Interleukin 2 up-regulates its own production

It has been previously reported that a combination pair of anti-CD2 monoclonal antibodies (mAb) T11(2)+T11(3) induces a strong proliferation of T cells, which does not require the involvement of accessory cells and exogenous interleukin 2 (IL-2). More recently, we have shown that the requirement for optimal T cell proliferation depends on the combination pairs of anti-CD2 mAb used. Among them, anti-GT2+T11(1) mAb do not allow optimal proliferation of TA4 helper cloned T cells due, at least in part, to a low level of IL-2 production. This observation offered us the opportunity to study the effect of IL-2 on its own production. We show here that stimulation of cloned TA4 cells with anti-GT2+T11(1) mAb induces only a marginal level of IL-2 production. By contrast, significantly higher levels of IL-2 activity are detected in the culture supernatant of TA4 cells preincubated with recombinant IL-2 (rIL-2) before stimulation with anti-GT2+T11(1) mAb. This effect is dose-dependent over a wide range (5 to 50 IU/ml) of rIL-2 concentrations added during preincubation time. In addition, it is not due to carryover of rIL-2 bound during the preincubation time, or to lesser IL-2 consumption by these cells, or to increasing numbers of IL-2-producing cells induced by exogenous IL-2. Moreover, the observation was confirmed with IL-2 mRNA. Although neither rIL-2 nor anti-GT2+T11(1) mAb alone could induce a significant production of IL-2, rIL-2 appears to up-regulate its own production when the TA4 cells are activated by the anti-CD2 mAb-mediated second signal.     

Formation of lymphocyte colonies under serum-free culture conditions in normal individuals and patients with chronic lymphocytic leukemia

This paper describes a culture system which supports the formation of B cell and some T cell colonies under serum-free conditions in peripheral blood samples of normal individuals and patients with chronic lymphocytic leukemia (CLL) of B cell type. In this system, serum is replaced by bovine serum albumin, transferrin, cholesterol, insulin and catalase or horseradish peroxidase. In addition, it is necessary to add staphylococcus protein A, mitomycin-treated T cells as feeders and phytohemagglutinin leukocyte-conditioned medium as a source of growth factors. The plating efficiency is greatly enhanced when normal cells are incubated with galactose oxidase prior to plating and when CLL cells are exposed sequentially to neuraminidase and galactose oxidase.     

Analysis of low concentrations of 5-bromo-2-deoxyuridine on sister chromatid exchanges in human lymphocytes

To determine a concentration of 5-bromo-2-deoxyuridine (BrdU) sufficient for sister chromatid differentiation (SCD), and yet having a minimal effect on the number of sister chromatid exchanges (SCEs), we assessed the effect produced on the number of SCEs by low concentrations (1, 3, and 10 micrograms/mL) of BrdU. SCD was not obtained in 19% of the 31 subjects with 1 microgram/mL of BrdU, while the differentiation was adequate for all samples treated with 3 and 10 micrograms/mL. We statistically analysed the effects of these three different doses and found no significant difference in the number of SCEs obtained with the doses of 1 and 3 micrograms/mL, but a significant difference was observed between these two concentrations and 10 micrograms/mL. We therefore suggest that the dose of 3 micrograms/mL, while sufficient to produce reliable differential staining, still permits an adequate evaluation of the base line of SCEs and appears to enhance the sensitivity of the test to evaluate between-individual variations. Our experiments also underline that SCE counts should include the centromere exchanges.     

Estrogen influences dolichyl phosphate distribution among glycolipid pools in mouse uteri

The steroid hormone 17 beta-estradiol dramatically induces uterine N-linked glycoprotein assembly [Dutt, A., Tang, J.-P., Welply, J. K., & Carson, D. D. (1986) Endocrinology (Baltimore) 118, 661-673]. To determine the role that dolichyl phosphate availability plays in this induction, we studied the effects of estrogen priming on the content of dolichyl phosphate and the distribution of dolichyl phosphate among various glycolipids in uteri. Dolichol-linked saccharides were metabolically labeled to equilibrium with either [3H]glucosamine or [3H]mannose and extracted from primary explants of uterine tissue. The amount of dolichol-linked saccharide was calculated from the specific radioactivity determined for the corresponding sugar nucleotides extracted from the tissues. The major dolichol-linked saccharides identified were mannosylphosphoryldolichol (MPD), oligosaccharylpyrophosphoryldolichol (OSL), and N,N'-diacetylchitobiosylpyrophosphoryldolichol (CBL). Estrogen increased the levels of MPD and OSL 4-fold; however, CBL levels did not change. After 3 days of treatment, the levels of these glycolipids were very similar to those in uteri from pregnant mice. Remarkably, MPD constituted 90-95% of dolichol-linked saccharides detected under all conditions. The tissue contents of total dolichyl phosphate and alkali-labile dolichyl phosphate, presumably MPD, were estimated by liquid chromatography. The levels of alkali-labile dolichyl phosphate determined in this way were in good agreement with the values estimated for MPD by metabolic labeling; moreover, alkali-labile dolichyl phosphate constituted 50-98% of the total dolichyl phosphate pool. The variations in MPD content depended upon the steroid hormone influence, most notably that of estrogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selection of microcarrier diameter for the cultivation of mammalian cells on microcarriers

The kinetics of mammalian cell growth in a microcarrier culture are affected by the distribution of cells on microcarriers. It has been shown previously that a critical cell number per microcarrier is required for the growth of FS-4 cells on microcarriers. It is advantageous to alter the cell distribution on microcarriers to allow for a larger fraction of microcarriers to acquire enough cells to initiate normal growth. This can be achieved by selecting the diameter of the microcarriers employed. It has also been shown previously that the critical cell number could be reduced by choosing a better culture medium to support low density growth. However, even if all cells inoculated into a culture are capable of growing to confluence, it is still necessary to select the microcarrier diameter ration ally to improve the growth kinetics. The method of selecting the microcarrier diameter is discussed. By employing a improved medium as well as using microcarriers of selected diameter, the multiplication ratio was in creased to 15- to 16-fold for FS-4 cells, as opposed to 3- to 4-fold typically obtained in a batch culture.     

Attachment and growth of mammalian cells on microcarriers with different ion exchange capacities

In the design of microcarriers for animal cell growth, the exchange capacity has been considered a critical factor. However, charge densities of microcarriers under culture conditions are not the same as the exchange capacities. Furthermore, the charge density requirement for optimum attachment is not necessarily the same as that required for optimum growth. We demonstrate that charge is not the sole factor affecting the attachment and growth of animal cells on microcarriers. We also show that supplemental serum in the growth medium has a negative effect on cell attachment to microcarriers.     

Expression of human lactotransferrin receptors in phytohemagglutinin-stimulated human peripheral blood lymphocytes. Isolation of the receptors by antiligand-affinity chromatography

In the resting rate, the human peripheral blood lymphocytes did not show detectable surface and intracellular receptors for human lactotransferrin. However, both types of lactotransferrin receptors were expressed during stimulation of lymphocytes with phytohemagglutinin. The appearance of receptors was time-dependent and the number of receptors reached a plateau after at least two days of mitogen stimulation. These results suggest that the presence of surface receptors on mitogen-stimulated lymphocytes is not consecutive to a modification of subcellular distribution but to an induction of biosynthesis of the receptors. As measured by incorporation of [3H]thymidine into DNA, addition of human lactotransferrin in a serum-free medium increased the proliferative activity of phytohemagglutinin-stimulated lymphocytes. Optimal enhancement of [3H]thymidine incorporation was obtained by adding 30% iron-saturated lactotransferrin at a concentration of 0.17 microM. Therefore, the role of lactotransferrin in the response of lymphocytes to mitogen stimulation appears to be similar to that previously described for serotransferrin. The lactotransferrin receptor was visualized using 125I-labeled lactotransferrin on nitrocellulose paper after electroblotting of the Triton X-100 extract of the phytohemagglutinin-stimulated lymphocytes as two protein bands of 100 and 110 kDa molecular mass. Purification of the lactotransferrin receptor from the Triton-X-100-soluble extract of stimulated lymphocytes was performed by antiligand-affinity chromatography. The binding of lactotransferrin to the purified receptors was reversible and dependent on concentration and pH.