Kerstin Lindahl-Kiessling

Separation of various blood cells in colloidal silica-polyvinylpyrrolidone gradients.

Abstract New methods are described for the separation of the cellular components of human blood. They can be separated into erythrocytes, polymorpho-nuclear leucocytes, mononuclear leucocytes and platelets by centrifugation in density gradients of colloidal silica and polyvinylpyrrolidone. The procedures are simple and the yields and resolution achieved higher than in methods currently available. The fractionations are made in media of physiological pH and osmolality. The cells are intact after the fractionation as measured by the fluorescein diacetate accumulation test, the trypan blue exclusion test and thymidine incorporation in the lymphocyte fraction after phyto-hemagglutinin stimulation. In the techniques described, between 1 and 200 ml of blood can be fractionated at a time. Alternatively, the different cell populations may be banded at the interface between silica solutions of different densities (on density cushions). The latter technique is especially suitable for working up larger quantities of blood.

Inability of UV-Irradiated Lymphocytes to Stimulate Allogeneic Cells in Mixed Lymphocyte Culture

Cells exposed to UV light are not able to induce DNA synthesis in allogeneic cells. HL-A typing before and after irradiation gives identical results. It is suggested that active cell metabolism and no

Mechanism of phytohemagglutinin (PHA) action: V. PHA compared with concanavalin A (Con A)

Abstract The stimulatory properties of PHA and Con A in short-term human lymphocyte cultures have been compared. PHA binds more strongly to the cells than does Con A. Much smaller quantities of PHA than of Con A suffice for maximal stimulation. The peak thymidine incorporation generally occurs 1 day earlier in PHA cultures. Removal of Con A with α-methylmannoside after up to 19 h in culture reduces the mitotic activity. Lymphocytes exposed to Con A for 8 h attain maximal thymidine incorporation if restimulated later on with PHA or ALG although neither stimulus equally timed is sufficient to cause DNA synthesis on its own. It has not been possible to demonstrate any definite effect of EDTA on the mitogenic properties of Con A although the binding to lymphocytes seems to be impaired. However, PHA cannot bind at all to lymphocytes in the presence of EDTA. Acidified and dialysed PHA in saline cannot induce thymidine incorporation in lymphocytes although it may be bound to the cells. It is suggested that multiple binding sites are required on the inducer molecule to achieve mitotic induction.

Increased frequency of acetaldehyde-induced sister-chromatic exchanges in human lymphocytes treated with an aldehyde dehydrogenase inhibitor

Acetaldehyde, the first metabolite of ethanol oxidation, in concentrations ranging from 100 microM to 400 microM caused a dose-dependent linear increase in the frequency of sister-chromatid exchanges (SCE) in cultured human peripheral lymphocytes. The SCE frequency was on an average 2-fold higher when the cells were exposed to the acetaldehyde after 24 h incubation instead of at the time of mitogen stimulation (0 h). When acetaldehyde was added together with the potent aldehyde dehydrogenase inhibitor 1-aminocyclopropanol (0.1 mM), the SCE response was significantly (p less than 0.05) increased. The present results indicate that acetaldehyde is metabolized within human lymphocytes, and, moreover, that alcohol consumption during treatment with drugs that inactivate aldehyde dehydrogenase may cause a further increased incidence of acetaldehyde-induced SCE and concomitant lesions.

SCE in B and T lymphocytes. Possible implications for Bloom's syndrome.

Conventional cultures of PHA-stimulated lymphocytes from human peripheral blood used in cytological studies contain B cells as well as T ce1ls.A~ both types are known to respond to the mitogen in heterogeneous cell cultures, B and T cells will be harvested mixed together. Consequently, their mitoses will be randomly selected for analysis. The proportions of the two subsets are bound to vary under different culture conditions. According to different authors, considerable variations in frequencies of sister chromatid exchanges (SCE) seem to exist not only between cells, but also between individuals, and even between repeated cultures a t similar BrdU concentrations. The aim of this study was to elucidate whether these variations could be due to differences between B and T cells.

Genotoxic effects of ethylene oxide and propylene oxide: a comparative study

The two alkylating agents ethylene oxide (EO) and propylene oxide (PO) were compared for genotoxic effectiveness in various test systems. The study was undertaken partly to shed light on the difference between the compounds found after chronic exposure of monkeys (Lynch et al., 1984) where EO but not PO was able to induce SCE and chromosomal aberrations. In the present study EO was found to be 5-10 times more effective than PO with respect to gene conversion and reverse mutation in Saccharomyces cerevisiae D7 and sister-chromatid conversion in S. cerevisiae RS112. In contrast, the abilities of the two compounds to induce point mutation in S. typhimurium strains and SCE in human lymphocytes were approximately equal. One possible cause of EO being more effective than PO in certain respects, discussed on the basis of inference from earlier studies, is an expected difference in ability to cause strand breaks via alkylation of DNA-phosphate groups.

On the purification of phytohemagglutinins from Phaseolus vulgaris seeds

Abstract Two phytohemagglutinins from Phaseolus vulgaris seed have been isolated. One of them, having a molecular weight considerably lower than that previously reported, has been more extensively characterized. Some studies of the affinity toward carbohydrates as well as of the tendency to aggregate are described. No dissociation of agglutinating and mitogenic parts of the molecule was obtained.

Polyploidy and Endoreduplication in Human Leukocyte Cultures Treated with β-Mercaptopyruvate

Treatment of cultured human peripheral blood leukocytes with β-mercaptopyruvate resulted in marked increases in polyploidy and endoreduplication in squash preparations of mitoses in the metaphase stage. Since β-mercaptopyruvate occurs in man as an intermediary metabolite in cysteine degradation, it might contribute to the development of polyploidy in vivo, especially in tumors lacking desulfurase enzymes.

Kinetics of lymphocyte stimulation in vitro by non-specific mitogens.

Abstract The role of mitogens during lymphocyte activation was studied with kidney bean leucoagglutinin, concanavalin A and kidney bean phytohemagglutinin. The mitogens were removed by treatment with appropriate antisera, which was demonstrated to remove also mitogens already attached to the cells. The process of lymphocyte activation in vitro can be divided into four distinct steps, three of which are mitogen-dependent and the fourth is mitogen-independent. The first step consists of attachment of the stimulatory molecules to the cell membrane. The second step consists of reaction between mitogen and an activating system. During these two phases the cells become preactivated. The establishment of a preactivated state involves at least some synthesis of cytoplasmic RNA. The preactivated state is reversible and during the third step of lymphocyte activation the final result of preactivation is determined. Depending on the presence or absence of mitogen the cells may remain preactivated for over 60 h, they may return to the resting state or they may proceed through the final stages of the proliferation cycle and eventually divide. This fourth step is independent of the presence or absence of mitogen. A prolonged contact between cells and mitogen is required during the mitogen-dependent steps. The process of lymphocyte activation by mitogen is thus continuously being regulated by the stimulatory molecules on the lymphocyte membrane, which may be of considerable significance also for in vivo immunologicai reactions at the cellular level.

The mechanism of phytohemagglutinin (PHA) action: II. The effect of certain enzymes and sugars☆

Abstract In an attempt to elucidate the nature of the binding of PHA to lymphocytes, these cells were exposed to different enzymes before cultivation with PHA. The degree of stimulation was measured as 14C-thymidine incorporation into the acid insoluble fraction. It appears that certain enzymes, known to act on cell surfaces, suppress the PHA response while others leave it unchanged or even enhance the PHA effect. Trypsin and neuraminidase belong to the former group. Several hours after enzyme digestion lymphocytes are refractory to PHA treatment but gradually they recover their potential, implying that a regeneration of the surface has to occur before the PHA response can be initiated. Common monosaccharides had little or no effect on the PHA stimulation.