Cell death induced by hydroxyapatite on L929 fibroblast cells.

Biomaterials intended for end-use application as bone-graft substitutes have to undergo safety evaluation. In this study, we investigated the in vitro cytotoxic effects especially to determine the mode of death of two hydroxyapatite compounds (HA2, HA3) which were synthesized locally. The methods used for cytotoxicity was the standard MTT assay whereas AO/PI staining was performed to determine the mode of cell death in HA treated L929 fibroblasts. Our results demonstrated that both HA2 and HA3 were not significantly cytotoxic as more than 75% cells after 72 hours treatment were viable. Furthermore, we found that the major mode of cell death in HA treated cells was apoptosis. In conclusion, our results demonstrated that these hydroxyapatite compounds are not cytotoxic where the mode of death was primarily via apoptosis.

Distribution of Border disease virus antigen in lymphocyte subpopulations in the peripheral blood of experimentally infected lambs.

Ten lambs were experimentally infected with Border disease virus and the distribution of viral antigen in lymphocyte subpopulations studied by flow cytometry. The virus was isolated in culture from the peripheral blood mononuclear cells (MNC) of all experimentally infected lambs for a mean period of 9.8 +/- 1.03 days. The peak virus titre of 3.26 log10 TCID50 per 10(6) MNC was attained Day 9 post-inoculation (pi). Viral antigen was present in peripheral blood lymphocytes of experimentally infected lambs as early as 24 h pi and continued to be detected up to Day 10 pi. The number of lymphocytes expressing viral antigen rose from 12.38 +/- 1.22% in samples taken Day 3 pi to 23.21 +/- 2.82% on those collected Day 7 pi, dropping gradually thereafter. During the peak period of infection, 12.46 +/- 2.09% of B cells, 37.71 +/- 10.96% of T cells and 52.33 +/- 8.27% of lymphocytes which were neither B nor T lymphocytes expressed viral antigen. Most of the infected lymphocytes expressed the OvCD5 (T cell) molecule. The virus affected all T cell subsets but the suppressor/cytotoxic (OvCD8+) cells appeared to be the main targets. During the peak period of infection, 54.20 +/- 6.16% of the infected T cells expressed the OvCD8 molecule, 31.58 +/- 7.12% were OvCD4+, and 12.67 +/- 6.50% were OvWC1+ (T-19+, gamma/delta).

Border disease virus antigens in lymphocyte subpopulations in the peripheral blood of persistently infected sheep.

Biotinylated virus-specific antibodies were used to detect Border disease virus antigen by flow cytometry in the mononuclear cells of the peripheral blood of 15 sheep persistently infected with Border disease virus. The viral antigen was present in 12.86 +/- 4.65% (mean +/- SD) of mononuclear cells (MNC). The percentage of MNC that contained viral antigen was higher in lambs than in adult sheep, with mean rates of 22.68 +/- 5.02% and 11.65 +/- 4.39%, respectively. Depletion methods were used to estimate the distribution of viral antigen in peripheral blood lymphocyte subsets. The viral antigen was present in T-cells (OvCD5+), B-cells (LCAp220+) and non-T- and non-B-cells. Depletion studies revealed that 5.39 +/- 2.47% of the cells expressing the LCAp220 epitope (B-cells), 23.38 +/- 11.38% of those expressing the OvCD5 epitope (T-cells) and 55.07 +/- 10.93% of those which were neither B- nor T-cells were positive for viral antigen. Most (57.18 +/- 5.41%) of the T-cells containing viral antigen were cytotoxic/suppressor (OvCD8), 25.63 +/- 2.97% were helper (OvCD4) cells and 12.24 +/- 3.21% expressed the gamma/delta (OvWC1) epitope.

Cytotoxic T cell responses in lambs experimentally infected with Border disease virus.

Mononuclear cells cytotoxic to a noncytopathic strain of Border disease virus (BDV) but not to uninfected cells were detected in the peripheral blood of experimentally infected lambs, 10 days after experimental infection, reaching peak levels of cytotoxicity 15 days post-inoculation. The specificity of the cytotoxic activity to BDV-infected cells was tested by including autologous targets infected with another virus (bovine respiratory syncytial virus, BRSV) and normal uninfected autologous targets. The cytotoxic T cells were virus-specific, as only autologous BDV-infected targets were lysed, whereas autologous targets infected with BRSV and uninfected targets were not lysed. The present results also suggest that the cytotoxic activity was largely MHC-restricted as the specific release from BDV-infected autologous targets was significantly higher than that of BDV-infected heterologous targets (P < 0.001) and the cytotoxicity against BDV-infected homologous targets was significantly reduced by the selective depletion of OvCD8+ T cells (P < 0.001).

Lymphocyte responses to viral antigens and phytohaemagglutinin in persistently viraemic sheep and lambs experimentally infected with Border disease virus.

Lymphocytes obtained from lambs 5 to 21 days after experimental infection with Border disease virus (BDV) showed significant blastogenic responses to live or inactivated BDV. Live virus stimulated significantly higher lymphocyte transformation (LT) responses than inactivated virus. Lymphocytes obtained from uninfected control and persistently infected lambs had no significant response to live or inactivated antigen. Lymphocyte responses to phytohaemagglutinin were significantly higher in control lambs than in experimentally infected lambs in samples obtained 5 to 10 days post-inoculation.

Replication of border disease virus in ovine lymphocytes and monocytes in vitro.

Adherent and non-adherent mononuclear cells obtained from the peripheral blood of normal sheep supported the in vitro replication of a non-cytopathic and a cytopathic strain of Border disease virus (BDV) with no apparent cytopathic effects. There was a significant rise in virus titres in adherent mononuclear cells (monocyte) and non-adherent (lymphocyte) cultures infected with both non-cytopathic and cytopathic strains of BDV 24 hours after inoculation. Peak virus titres of 5.36 log10 TCID50 ml-1 were recorded in adherent samples incubated for 48 hours while peak titres of 6.17 log10 TCID50 ml-1 were recorded in lymphocyte culture after 72 hours of incubation. Both the non-cytopathic and the cytopathic strains of BDV produced significantly higher titres in non-adherent (lymphocyte) cultures than in adherent (monocyte) cultures (P < 0.001) but the replication in adherent cells was faster than in nonadherent cell cultures. The addition of virus on both types of mononuclear cell cultures had no effect on cell viability but it had a significant inhibitory effect on the blastogenic responses of lymphocytes to phytohaemagglutinin.

Effects of experimental infection with border disease virus on lymphocyte subpopulations in the peripheral blood of lambs.

Experimental infection with Border disease virus was characterised by significant changes in the total numbers of leucocytes and neutrophils, and in the proportions and numbers of the different lymphocyte subpopulations. Three days after experimental infection there was significant leucopenia due to lymphocytopenia and neutropenia (P < 0.001). The lymphocytopenia and neutropenia lasted for up to seven days after inoculation. The lymphocytopenia was due to a reduction in the number of both T cells and B cells. During the early period of infection, the reduction in T cells was mainly due to a reduction in the number of OvCD4+ and T-19+ lymphocytes as the number of circulating OvCD8+ cells was not significantly affected. The cells expressing the OvCD4 and OvWC1 epitopes returned to pre-inoculation values 10 and 14 days after inoculation, respectively. In contrast, during the same period, the number of T cells expressing the OvCD8 molecule became significantly higher than the corresponding pre-inoculation values. There were no significant changes in all the T cell subsets in the control lambs.