Preferential cytotoxic effect of Newcastle disease virus on lymphoma cells.

Susceptibility of lymphoma cells (Daudi, HD-Mar) to Newcastle disease virus toxicity was found to be higher than that of lymphoblastoid cells (Milstein) and of resting peripheral blood lymphocyte (PBL). Phytohemagglutinin- and/or pokeweed-mitogen-activated PBL however, exhibited, elevated sensitivity, similar to that of lymphoma cells. The level of cytotoxicity was monitored by cell viability, inhibition of DNA synthesis and release of 51Cr. When Daudi cells were mixed with PBL they were significantly more sensitive to the killing effect of the virus (70% mortality compared to 30% 30 h after infection, P < 0.05). The degree of sensitivity to viral cytotoxicity was unrelated to the efficacy of adsorption, which was similar for all cell lines as shown by immunofluorescent staining and flow cytometry. Also an influenza strain A/PR/8/34 (H1N1) adsorbed but did not affect the viability of any of the cells tested. Our results demonstrate that New-castle disease virus caused preferential damage to lymphoma cells as compared to non-cancerous normal cells.

Adherence of Candida albicans to epithelial cells: studies using fluorescently labelled yeasts and flow cytometry.

Candida albicans adherence to epithelial cells is the first step in the infectious process, but in spite of its importance, current methods for the quantitative measurement of adherence of C. albicans to epithelial cells in vitro have some serious limitations. They are based on filtration assays and either microscopic or radiometric analysis. The adherence reaction is usually carried out with a large excess of yeasts (100-fold) over epithelial cells in order to perform the microscopic analysis, which is slow, subjective and limited to 100-200 cells and thus lacks statistical power. The radiometric analysis fails to measure individual cells. A method for measuring yeast adherence that overcomes these problems has been developed. It is based on labelling the yeasts with the fluorogenic marker 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF) prior to the adherence reaction, and analysing 10(4) epithelial cells by flow cytometry, while nonbound yeasts are excluded by gating. Two subpopulations of buccal epithelial cells (BECs) which differ in their mean fluorescence intensities per cell (MFIs) were observed: one with MFI which did not exceed nonspecific fluorescence, and the other with MFI as high or higher than the MFI of labelled yeasts. The two subpopulations represent yeast-free and yeast-binding epithelial cells, respectively, and the MFI increment of the BECs is a quantitative measure of the extent of yeast adherence. Control experiments confirming previously described basic features of adherence, such as enhanced adherence at increasing yeast excess, diminished adherence of trypsin-treated or heat-inactivated yeasts, and the differential adherence of various Candida species, supported the validity of the assay. The possibility of studying adherence reliably at low yeast:epithelial cell ratios, which better mimic adhesion as it occurs in vivo, is an important advantage of the assay. New findings, using this method, included the observation that exfoliated BECs from diabetic patients exhibited the same capacity for C. albicans adherence as cells from healthy controls, and that epithelial cells from early human ontogenic stages had a significantly lower adherence level than those from later stages.

The interaction between cytotrophoblasts and their derived tumor cells.

Previous experiments demonstrated that human cytotrophoblasts and cells of the choriocarcinoma cell line JAr interact in vitro. As a result of this interaction there is an increased synthesis of CG and hPL, probably as a result of the increased CG and hPL synthesis by the cytotrophoblasts. In the present investigation we studied this interaction in greater detail and found that both cytotrophoblasts and JAr cells undergo changes in their biological properties as a result of this interaction. JAr cells and cytotrophoblasts cocultured for 72 hr were fractionated according to their size by centrifugal elutriation. The number of cells in the fraction which contain the largest cells was very significantly increased as a result of the coculture. This increase was due to an increase in the number of cells of both cell types. This fraction was the most active one in the synthesis of CG and hPL. The synthesis of DNA by the JAr nuclei in this fraction of the cocultured cells was almost completely inhibited but in the parallel fraction of the JAr cells cultivated alone the level of DNA synthesis was equal to that of all other JAr cell fractions. Heterokaryons are formed in the coculture. In these heterokaryons a factor which inhibits DNA synthesis in the cytotrophoblasts may inhibit DNA synthesis in JAr nuclei and at least be partly responsible for the inhibition of DNA synthesis observed.

Thymocyte maturation following interaction with thymus-derived macrophages.

Murine C57BL/6 thymocytes were cultivated together with syngeneic thymus-derived macrophages (TDM phi) for up to 96 hr to determine whether TDM phi participate in thymocyte maturation. The expression level of H-2b and Thy-1.2 antigens served as thymocyte differentiation surface markers as analyzed by flow cytometry. Indirect immunofluorescent staining profiles of the thymocytes demonstrate a dramatic increase in H-2b expression and a profound decrease in Thy-1.2 expression during cultivation with TDM phi. A similar phenomenon was observed when enriched populations of immature thymocytes were cocultivated with TDM phi. These changes were not observed when thymocytes were cultivated alone or with trypsin-treated TDM phi; neither were they observed when cortisone-resistant thymocytes manifesting mature characteristics were cultivated together with TDM phi. These findings suggest that interaction of thymocytes with TDM phi, involving binding and engulfment, results in the appearance of mature thymocyte subsets.

Cytofluorometric analysis of thymic interdigitating cells from C57BL/6 mice prior and after leukemogenic X-irradiation.

The thymus is populated by various Ia+ cell populations, including epithelial cells, macrophages and dendritic cells. Thymic cell suspensions were stained with an anti-Ia antibody and shown by cytofluorometry to contain a small number of strongly Ia+ cells characterized by a large diameter. The cell population was separated with the aid of the fluorescence-activated cell sorter (FACS) and characterized. They were shown to express high levels of membranal Ia antigens; they demonstrated ATPase activity and displayed the ultrastructural features characteristic of the previously described thymic interdigitating cells. C57BL/6 mice were submitted to various regimens of X-irradiation. Whereas exposure to a single dose of X-irradiation was followed by an increase in the percentage of strongly Ia+ cells, exposure to a leukemogenic regimen of fractionated X-irradiation led to a decrease in the percentage and absolute numbers of these cells in the thymus. Of the C57BL/6 mice that were irradiated with fractionated X-irradiation, 77% developed leukemia. Intravenous injection of syngeneic bone marrow one day following the last irradiation or protection of the femur during irradiation prevented both the appearance of leukemia and the disappearance of interdigitating cells. Therefore an inverse correlation between the presence of thymic dendritic cells and the incidence of leukemia in C57BL/6 mice could be demonstrated. These findings are discussed in relation to the putative role of dendritic cells in the thymus.

Flow cytofluorometric analysis of the uptake of the fluorescent fatty acid pyrene-dodecanoic acid by human peripheral blood cells.

The fluorescence activated cell sorter (FACS) was used for measuring the uptake of the fluorescent fatty acid derivative 12-(1-pyrene) dodecanoic acid (P12) by human peripheral blood cells. The results indicate that blood cells differ widely in their ability to take up P12, with polymorphonuclear cells showing the greatest uptake, followed by lymphocytes, platelets, and RBCs. These differences in P12 uptake provide a potential additional parameter for differential cell counting. Using the ability of the FACS to "gate out" nonrelevant cells, it was possible to measure the rate of P12 uptake by each respective cell type even when admixed with other cells. Thus elaborate physical separation procedures could be avoided, and contaminating cells did not influence the results. Differences in P12 uptake were also utilized to separate blood cells into pure subpopulations of specific cell types.

Experimental autoimmune anemia induced with haptenated syngeneic mouse red blood cells and low dose X-irradiation.

This paper describes an experimental model of autoimmune anemia induced with haptenated syngeneic mouse red blood cells (MRBC). A strain mice immunized with penicillinated MRBC (PEN-MRBC) generated IgM anti-MRBC autoantibody response and a very low level of the corresponding IgG response. The IgG anti-MRBC autoantibody response was augmented when mice were X-irradiated (250 rad) prior to immunization with PEN-MRBC, suggesting that a radiosensitive suppressive mechanism controls the IgG autoantibody response. Both the IgM and the IgG secondary anti-MRBC autoantibody responses emerged in mice that had been injected with PEN-MRBC 2 months before a second PEN-MRBC injection. Low dose X-irradiation (250 rad) of A mice without subsequent immunization induced an early IgG anti-MRBC autoantibody response and late IgM response, suggesting the existence of antierythrocyte autoreactivity in normal animals which is controlled by a radiosensitive suppressor mechanism. The anti-MRBC autoantibody response was not only induced by PEN-MRBC. Multiple injections of A mice with trinitrophenylated MRBC also induced autoantibodies against erythrocytes. The IgG anti-MRBC autoantibody response was associated with anemia, but it is not yet known if the two events are related or independent. Autoantibodies of X-irradiated mice immunized with PEN-MRBC were linearly titrated and partially absorbed with MRBC. Less efficient absorption was achieved with human and sheep red blood cells. Fifty percent of splenocytes from X-irradiated mice injected with PEN-MRBC expressed IL-2 receptor 3 days after the injection, whereas the number of cells expressing this receptor earlier or later was significantly less. The various applications of this autoimmune model are discussed.

A rapid procedure for flow cytometric DNA analysis in cultures of normal and transformed epidermal cells.

A simple, rapid, and highly reproducible procedure for flow cytometric DNA analysis has been adapted for studying cell cycle kinetics in epidermal cell cultures. The preparation of cell nuclei and their staining with the fluorescent dye propidium iodide were performed directly on the culture dish, without prior suspension and fixation of the cells. Singly dispersed nuclei were produced by mild trypsinization of cells in the presence of the nonionic detergent Nonidet P-40 and spermine. The culture dishes could be kept frozen for prolonged periods of time before trypsinization and staining, without affecting either the recovery of nuclei or the cell cycle distribution profiles. This remarkable stability of cell nuclei greatly simplified the analysis of multiple samples in cell cycle kinetic studies. This method was used to analyze the cell cycle distribution in cultures of normal and transformed mouse epidermal cells, human colon carcinoma cells, primary bovine aortic endothelial cells, and fibroblastic and myogenic cell lines. This procedure should be very useful in studying growth kinetics, differentiation, and transformation of epidermal as well as other adherent cell types.

Use of the fluorescence activated cell sorter for studying uptake of fluorescent fatty acids into cultured cells.

A procedure is described for the continuous monitoring and recording of fatty acid transport into cultured cells. Uptake of the fluorescent fatty acid derivative, 12-(1-pyrene)dodecanoic acid [P12] by a suspension of human promyelocytic leukemia (HL-60) cells was analyzed and recorded by the Fluorescence Activated Cell Sorter (FACS). A major advantage of the FACS is its ability for measuring the fluorescence of the cells only, without interference by that of the fatty acids dissolved or dispersed in the medium or complexed with serum albumin. The time-dependent fluorescence increase due to uptake of the acid by the cells could thus be monitored and recorded directly without a need for sedimenting, washing and extracting the cellular lipids. This procedure provided an accurate measurement of the kinetics of uptake of the fatty acid into the cells and permitted study of the effect of various parameters such as temperature, glucose, albumin or serum. The results indicated a biphasic uptake of P12 into the cells. The first, a rapid, energy-independent phase, lasted 3-4 min. This was followed by a slower uptake which was directly related to the metabolic utilization of the acid in the cells. This second phase represents the overall process of translocation across the cell membrane, activation to acyl coenzyme A and incorporation into the cellular neutral lipids and phosphoglycerides. When compared to HL-60, murine erythroleukemia (MEL) cells took up considerably lesser quantities of P12. This was utilized for setting up a model system, in which mixtures of these two respective cell types could be identified and separated from each other on the basis of their relative rates of uptake of the fluorescent fatty acid.

Uptake of fluorescent fatty acids by erythroleukemia cells. Effect of differentiation.

The uptake of a fluorescent derivative of a fatty acid (FDFA), 12-(1-pyrene) dodecanoic acid (P12) by murine erythroleukemia (MEL) cells was studied. Because of the intense fluorescence of the pyrene ring, the association of P12 with intact cells could be analysed using a fluorescence microscope or a fluorescence-activated cell sorter (FACS), and the incorporation of P12 into cellular lipids could be quantified, following their extraction in a spectrofluorimeter. These procedures indicated that P12 uptake and intracellular utilization are reduced, following induction of erythrodifferentiation by dimethylsulfoxide (DMSO) or hexamethylene-bis-acetamide (HMBA). The differences in the fluorescence observed following exposure to P12 permitted us to separate a mixture of differentiated and undifferentiated cells into two distinct cell subpopulations; the high fluorescence population consisted mainly of undifferentiated cells, and the low one of differentiated cells. The results of this study suggest that fluorescent fatty acids are useful for distinguishing between and sorting cells at different stages of differentiation.

Parameters affecting the in vitro maturation of human monocytes to macrophages.

In vitro maturation of human monocytes to macrophages was characterized by morphological criteria, cell size and lysosomal enzymes activity. Purified populations of monocytes were maintained in culture at either adherent or nonadherent conditions and their maturation to macrophages was observed in both cases. The addition of external factors such as hydrocortisone and vitamin D3 inhibited monocyte maturation. In the absence of external factors, nonadherent monocytes were inhibited in their maturation for up to 10 days when plated at crowded cell concentrations. In addition, the presence of human serum in the culture media had a higher inhibitory activity than similar concentrations of fetal calf serum. Supernates from crowded macrophages were also inhibitory for monocyte maturation. We suggest the possibility that cell crowding, as well as soluble factors found in the serum and probably secreted by macrophages, participate in the regulation of monocyte development by inhibiting their maturation. Once released from this inhibitory signal or environment, the monocytes mature to macrophages.

Replacement of 5-methylcytosine by cytosine: a possible mechanism for transient DNA demethylation during differentiation.

In an earlier study it was discovered that when Friend erythroleukemia cells (FELC) were exposed to a variety of chemical agents capable of inducing differentiation, their DNA underwent genome-wide transient demethylation. In an attempt to elucidate the biochemical mechanism responsible for this phenomenon we have induced FELC with 5 mM hexamethylenebisacetamide and labeled the DNA in vivo with a density label, 5-bromodeoxyuridine, and a radioactive label, deoxy[5-3H]cytidine. Newly replicated DNA (heavy-light) was separated from parental DNA (light-light) by isopycnic centrifugation. Incorporation of deoxy[5-3H]cytidine into light-light duplex DNA has been observed only in induced cells concomitantly with the demethylation of the DNA, whereas, in parallel experiments, deoxy[G-3H]adenosine was not incorporated into light-light DNA. It was also found that the labeling of light-light DNA with deoxy[5-3H]cytidine is transient since the 3H label was removed from the DNA during the period of de novo DNA methylation that follows the demethylation. These results, taken together, strongly suggest that the demethylation of the DNA during differentiation is achieved by an enzymatic mechanism whereby 5-methylcytosine is replaced by cytosine.

Cell cycle-dependent regulation of eukaryotic DNA methylase level.

DNA methylase activity in the nuclei of somatic cells arrested at G0 increased markedly when the cells were subjected to a mitogenic stimulus. Treatment of mouse splenocytes with Concanavalin A resulted in about 20-fold increase in methylase activity within 20 h starting 12-15 h after Concanavalin A addition. The methylase level in rat liver was elevated approximately 3-fold at about 20-h posthepatectomy. A detailed time course of the increase in methylase activity with respect to the cell cycle revealed that the onset of this event coincided with the entry of the cells into S phase. In both systems, the extent of methylation in CpG sequences is not altered significantly even under conditions of active DNA synthesis which is induced by the mitogenic effect. These results suggest that the cell responds to the mitogenic stimulus by adjusting the DNA methylase activity to enable conservation of the methylation level in DNA.

Monoclonal autoantibodies to histones from autoimmune NZB/NZW F1 mice.

Fusion of spleen cells from autoimmune NZB/NZW female mice with drug-resistant myeloma cells (clones NSI/1, X63-Ag8.653 and NSO/1) produced hybrid clones which secreted antibodies to various nuclear components. Roughly 50% of the anti-nuclear hybridomas produced antibodies reacting with DNA, 20% with RNA and 30% reacted with other nuclear antigens. Two hybridomas of the latter group were cloned and studied in detail. They secreted antibodies which produced bright fluorescence staining of nuclei and metaphase chromosomes. The specificity of the antibodies was determined by testing them in an enzyme-linked immunosorbent assay and a radioimmunoassay against individual acid- and salt-extracted histones, against histones mixed two and three at a time and against histone complexes isolated as such from chromatin. One of the monoclonal antibodies was specific for histone H2B and reacted with the histone free in solution or when present as a H2A-H2B complex. The second monoclonal antibody recognized a specific conformation in the H3-H4 complex that was present only when the complex was obtained from chromatin by salt extraction. The same conformation, however, could be induced by adding histone H2B to a mixture of acid-extracted H3 and H4. Our findings show that the autoimmune syndrome in NZB/NZW mice resembles human systemic lupus erythematosus not only in the incidence of antibodies to DNA and RNA, but also in the production of autoantibodies to histones.

Nuclear structure: determination of the fate of the nuclear envelope in Drosophila during mitosis using monoclonal antibodies.

Libraries of monoclonal antibody against nuclear proteins of Drosophila melanogaster have been established recently to investigate nuclear structure and function. Some of the antibodies have been characterized as being directed against the nuclear envelope. Further studies detailed in this paper describe the fate of the nuclear envelope during mitosis. Indirect immunofluorescence staining of whole developing Drosophila embryos has been used as a system in which nuclear events can be studied both synchronously and in a longitudinal gradient of mitotic structures. The results show a pattern of breakdown and reconstruction of the nuclear envelope in which the antigen is always present in particulate structures. In addition, the processes of antigen rearrangement are shown to be spatially determined throughout mitosis.

Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate.

n-Prop]yl gallate (0.1 to 0.25 molar, in glycerol) reduces by a factor of 10 the rate of fading of fluorescence of cell structures labeled with tetramethylrhodamine or fluorescein-conjugated antibodies. Hence, prolonged photographic exposure of immunofluorescently labeled cells in the fluorescence microscope yields images with increased sensitivity, making feasible multiple data collection, as with serial optical sectioning.

On the mechanism of the glucose-induced ATP catabolism in ascites tumour cells and its reversal by pyruvate.

Addition of glucose to Ehrlich-Landschütz ascites tumour cells preincubated for 30-60 min in phosphate-buffered Krebs-Ringer salt solution ("starved cells") resulted within 1-2 min in an approx. 90% decline of their ATP content and a massive accumulation of fructose 1,6-bisphosphate. These alterations, which took place under both aerobic and anaerobic conditions, were followed by a gradual spontaneous recovery with restoration of normal ATP and fructose 1,6-bisphosphate values. The transient derangement of the energy metabolism after glucose addition to starved ascites tumour cells by preventable by simultaneous addition of pyruvate or 2-oxobutyrate, or by preincubating the cells in the presence of glucose. The protective effect of pyruvate was duplicated by addition of phenazine methosulphate or NAD+ to the incubation medium. The data seem to warrant the conclusion that the glucose-induced ATP depletion is determined by a blockade of glycolysis at the stage of glyceraldehyde phosphate dehydrogenase caused by the failure of the cells to oxidize the NADH produced in the same reaction. The continued unrestrained action of 6-phosphofructokinase results in accumulation of fructose 1,6-bisphosphate, which constitutes a trap for the high-energy phosphate bonds of ATP. The primary metabolic disturbance appears to consist of a transient inhibition of pyruvate kinase with the resultant inability of the cells to maintain an unimpaired supply of pyruvate, as required for the lactate dehydrogenase-mediated oxidation of NADH. The regulatory mechanism underlying this phenomenon is discussed.

Inhibition of peptide chain initiation in lysates from ATP-depleted cells.I. Stages in the evolution of the lesion and its reversal by thiol compounds, cyclic AMP or purine derivatives and phosphorylated sugars.

Anaerobic incubation of rabbit reticulocytes at 37 degrees C in Krebs-Ringer solution supplemented with hemin but devoid of glucose resulted at the end of 1-2h in a drastic decline of their ATP content and an attendant arrest of protein synthesis. Subsequent provision of glucose and reoxygenation of the cells was followed by a rapid replenishment of the ATP pool, while resumption of protein synthesis was markedly delayed. This lag period could be considerably reduced by addition of 5-10 mM adenine or 2,6-diaminopurine to the incubation medium. Lysates prepared from ATP-depleted cells exhibited disaggregation of the polysomes and an inhibition of the nedogenously coded protein synthesis, when tested in a cell-free system supplied with an adequate ATP generator. Both alterations increased in severity with the progressive decay of the intracellular ATP pool. The early phase of partial inhibition following a 40-70% decrease of the cellular ATP level was fully reversible by fortifying the cell-free preparation with dithiothreitol or a suitable NADPH-generating system. Aternative, the inhibition could be also overcome by millimolar amounts of adenine, 2,6-diaminopurine and a variety of other purine derivatives or cyclic AMP. The effect of these compounds was unrelated to the endogenous cyclic AMP pool. Joint addition of both dithiothreitol and cyclic AMP or adenine was necessary for relieving the initiation block in lysates derived from cells depleted of 80-90% of their ATP content. On further aggravating the conditions of energy starvation, an additional requirement for phosphorylated sugars, e.g. glucose 6-phosphate or fructose 1,6-diphosphate, became apparent. ATP depletion brought about by exposing the cells to Antimycin A or 2,4-dinitrophenol resulted in a lesion which was indistinguishable from that induced by anaerobic incubation. On the other hand, energy deprivation in cell-free lysates from untreated reticulocytes, preincubated in the absence of an ATP-generating system failed to duplicate the deleterious effect of intracellular ATP depletion. Some aspects bearing on the biochemical mechanism of the lesion and its reversal are discussed in the light of the available data.

Inhibition of peptide chain initiation in lysates from ATP-depleted cells. II. Studies on the mechanism of the lesion and its relation to similar alterations caused by oxidized glutathione and hemin deprivation.

The impairment of peptide chain initiation in lysates from ATP-depleted rabbit reticulocytes is accompanied by a loss of their ability to form the 40 S methionyl-tRNAfMet complex with a resultant failure to promote the AUG codon-dependent combination of the complex with the 60 S ribosomal subunit. These partial initiation reactions, as well as the overall protein-synthetic activity of the defective lysates could be restored by addition of a 0.5 M KC1 ribosomal extract or normal postribosomal supernatant or a 40-70% (NH4)2-SO4 fraction derived from it. Alternatively, reactivation of the impaired lysates could be achieved by supplementation with millimolar amounts of cyclic AMP or certain purine derivatives. The same subcellular fractions, as well as cyclic AMP or purine derivatives were also capable of overcoming the inhibition caused by incubating reticulocyte lysates in the presence of oxidized glutathione or in the absence of hemin. Severe intracellular ATP deprivation resulted in accumulation of a soluble translational inhibitor in the postribosomal fraction, thus resembling the parallel phenomenon described in hemin-deprived lysates. The striking similarities between the three kinds of inhibition studied by us point to an identical site of the underlying biochemical lesion, despite the different mechanisms mediating their induction.