Immunoregulation by breast milk cells.

Although B cells capable of synthesizing IgG and IgM have been identified in human milk, only IgA synthesis is measured in vitro. These data suggest that milk lymphocyte differentiation is a regulated process and that there may be a specific milk cell factor capable of stimulating differentiation of IgA-bearing B cells. To investigate this possibility lymphocyte/ macrophages from early (≤5 days) and late (≥8 days) milk were incubated and subsequently small aliquots of their cell-free culture media were added to peripheral blood lymphocyte cultures. The release of IgA, IgG, and IgM by the blood lymphocytes in culture was quantitated using double-antibody (Ab) competitive radioimmunoassays. The cell-free media from early (colostral) milk cell cultures significantly stimulated (P < 0.0001) IgA synthesis and had no effect on the production of IgG or IgM. There was no effect on immunoglobulin production when the milk cell supernate came from cells isolated from more mature milk. Therefore, it is postulated (i) that a soluble mediator(s) of immunologic regulation is released by human milk cells, (ii) that this factor(s) at least in part, explains the peculiar immunologic behavior of human milk cells in vitro, (iii) that this factor(s) is released in greater amounts by colostral cells than by cells in mature milk, and (iv) that human colostrum may play a role in affecting active local immunity in the gastrointestinal tract of the recipient newborn.     

Optical guiding by photoreceptor cells.

The potential for any morphological part of a photoreceptor cell to function as an optical waveguide is determined 1) by the dimensionless parameter V as shown by Snyder (IEEE Trans., Microwave Theory Tech. 17: 1133, 1969) and by Snyder, Pask and Mitchell (J. Opt. Soc. Am. 63: 59, 1973), and 2) by the nature of the optical excitation for that part of the cell. The effects of these two factors are considered for the myoids and outer segments of vertebrate photoreceptor cells and for the crystalline tracts and rhabdomers of invertebrate photoreceptor cells. Examples of both guiding and nonguiding myoids, outer segments, rhabdomers and tracts are cited and some consequences for vision are analyzed.     

Immobilization of microbial cells by adsorption.

Immobilized cells cover a wide area of applications and are essential components of many biotechnological processes. In general it can be distinguished between two immobilization methods: (1) entrapment into polymers and (2) natural adsorption onto porous and inert support materials. The immobilization by adsorption is discussed by the following criteria: biomass loading, strength of adhesion, enzymatic stability/specific activity of the biocatalyst, effectivity/reaction engineering and operational stability.     

Uptake of dolichol by Vero cells.

Characterization and kinetics of dolichol uptake by a Vero cell line are reported. Vero cells incorporate dolichol in a time- and dose-dependent manner. Optimal uptake is found at 37 degrees C and at a pH of 7.4. In contrast to cholesterol, an inhibitory effect on the dolichol incorporation is found for farnesol, geraniol, and retinol. Long chain polyprenols were slightly stimulatory. The translocation seems not to be highly energy dependent. The lack of substantial inhibition by chloroquine does not plead for a receptor-mediated endocytosis. Incorporated dolichol was distributed over both membranes and supernatant fractions, paralleling the distribution of the lysosomal marker beta-N-acetylhexosaminidase. The incorporated dolichol is subject to a fast efflux process, which is potentiated by the presence of lipid acceptors in the extracellular medium.     

Lymphocyte transformation induced by autologous cells.

Human peripheral blood T lymphocytes are stimulated to proliferate when cultured with autologous B-lymphoblastoid cell lines, autologous mitogen-induced lymphoblasts, or autologous non-T blood lymphocytes. This reaction, the autologous mixed lymphocyte reaction, has attributes of an immune response possessing both memory and specificity. The capacity to stimulate autologous T lymphocyte proliferation depends on the lineage of the lymphoid cell and not on its establishment in continuous culture or carriage of the EB viral genome. The determinant on non-T lymphocytes which stimulates the autologous mixed lymphocyte reaction appears to be an Ia determinant. Thus, allogeneic graft rejection and the allogenic mixed lymphocyte reaction are very likely extensions of an immune response expressed within the host.     

Carrot cells detoxify N-phosphonoacetyl-L-aspartate by esterification.

Unlike bacterial and mammalian cells, carrot cells are able to tolerate N-phosphonoacetyl-L-aspartate (PALA), a potential inhibitor of pyrimidine biosynthesis, by detoxifying the compound. Anion-exchange chromatography showed that detoxified PALA was less negatively charged than PALA, and allowed detoxified PALA to be isolated. Incubation of detoxified PALA with a low-specificity carboxylic-ester hydrolase fully restored the ability to inhibit aspartate transcarbamoylase, the target enzyme, indicating that the detoxification involves the formation of carboxylic ester. G.1.c. analysis of the alcohol products of enzymic hydrolysis, and of their ratio to PALA, showed that the detoxification produced a mixture of mono- and di-carboxylic esters and of methyl and ethyl esters. The detoxification mechanism showed considerable specificity towards PALA, since the analogous carboxy groups of succinate were not modified in the same way. Succinate was depleted much more slowly, no succinate esters could be detected, and the presence of a 10-fold excess of succinate did not inhibit the esterification rate of PALA. The possible significance of these results is discussed.     

Liposome-mediated DNA uptake by sperm cells.

To investigate the potential use of sperm cells as vectors to transfer exogenous DNA via the fertilization of oocytes into the germ line of mice, we have used liposomes to transfect DNA into the sperm head. Although the DNA transfer into sperm mediated by liposomes was very efficient and no obvious reduction in the fertilization frequency of oocytes could be detected, we were unable to generate transgenic mice by this method.     

Macrophage-like suppressor cells in rats. I. Inhibition of natural macrophage-like suppressor cells by red blood cells

When trinitrobenzenesulfonic acid (TNBS), the reactive form of trinitrophenyl (TNP) hapten, is injected into a mouse, a brief intrinsic B-cell tolerance to TNP has been shown to result. Yet antigen-binding cells (ABC) with receptors for TNP persist in the TNBS-treated animal.After treatment with Pronase under conditions preserving cell recovery and viability, 80–90% of TNP-ABC failed to bind antigen. After 2 hr in vitro, Pronase-treated 4-day immune TNP-ABC displayed significant recovery of antigen binding, whereas nonimmune TNP-ABC performed the same feat by 18 hr. However, TNP-ABC tested 2 to 11 days after TNBS failed to replace digested receptors by 18 hr in vitro. Thirty days after TNBS, they had recovered this ability. This defective receptor replacement by TNP-ABC was not reversed by colchicine, and was not shared by the sheep-erythrocyte ABC of the same animals, which replaced receptors normally. When challenged with antigen (TNP-sheep erythrocytes) simultaneously with TNBS, recovery by 2 hr was evident on Day 11. When challenged with antigen 4 days after TNBS, receptor regeneration had returned to normal by the next day, and partial recovery of the anti-TNP plaque-forming cell response was evident 4 days later.Thus, the inability to replace receptors and immune unresponsiveness coincides in time, so that a causal relationship between these two defects may be hypothesized. This result contrasts with the membrane locking defect, previously described in the TNP-ABC of TNBS-treated animals, which far outlasted the unresponsive state.     

Catecholamine secretion by isolated adrenal cells.

Isolated adrenal cells were prepared by collagenase digestion of guinea pig adrenal glands. Acetylcholine stimulates the secretion of catecholamines by these isolated adrenal cells. Acetylcholine-stimulated catecholamine secretion is inhibited by cholinergic blocking agents (atropine and hexamethonium) and by local anaesthetics (tetracaine), and is dependent upon the concentration of Ca2+ in the incubation medium. In the presence of Ca2+, catecholamine secretion is also stimulated by two divalent cation ionophores, A23187 and X-537A. Cyclic nucleotides and 5'-nucleotides cause a small, non-specific stimulation of catecholamine secretion. These results indicate that isolated adrenal cells are a useful system in which to study catecholamine secretion, and support the hypothesis that increased Ca2+ entry into chromaffin cells is a sufficient stimulus for catecholamine secretion.     

Granulocyte differentiation by Friend leukemia cells.

Friend leukemia cells growing in suspension culture are thought to represent a population of primitive erythroid cells which have undergone malignant transformation. We have found that when growing in vivo or in plasma clots in vitro, these suspension culture cells can exhibit morphologic and enzymatic properties which are characteristic of primitive granulocytic cells. The microenvironment in which the tumor cells grow plays a major role in determining the direction of differentiation of these leukemia cells. Hence it appears likely that the Friend cell is in fact a neoplastic pluripotent hematopoietic stem cell.     

Radiosensitization of mammalian cells by diamide.

The effect of diamide on the radiosensitivity of T-cells was investigated under oxic and anoxic conditions. The compound was found to sensitize the cells under both conditions. Under oxic conditions, exposure for 10 min before and during irradiation to 0.1, 0.5 and 1.0 mM diamide produced dose-modifying factors of 0.81, 0.60 and 0.55, respectively. Under anoxic conditions, exposure for 10 min before and during irradiation to 0.5 mM produced a dose-modifying factor of 0.34. When the cells in oxic conditions were exposed for just 20 min before irradiation, the sensitizing effect was smaller, but some sensitization effect was still apparent after a 120 min interval between diamide treatment and irradiation. Diamide also sensitized the cells after irradiation, but this effect was less than when it was present during irradiation. The presence of whole rat-blood in the incubation medium prevented sensitization. No sensitization could be detected in the whole animal. It is proposed that sensitization is due to lack of capacity for repair of radicals by hydrogen transfer and biochemical repair processes.     

Catecholamine secretion by hamster adrenal cells.

Abstract— Suspensions of isolated adrenal cells were prepared by digesting hamster adrenal glands with collagenase, and the secretion of catecholamine from these cells was studied. Acetylcholine (ACh) produces a dose-dependent increase in catecholamine secretion; half-maximal secretion is produced by 3 μm -ACh, and maximal secretion by 100 μm -ACh. The cholinergic receptor in these cells appears to be nicotinic, since catecholamine secretion is stimulated by the nicotinic agonists nicotine and dimeth-ylphenylpiperaziniurn, but not by the muscarinic agonists pilocarpine or oxotremorine. ACh-induced catecholamine secretion is inhibited by hexamethonium, tubocurarine, and atropine, but is not inhibited by α-bungarotoxin. ACh-induced catecholamine secretion is dependent upon the presence of extracellular Ca²⁺, and appears to occur by exocytosis, since the release of catecholamine is accompanied by the release of dopamine β-monooxygenase, but not of lactate dehydrogenase. These biochemical studies complement the morphological evidence for exocytosis in hamster adrenal glands, and indicate that catecholamine secretion from hamster chromaffin cells is similar to that from chromaffin cells of other species.     

Enucleation of mammalian cells by cytochalasin B. II. Formation of hybrid cells and heterokaryons by fusion of anucleate and nucleated cells

The development of methods for the formation of hybrid cells and heterokaryons by virus-induced fusion of chemically-enucleated cells and nucleated cells has been described. Heterokaryons and hybrid cells formed by fusion of anucleate mouse peritoneal macrophages (MPM) and nucleated mouse L and human HEp-2 cells were identified by mixed haemadsorption, by their sensitivity to trypsin and by their capacity to ingest antibody-coated sheep red blood cells. The expression of macrophage markers in these cells declined rapidly after fusion. Hybrid cell and heterokaryon formation was identified in mixed cultures of anucleate L cells and nucleated MPM, and was accompanied by the reactivation of DNA synthesis in the macrophage nuclei. Other hybrids and heterokaryons were formed by virus-induced fusion of anucleate MPM and nucleated chick embryo erythrocytes and anucleate L cells and nucleated HEp-2 cells. The value of anucleate-nucleate cell hybrids in the study of metabolic and genetic regulation in mammalian cells is discussed.     

Primary anti-trinitrophenyl memory cells investigated by plaque-forming cells and ELISA.

Primary anti-trinitrophenyl antibody production was investigated from spleen cells of mice immunized with trinitrophenylated-keyhole limpet hemocyanin, using the plaque-forming cell method and ELISA. Cells taken 5 days after antigen injection do not produce IgE, but do produce IgM and IgG1 anti-trinitrophenyl antibodies as demonstrated by plaque-forming cells. Substantial increase of IgM, IgG1, and IgE antibody production was seen from cells taken 7 days after immunization, followed by a rapid decline. By ELISA it was seen that cells taken 3 days after immunization already produce small amounts of anti-trinitrophenyl antibodies. Presence of antigen from the start of the cultures did not increase antibody production from cells taken 3 days after immunization, but potentiated antibody secretions from cells taken 5 days or later after immunization. This potentiation was interpreted as recruitment of antibody-forming cells from early memory B cells. The presence of IL-4 from the start of the cultures had no appreciable effect. Cell sorting with specific antibody-coated magnetic beads showed that plaque-forming cells from nonsorted cells, membrane IgE+ or membrane IgE- cells secreted similar amounts of anti-trinitrophenyl IgG1 and IgE antibodies. No difference in anti-trinitrophenyl IgM, IgG1, or IgE production was found in controls; cells sorted negatively or positively for CD23. The data show that memory B cells can be demonstrated already on Day 5 after immunization, and their antigen-induced antibody secretion is IL-4 dependent.     

Regulation of antibody secretion by hybridoma cells. I. Suppression of antibody secretion by coculture of hybridoma cells with idiotype-induced suppressor cells

In this study, we have demonstrated that antibody secretion by hybridoma cell lines can be down-regulated by idiotype-specific immune spleen cells or by nylon wool nonadherent spleen cells. This suppression of antibody secretion can be abolished by treating the idiotype-specific immune spleen cells with anti-Thy 1.2 plus complement. The hybridoma we used for most of our experiments secretes IgM specific for the cross-reacting haptens 2,4,6-trinitrophenyl (TNP) and 2,4-dinitrophenyl (DNP). Suppression was achieved by direct coculture of hybridoma cells with immune cells from animals which were injected with affinity-purified hybridoma antibody-coupled syngeneic spleen cells. The suppressed and control cultures contained similar numbers of viable hybridoma cells, suggesting that a simple cytotoxic effect is not responsible. Idiotype specificity was established in experiments showing that two idiotype immune animals immunized with antibody from two different IgM anti-TNP hybridomas could suppress the hybridoma to which they were immunized but could not affect the other hybridoma. Immune spleen cells required 3-4 days of coculture with hybridoma cells before maximum suppression was achieved. The kinetics of the response suggest that the final effector suppressor cell is generated during the coculture period and that a second signal, perhaps a product of the hybridoma cells, may be required.