Protective Effect of a Polyherbal Aqueous Extract Comprised ofNigella sativa(Seeds),Hemidesmus indicus(Roots), andSmilax glabra(Rhizome) on Bleomycin Induced Cytogenetic Damage in Human Lymphocytes

This study was carried out to determine the chemoprotective potential of a polyherbal aqueous decoction comprised ofNigella sativa(seeds),Hemidesmus indicus(roots), andSmilax glabra(rhizome) against bleomycin induced cytogenetic damage in human lymphocytes. Isolated peripheral blood lymphocytes (PBLs) were exposed to bleomycin at a dose of 40 µg/mL for 2 hrs in the presence or absence of different doses of the decoction (100, 300, and 600µg/mL). Modulatory effect of the decoction on bleomycin induced cytogenetic damage was evaluated by (a) degree of chromosomal aberrations (CA), (b) formation of micronuclei (MN), and (c) induction ofγH2AX foci in lymphocytes exposed to bleomycin. Lymphocytes pretreated with the decoction showed that a significant reduction (p<0.05) in bleomycin induced (a) stable and unstable chromosome aberrations (CA), (b) MN formation, and (c) formation ofγH2AX foci, when compared to lymphocytes treated only with bleomycin. The decoction by itself did not induce any significant cytogenetic damage in PBLs. Overall results of the present study confirm that the decoction can attenuate the cytogenetic damage mediated by bleomycin in human PBLs.

Genotoxic and cytotoxic effects of flumetralin in human peripheral blood lymphocytes in vitro

Flumetralin, a synthetic plant growth regulator with herbicidal activity belonging to the 2,6-dinitroaniline class of chemicals, has been evaluated for its ability to induce genotoxicity in human peripheral blood lymphocytes (PBLs). The potential genotoxic and cytotoxic effects of flumetralin were investigated in vitro by chromosome aberration (CA) and cytokinesis-block micronucleus assays. Human PBLs were treated with 125, 250, 500, and 1000 µg/mL flumetralin for 24 and 48 h. Flumetralin statistically significantly increased the frequency of structural CAs at the three highest concentrations (250, 500, and 1000 µg/mL) for both treatment periods (24 and 48 h) when compared with both the negative and solvent controls. In addition, micronucleus formation was significantly induced at higher concentrations (250, 500, and 1000 µg/mL) for 24 h and at 125 and 500 µg/mL of flumetralin for the 48-h treatment period compared with the controls. Because of the excessive cytostatic effects of flumetralin, binuclear cells could not be detected sufficiently at the highest two concentrations (500 and 1000 µg/mL) for the 48-h treatment period. Furthermore, flumetralin significantly decreased the mitotic index and nuclear division index for all concentrations and treatment times compared with the control groups. The present results indicate that flumetralin was clastogenic and cytotoxic/cytostatic to human PBLs. This study presents the first report of the genotoxic and cytotoxic properties of flumetralin.

Engraftment of human peripheral blood lymphocytes in normal strains of mice

Transplantation of bone marrow from SCID mice into lethally irradiated normal mice can potentially endow the normal recipients with characteristics typical of the immune-deficient SCID mouse. In the present study, we investigated whether intraperitoneal grafting of human peripheral blood lymphocytes (PBLs), which has been documented in the SCID mouse, can also be achieved in irradiated BALB/c mice radioprotected with SCID bone marrow. Evaluation of different radiation protocols suggested that, considering the quality of engraftment and rate of survival, optimal results were obtained with split dose total body irradiation (TBI; 4 Gy followed 3 days later by 10 Gy). Monitoring of mouse T cells in peripheral blood indicated an inverse correlation between the presence of such cells and the engraftment of human CD45+ cells in the peritoneum. Also, engraftment of human PBLs in nude BALB/c mice, conditioned with the same radiation protocol, was significantly higher than that achieved in their normal counterparts. Further improvement of human PBL engraftment was found when the mice were thymectomized 2 weeks before conditioning with split TBI. After transplantation of 80 x 10(6) human PBLs in such recipients, a marked engraftment of human T cells and B cells in the peritoneum cavity could be detected for at least 2 months, whereas significant amounts of human Ig could be detected for more than 3 months. Migration of human PBLs into internal organs such as spleen, liver, kidney, and lungs (and into thymus in nonthymectomized mice) was found within a few days of grafting and also persisted for 2 to 3 months. The majority of the engrafted lymphocytes were single-positive CD4+ and CD8+ T lymphocytes, about 50% of which were activated, as judged by their expression of HLA- DR. Staining with anti-CD25 antibody was lower compared with that found with anti-HLA-DR. CD20+ B cells were detected in all of the above- mentioned internal organs, but were mainly concentrated in the spleen. CD14+ monocytes could be detected only during the first week posttransplant of PBLs. Total human Ig in peripheral blood reached an average of 2.8 mg/mL 14 days posttransplant, and continued to be significant for several months. In vitro transformation by Epstein-Barr virus of human B cells from different tissues could be established 30 days after transplantation and led to outgrowth of two IgG+ cell lines, two IgM+ cell lines, and one IgA+ cell line producing 0.6 to 4.2 micrograms/mL human Ig in the supernatant.

Engraftment of human peripheral blood lymphocytes in normal strains of mice

Transplantation of bone marrow from SCID mice into lethally irradiated normal mice can potentially endow the normal recipients with characteristics typical of the immune-deficient SCID mouse. In the present study, we investigated whether intraperitoneal grafting of human peripheral blood lymphocytes (PBLs), which has been documented in the SCID mouse, can also be achieved in irradiated BALB/c mice radioprotected with SCID bone marrow. Evaluation of different radiation protocols suggested that, considering the quality of engraftment and rate of survival, optimal results were obtained with split dose total body irradiation (TBI; 4 Gy followed 3 days later by 10 Gy). Monitoring of mouse T cells in peripheral blood indicated an inverse correlation between the presence of such cells and the engraftment of human CD45+ cells in the peritoneum. Also, engraftment of human PBLs in nude BALB/c mice, conditioned with the same radiation protocol, was significantly higher than that achieved in their normal counterparts. Further improvement of human PBL engraftment was found when the mice were thymectomized 2 weeks before conditioning with split TBI. After transplantation of 80 x 10(6) human PBLs in such recipients, a marked engraftment of human T cells and B cells in the peritoneum cavity could be detected for at least 2 months, whereas significant amounts of human Ig could be detected for more than 3 months. Migration of human PBLs into internal organs such as spleen, liver, kidney, and lungs (and into thymus in nonthymectomized mice) was found within a few days of grafting and also persisted for 2 to 3 months. The majority of the engrafted lymphocytes were single-positive CD4+ and CD8+ T lymphocytes, about 50% of which were activated, as judged by their expression of HLA- DR. Staining with anti-CD25 antibody was lower compared with that found with anti-HLA-DR. CD20+ B cells were detected in all of the above- mentioned internal organs, but were mainly concentrated in the spleen. CD14+ monocytes could be detected only during the first week posttransplant of PBLs. Total human Ig in peripheral blood reached an average of 2.8 mg/mL 14 days posttransplant, and continued to be significant for several months. In vitro transformation by Epstein-Barr virus of human B cells from different tissues could be established 30 days after transplantation and led to outgrowth of two IgG+ cell lines, two IgM+ cell lines, and one IgA+ cell line producing 0.6 to 4.2 micrograms/mL human Ig in the supernatant.

A unique V-J-C-rearranged gene encodes a gamma protein expressed on the majority of CD3+ T cell receptor-alpha/beta- circulating lymphocytes.

We have recently described an mAb, anti-Ti gamma A, that recognizes an antigenic determinant carried by a TCR gamma chain. This antibody binds to approximately 3% of human PBLs and delineates a CD2+, CD3+, TCR-alpha/beta-, CD4-, CD8+/-, CD5+, NKH1-, and HLA class II- subset. The present study was designed to identify the gene encoding the Ti gamma A epitope. A first analysis was carried out on a previously characterized TCR gamma + fetal-cloned cell line termed F6C7. It was found that F6C7 cells have one gamma rearrangement on each chromosome: one joins V gamma 3 to J gamma 1, and the second joins V gamma 9 to J gamma P. Because only the latter allele appeared to be transcribed in the F6C7 lymphocytes, these data strongly suggested that anti-Ti gamma A mAb is specific for either a V gamma 9 or a V gamma 9-J gamma P-encoded peptide. To confirm this point, we studied an additional series of 13 randomly selected Ti gamma A+ cloned cells derived from peripheral blood of three distinct adult individuals. Each one of these lymphocytes was shown to both possess and transcribe a V gamma 9-J gamma P-C gamma 1-rearranged gene. It is therefore concluded that a predominant subpopulation of CD3+ TCR-alpha/beta- human circulating T lymphocytes (namely, the subset defined by anti-Ti gamma A mAb) surface expresses a gamma protein with a limited potential of variability from one cell to another.