Edaravone protects human peripheral blood lymphocytes from γ-irradiation-induced apoptosis and DNA damage

Radiation-induced cellular injury is attributed primarily to the harmful effects of free radicals, which play a key role in irradiation-induced apoptosis. In this study, we investigated the radioprotective efficacy of edaravone, a licensed clinical drug and a powerful free radical scavenger that has been tested against γ-irradiation-induced cellular damage in cultured human peripheral blood lymphocytes in studies of various diseases. Edaravone was pre-incubated with lymphocytes for 2 h prior to γ-irradiation. It was found that pretreatment with edaravone increased cell viability and inhibited generation of γ-radiation-induced reactive oxygen species (ROS) in lymphocytes exposed to 3 Gy γ-radiation. In addition, γ-radiation decreased antioxidant enzymatic activity, such as superoxide dismutase and glutathione peroxidase, as well as the level of reduced glutathione. Conversely, treatment with 100 μM edaravone prior to irradiation improved antioxidant enzyme activity and increased reduced glutathione levels in irradiated lymphocytes. Importantly, we also report that edaravone reduced γ-irradiation-induced apoptosis through downregulation of Bax, upregulation of Bcl-2, and consequent reduction of the Bax:Bcl-2 ratio. The current study shows edaravone to be an effective radioprotector against γ-irradiation-induced cellular damage in lymphocytes in vitro. Finally, edaravone pretreatment significantly reduced DNA damage in γ-irradiated lymphocytes, as measured by comet assay (% tail DNA, tail length, tail moment, and olive tail moment) (p < 0.05). Thus, the current study indicates that edaravone offers protection from radiation-induced cytogenetic alterations.     

Analysis of chromosome aberrations in human peripheral lymphocytes induced by in vitro α-particle irradiation

Irradiation of human lymphocytes by α-particles under different conditions has been seen to be substantially more effective in the induction of dicentric chromosomes than irradiation by ψ-rays. However, the relative biological effectiveness (RBE) determined in these studies RBE are likely to be due in part to differing exposure conditions. Therefore, a technique designed to insure iniformity of irradiation was developed in the present study, and complications due to the cell cycle kinetics were controlled. After stimulation with phytohaemagglutinin (PHA), separated lymphocytes were allowed to attach for 3 h to the thin foil bottom of an irradiation chamber. Cell monolayers were exposed with α-particles from²⁴¹Am. Strong over-dispersion was noted for the cell-to-cell variance of the number of dicentrics. The dose response of dicentrics was linear, with a yield of 0.27 dicentrics per cell and per Gy. This corresponds to a low dose RBE of 15 relative to¹³⁷Cs γ-ray exposure under the same experimental conditions.     

Helicobacter pylori induces activation of human peripheral γδ+ T lymphocytes

Helicobacter pylori is a gram-negative bacterium that causes gastric and duodenal diseases in humans. Despite a robust antibody and cellular immune response, H. pylori infection persists chronically. To understand if and how H. pylori could modulate T cell activation, in the present study we investigated in vitro the interaction between H. pylori and human T lymphocytes freshly isolated from peripheral blood of H. pylori-negative donors. A direct interaction of live, but not killed bacteria with purified CD3+ T lymphocytes was observed by microscopy and confirmed by flow cytometry. Live H. pylori activated CD3+ T lymphocytes and predominantly γδ+ T cells bearing the TCR chain Vδ2. Upon interaction with H. pylori, these cells up-regulated the activation molecule CD69 and produced cytokines (such as TNFα, IFNγ) and chemokines (such as MIP-1β, RANTES) in a non-antigen-specific manner. This activation required viable H. pylori and was not exhibited by other gram-negative bacteria. The cytotoxin-associated antigen-A (CagA), was at least partially responsible of this activation. Our results suggest that H. pylori can directly interact with T cells and modulate the response of γδ+ T cells, thereby favouring an inflammatory environment which can contribute to the chronic persistence of the bacteria and eventually to the gastric pathology.     

Azathioprine, a clastogen in human somatic cells? Analysis of chromosome damage and SCE in lymphocytes after exposure in vivo and in vitro

Chromosomal analyses in lymphocytes of 28 patients with multiple sclerosis were carried out before, during and after Azathioprine (Aza) therapy. Only a higher incidence of gaps was found in treated patients than in a group of healthy persons but not in comparison with untreated patients. Similarly, no significant clastogenic effect was observed in vitro after short-term and long-term treatment of unstimulated and stimulated lymphocytes with concentrations of 1--100 microgram Aza per ml. Treatment of cultures with 0.0001--4.0 microgram/ml did not yield increased SCE frequencies. The absence of any significant clastogenic effect of therapeutic doses of Aza on human somatic cells is deduced from an evaluation of previously published data and from the present results.     

Azathioprine,a clastogen in human somatic cells? Analysis of chromosome damage and SCE in lymphocytes after exposure in vivo and in vitro

Chromosomal analyses in lymphocytes of 28 patients with multiple sclerosis were carried out before, during and after Azathioprine (Aza) therapy. Only a higher incidence of gaps was found in treated patients than in a group of healthy persons but not in comparison with untreated patients. Similarly, no significant clastogenic effect was observed in vitro after short-term and long-term treatment of unstimulated and stimulated lymphocytes with concentrations of 1–100 μg Aza per ml. Treatment of cultures with 0.0001–4.0 μg/ml did not yield increased SCE frequencies. The absence of any significant clastogenic effect of therapeutic doses of Aza on human somatic cells is deduced from an evaluation of previously published data and from the present results.     

Sex-specific selection on the human X chromosome?

Genes involved in major biological functions, such as reproductive or cognitive functions, are choice targets for natural selection. However, the extent to which these genes are affected by selective pressures remains undefined. The apparent clustering of these genes on sex chromosomes makes this genomic region an attractive model system to study the effects of evolutionary forces. In the present study, we analysed the genetic diversity of a X-linked microsatellite in 1410 X-chromosomes from 10 different human populations. Allelic frequency distributions revealed an unexpected discrepancy between the sexes. By evaluating the different scenarios that could have led to this pattern, we show that sex-specific selection on the tightly linked VCX gene could be the most likely cause of such a distortion.     

Cytoprotective effects of 6′-O-galloylpaeoniflorin against ultraviolet B radiation-induced cell damage in human keratinocytes

The cytoprotective effects of 6′-O-galloylpaeoniflorin against injury and death of human HaCaT keratinocytes resulting from ultraviolet B radiation were investigated. 6′-O-galloylpaeoniflorin exhibited the capacity to scavenge intracellular reactive oxygen species (ROS) generated by ultraviolet B radiation. 6′-O-galloylpaeoniflorin also attenuated ultraviolet B-induced oxidative macromolecular damage to DNA, lipids, and proteins, decreasing the number of DNA strand breaks, the level of 8-isoprostane (a biomarker of lipid peroxidation), and the level of protein carbonylation. Moreover, 6′-O-galloylpaeoniflorin rescued HaCaT cells from ultraviolet induced cell death, by downregulating the mitochondrial apoptotic pathway. Taken together, these results indicate that 6′-O-galloylpaeoniflorin has the potential to be developed as a medical agent against ROS-mediated skin diseases.     

Do mycoplasmas cause human cancer?

A linkage between mycoplasmas and malignancy was mainly proposed in the 1960s when human-associated mycoplasmas were becoming of interest given the novel characterization of the human respiratory pathogen Mycoplasma pneumoniae. Associations with leukemia and other malignancies, however, were largely ascribed to tissue-culture contamination, which is now recognized as a significant potential problem in molecular biology circles. A few epidemiological studies, however, continue to raise concern over such a linkage. As well, in vitro data have demonstrated the potential for some mycoplasmas to induce karyotypic changes and malignant transformation during chronic tissue-culture infestation. As cellular and molecular mechanisms for such transformation become studied, a resurgence of interest in this area is inevitable. A role for mycoplasmas in malignancy of any sort is conjectural, but there remains a need to continue with focussed epidemiological and laboratory investigations.     

Do human eggs attract spermatozoa?

A key process in human fertilization is bringing the two gametes together, so that the complex molecular events involved in sperm and egg interaction can begin. Does nature allow fertilization to occur only as a consequence of a chance collision, or is there a precontact sperm-egg communication? This review summarizes the bioassays used in testing human spermatozoa for chemotaxis, emphasizing the necessity to distinguish between chemotaxis and other accumulation-causing processes, and the results obtained. It demonstrates that human sperm chemotaxis to a follicular factor(s) does occur, at least in vitro, and that only capacitated spermatozoa are chemotactically responsive. Substances that have been proposed as attractants for human spermatozoa are reassessed. The potential role of sperm chemotaxis in vivo is discussed. Faulty precontact sperm-egg communication may be one of the causes of male infertility, female infertility, or both. On the other hand, interfering with human sperm chemotaxis may represent an exciting new approach to contraception. BioEssays 21:203–210, 1999. © 1999 John Wiley & Sons, Inc.     

E rosette formation at 37°:A property of mitogen-stimulated human peripheral blood lymphocytes

Peripheral blood lymphocytes from healthy humans formed stable E rosettes with sheep erythrocytes (SRBC) at 37°C after culture with phytohemagglutinin or the divalent cation ionophore A23187. Cells manifesting this phenomenon exhibited “blast” morphology, appeared by 16 hr of culture, increased dramatically in percentage and absolute number by 62 hr, and persisted in large numbers for the duration of culture (182 hr). Unstimulated lymphocytes formed rosettes at 4°C but not at 37°C. Increased “stickiness” due to surface-bound lectin mitogen was not the cause of rosette formation at 37°C.Formation of E rosettes at 37°C has previously been considered a property of lymphocytes less differentiated than the circulating T cell (e.g., thymocytes, leukemic lymphoblasts). The present findings indicate that this property can be “reexpressed” during blastogenesis in culture.This observation also demonstrates technical problems associated with the use of SRBC to quantitate lymphocytes with complement receptors (B cells) by the EAC rosette assay in culture. False positives resulted from 37°C E rosette formation, but this was overcome by replacing the SRBC with guinea pig erythrocytes in the EAC assay.     

Do NK cells regulate human autoimmunity?

Natural killer cells have been shown to regulate autoimmune responses under experimental conditions in animals. However, a similar role for human NK cells has not been investigated, although NK cells constitute a significant fraction of the infiltrating cells in a range of autoimmune diseases. This review investigates the evidence, both theoretical and experimental, for the involvement of these cells in human immunopathology.     

Do fish lymphocytes secrete interferon-γ?

Macrophage activating factor (MAF)-containing supernatants, generated by mitogen (Con A/PMA) stimulation of rainbow trout leucocytes, were found to confer viral resistance (interferon, IFN, activity) on a rainbow trout epithelial cell line challenged with infectious pancreatic necrosis virus. Both the MAF and IFN activities co-eluted by HPLC size exclusion chromatography and showed similar sensitivities to acid (pH 2), temperature (60° C) and trypsin. The mode of induction of this IFN, its acid and temperature sensitivity and its possible MAF activity suggest that fish leucocytes can secrete an IFN-γ like molecule.     

The dose response of chromosome aberrations in human lymphocytes induced in vitro by very low-dose γ rays

This paper considers the dose-effect relationship for unstable chromosome aberration yields in human lymphocytes in very low-dose range. Data are presented for (60)Co γ-ray doses of 0, 10, 20, 40 and 1000 mGy. More than 5,000 metaphases were scored for each data point at the very low doses, and each cell was double-checked using a semi-automated metaphase finding/relocation system. Aberration yields of dicentrics plus centric rings followed an excellent linear dose response down to zero dose; the yields were significantly above the control frequency from 20 mGy.     

Effects of 5-hydroxymethyl-2′-deoxyuridine and 3-aminobenzamide on chromosome aberrations in cultured human lymphocytes

Effects of 5-hydroxymethyl-2′-deoxyuridine (HmdUrd, a thymidine analog) and 3-aminobenzamide (3AB) on chromosome aberrations in cultured human lymphocytes were studied. The results show that HmdUrd is an effective clastogen in human peripheral lymphocytes. When cells were treated with HmdUrd and 3AB, a synergistic effect on chromatid gaps, breaks and exchanges was found. These findings support the hypotheses that 5-hydroxymethyluracil (HmuRa) residues in DNA are formed and then removed by the action of 5-HmUra-DNA glycosylase (Teeber et al., 1984) and that 3AB interferes with the completion of DNA repair following HmUra excision.     

Binding of stimulated human peripheral blood lymphocytes to Sepharose-concanavalin A is not reversed by methyl-α-mannoside

Concanavalin A (con A) bound to Sepharose beads stimulates human peripheral blood lymphocytes and as with soluble con A, DNA synthesis can be prevented by the addition of methyl-α-mannoside (MAM) 1 hr after exposure to mitogen. In contrast to the response of cells stimulated with soluble con A, addition of MAM 24 hr after the start of incubation with Sepharose bound mitogen does not prevent a second round of DNA replication as determined in bromodeoxyuridine density transfer experiments indicating that MAM does not dissociate the Sepharose-con A responding cell complex. We infer that within 24 hr, lymphocytes develop associations with con A-Sepharose beads at non MAM dissociable sites.     

Enhancement of the antibody-dependent cellular cytotoxicity of human peripheral blood lymphocytes with interleukin-2 and interferon α

Antibody-dependent cellular cytotoxicity (ADCC) is regarded as an important mechanism by which monoclonal antibodies (mAb) can exert an antitumour effect in vivo. It may be possible, therefore, to enhance the therapeutic efficacy of mAb by cytokines that are able to enhance the ADCC of human CD3^–, CD56⁺, CD16⁺ natural killer (NK) cells. We investigated in vitro the effects of recombinant interferon (rIFN) and recombinant interleukin 2 (rIL-2), alone or in combination, on the ADCC of human peripheral blood NK cells. Both cytokines enhanced the ADCC of the human effector cells. rIFN induced a maximally increased ADCC after an exposure of human effector cells to 20 IU/ml for 15–30 min, while rIL-2 induced optimal ADCC after incubation of the cells for 2 days in 20–50 U/ml. We now show that activation of the NK cells with a combination of rIL-2and rIFN induced significantly higher levels of ADCC than either cytokine alone. The highest ADCC was induced if the cells were first exposed to rIL-2 before rIFN was added to the culture. Culture of NK cells in medium or rIL-2 decreased the expression of FcRIII (CD16), indicating that intensity of CD16 expression and level of ADCC are not directly correlated, although blocking experiments with a mAb directed against CD16 showed that this FcR was essential for ADCC of the human effector cells.Supported by a grant from the Dutch Cancer Society (grant NKI-84-14)