8 hours and followed by immediate irradiation the radiation enhancement ratio (Table 1) is 1.07 to 1.14 and 1.04 to 1.22, respectively, if dFdC concentration is > or = 0.01 to 0.03 mumol/l. Further increase of the irradiation effect is demonstrated in cells exposed to > or = 0.003 to 0.03 mumol/l dFdC for 16 and 24 hours (radiation enhancement ratio 1.08 to 2.0 and 1.08 to 2.48, respectively) (Figure 3). If irradiation is applied 12 hours after 24-hour-exposure (0.01 and 0.03 mumol/l) the enhancement ratio was 1.18 and 1.7, respectively (Figure 4). CONCLUSIONS: In cell cultures the assays combining irradiation with dFdC demonstrate that dFdC is a potent radiation sensitizer of HeLa-cells. The effect of irradiation on cells pre-treated with non- and hardly cytotoxic concentrations of dFdC is increased in dependence of dose and time of exposure.

Selective inhibition of the generation of T suppressor cells of contact sensitivity in vitro by interferon.

The T suppressor (Ts) cell response in contact sensitivity is preferentially inhibited by murine interferon-alpha, beta (IFN-alpha, beta) in vivo. Previous studies in vivo have suggested that IFN exerts its effect directly on the Ts subpopulation rather than through an effect on antigen-presenting macrophages. Nevertheless, the mechanism of this selective blockade remained unclear. To better define the mechanism(s) of inhibition of suppression by IFN-alpha, beta, we determined whether IFN acted on lymphocytes, macrophages, or both. Antigen-specific T effector cells of delayed-type hypersensitivity (TDH) and Ts cells were induced in vitro by co-culture of spleen lymphocytes with bone marrow-derived antigen-presenting macrophages (BM-MA) pulse-labeled with 2,4-dinitrobenzene sulfonate (DNBSO3). TDH or Ts activity was demonstrated by transfer of the lymphocytes into naive recipient BALB/c mice after 3 days of culture. BM-MA cultured for 5 to 7 days (BM-MA d5-7) before labeling preferentially activated TDH cells (Thy-1+, Lyt-1+2-); 10- to 14-day-old BM-MA (BM-MA d10) induced Ts cells (Thy-1+, Lyt-2+), as previously shown. Treatment of the spleen lymphocyte suspension with pure mouse IFN-alpha, beta at a dose of 10(3) U/10(8) cells completely blocked the induction of Ts cells but had no effect on the induction of TDH cells. Pretreatment of the antigen-presenting BM-MA for 24 hr with IFN (10(2) U/3 X 10(5) cells) had no effect on the induction of Ts and TDH cells. Cultivation of lymphocytes on a DNP-BM-MA d6 monolayer did not result in the induction of Ts cells; however, in the presence of a goat anti-murine IFN-alpha, beta antibody, Ts cells were induced. This finding indicates that the spontaneous release of IFN-alpha, beta in those cultures prevented the induction of Ts cells. These results confirm our previous observation that Ts cells are more easily blocked by IFN-alpha, beta than TDH cells, and demonstrate that IFN affects the Ts subpopulation not via modulation of the antigen-presenting macrophages. IFN-alpha, beta-producing, antigen-presenting, or accessory cells may therefore prevent the activation of this type of Ts cell.