Abstract: Using a quantitative electron microscopic autoradiographic technique, we have localized the initial binding step of 125I-labeled epidermal growth factor (125I-EGF) to the plasma membrane of the human fibroblast. After initial binding, labeled EGF is internalized progressively by the cell in a time- and temperature-dependent fashion; when cell-associated radioactivity comes to steady state, approximately 1/3 of the autoradiographic grains are related to the plasma membrane and approximately 2/3 have been internalized. Under these conditions the internalized grains are almost exclusively related to lysosomal structures. When 125I-EGF associates with the cells for 2 hr at 4 degrees or 2 min at 37 degrees, 34% of grains localize to coated regions of the membrane. These coated regions make up less than 2% of the membrane surface. These data directly confirm kinetic studies and suggest that saturable binding of EGF is followed by adsorptive pinocytosis and cellular degradation of the ligand and possibly its cell surface receptor.

Abstract: We have previously shown that a subpopulation of nylon wool nonadherent, lymphoid cells in normal mice binds selectively to tumor cell targets that are sensitive to killing by natural killer (NK) cells. In the present report these target-binding cells (TBC) were enriched or depleted by velocity sedimentation and the isolation of single target-effector conjugates indicated that the majority of TBC killed their targets. Furthermore, the frequency of TBC correlated well (r = 0.86) with lysis in a population during kinetics experiments. TBC resembled small, resting lymphocytes with membrane specializations in the area of target cell contact as indicated by electron microscopy and cytochemical techniques. Target cell binding occurred before lysis in kinetics studies and regenerated in parallel with the lytic potential of a trypsinized population devoid of surface Ig or θ-bearing cells. Cell contact was prevented in the presence of EDTA whereas metabolic inhibitors or inhibitors of serine proteases suppressed lysis with no effect on the frequency of TBC. Conversely, an interferon-inducing agent elevated NK-mediated cytolysis with no effect on the level of TBC. These results suggest that i) the majority of TBC that we detect represents the NK cell and ii) lysis and binding are independently controlled events. A tentative model of NK-mediated lysis is proposed.

Abstract: The mitogenicity, ability to induce immune interferon, and relationship between interferon synthesis and cell proliferative response were studied using human peripheral lymphocytes stimulated by staphylococcal enterotoxin A (SEA), phytohemagglutinin-P (PHA-P), and concanavalin A (ConA) Maximum cell proliferative responses ([(3)H]thymidine incorporation) and protein synthesis ((14)C-amino acid incorporation) occurred on days 3 and 4, respectively, after stimulation by each of the three mitogens Maximal immune interferon levels were found 3 or 4 days after mitogen stimulation SEA-treated cultures produced approximately three times more interferon than did cultures stimulated with PHA-P or ConA Furthermore, SEA stimulated maximal cell proliferation over a much broader concentration range than did PHA-P and ConA (SEA, 10(-5) to 10(2) mug/ml; PHA-P, 10(1) to 10(2) mug/ml; ConA, 10(1) to 10(15) mug/ml) Interferon was also produced at maximal or near maximal levels over a broad concentration range of SEA (10(-2) to 10(2) mug/ml) Also, we found that inhibition of mitogen-induced DNA and protein synthesis to control levels by mitomycin C or cytosine arabinoside partially reduced interferon production The DNA inhibitor studies indicate that immune interferon synthesis occurs maximally in association with at least some proliferative response and that submaximal levels of interferon production occur in mitogen-treated cultures in the absence of detectable proliferation The ability of SEA to stimulate maximal DNA and immune interferon synthesis at concentrations of 35 x 10(-13) M and 35 x 10(-10) M, respectively, puts it in a potency range similar to that of hormones Thus, SEA may play an important role in gut immunity and Staphylococcus aureus infections at concentrations well below those required for emetic effects

Abstract: Human lymphocytes secrete high levels of interferon a few hours after being cultured with certain tumorderived or virus-transformed cell lines. Interferon and interferon inducers (e.g., viruses, inducer cell lines, synthetic inducers), after short incubation with lymphocytes, increase several-fold the cytotoxicity of human natural killer cells. When lymphocytes are tested as effector cells against interferon-inducing target cell lines in an 18-hr test of spontaneous cell-mediated cytotoxicity, the enhancing effect of the interferon released in the culture medium is responsible for 80 to 90% of the total cytotoxicity observed. The ability or inability of different target cell lines to induce interferon is responsible for the apparent difference in selectivity of the cytotoxicity against various targets when fresh lymphocytes, cultured lymphcoytes, or interferon-activated lymphocytes are used as effector cells. Moreover, some of the apparently specific results obtained in competitive assays with unlabeled target cells are also dependent on the ability or inability of the competitor and target cells to induce interferon. Interferon does not increase the antibody-dependent cytotoxicity mediated by human lymphocytes, which suggests that spontaneous and antibody-dependent cell-mediated cytotoxicity are mediated by different effector cells or that a unique class of effector cells can mediate cytotoxicity with two independent mechanisms. The inability of rabbit F(ab′) 2 fragment antihuman IgG to inhibit spontaneous cell-mediated cytotoxicity, renders unlikely the possibility that the activity of the human natural killer cells is dependent on cytophilic anti-target cell antibodies adsorbed in vivo to the effector lymphocytes.

Abstract: The relative roles of blood cell products and plasma factors on endothelial cell proliferation were evaluated by studying the proliferative response of human umbilical vein endothelial cells to cell free plasma derived serum (CFPDS), whole blood serum (WBS), platelet released factors, fibroblast growth factor and macrophage conditioned medium in vitro. Human adult arterial smooth muscle cells were treated in a similar manner for comparison. The rate of endothelial cell proliferation was directly related to the concentrations of both WBS and CFPDS. Growth rate in WBS was marginally greater than that observed in CFPDS during early culture, however, similar confluent densities were achieved. The addition of platelet released factors to CFPDS did not further stimulate endothelial cell proliferation. In contrast smooth muscle cells were quiescent in CFPDS despite increasing serum concentrations, but proliferated actively in response to platelet released factors. Both human macrophage conditioned medium and fibroblast growth factor increased endothelial cell proliferation significantly when compared with CFPDS alone. It is concluded that endothelial cell proliferation in preconfluent cultures is dependent on plasma factors while human vascular smooth muscle cells also require cell derived mitogens such as platelet growth factor to proliferate. The release of a substance by human macrophages mitogenic for endothelial cells may be involved in endothelial cell proliferation in vivo.

Abstract: Groups of inside cells (ICs) and inner cell masses (ICMs) were isolated from individual mouse embryos between the late morula and 3 1/2-day expanded blastocyst stages using a modified immunosurgical procedure, and their purity and developmental potential were assessed in vitro. Several different techniques failed to detect the presence of viable contaminating outside cells on ICs isolated from any of the stages studied. The numbers of inside cells isolated from the earlier stages, counted in air-dried preparations, were considerably higher than previous estimates from serial sections; whereas the numbers isolated from expanded blastocysts were in reasonable agreement. Thus the proportion of inside cells recovered by immunosurgery decreases over this period of development. In view of the evidence that inside cells divide at a faster rate than outside cells at these stages, it is argued that there may be an outward movement of inside cells capable of forming trophectoderm, during expansion of the blastocyst. ICs and ICMs in vitro were observed to develop in one of two distinct ways according to the stage at which they were isolated. ICs from late morulae and some early cavitating blastocysts formed blastocyst-like vesicles over a period of 24--36 h in culture. The presence of trophectoderm cells in these vesicles was confirmed by the persistence of giant cells after ectopic transfer. In contrast ICs from a minority of early cavitating blastocysts, and all ICMs from 3 1/2-day expanded blastocysts did not form vesicles, but proliferated endoderm-like cells. Thus at least some inside cells do not appear to lose the capacity to form trophectoderm and do not become committed to an ICM fate until after the initial formation of the blastocoel cavity.

Abstract: The problem of estrogen-promoted tumor cell growth has been studied extensively in an attempt to establish the direct mitogenic role of these steroid hormones. We have developed cell lines from three estrogen-responsive tumors or cell populations: the H-301 kidney tumor cells established from a parent estrogen-dependent hamster kidney tumor, the GH3/C14 rat pituitary tumor cell line established as a subline of the original GH3 population, and the MTW9/PL mammary cell line developed from a parent estrogen- and prolactin-responsive MT-W9A carcinogen-induced rat tumor. With all three of these cell lines, we have encountered a paradox: although estrogens are obligatory for tumor formation in vivo, no direct mitogenic effect of estrogens can be shown in culture when assayed by an increase in cell number. We have thus considered the possibility that estrogens may induce growth factors in vivo that are then responsible for tumor formation by the three cell lines described. Experiments presented in this report show that extracts of rodent uterus, kidney, or liver contain growth activity for these three tumor cell lines, that estrogen treatment causes an increase in tissue content of these activities, and that the estrogen-induced activities are specific for the estrogen-responsive cells. These studies suggest that estrogen-responsive tumor growth in vivo includes the mechanism of estrogen leads to uterus, kidney, or liver leads to specific growth factors leads to estrogen-responsive tumor cells.

Abstract: A SERIES of 2–5 linked oligoadenylic acid triphosphate (2–5A) inhibitors of cell-free protein synthesis are formed from ATP by an enzyme (2–5A synthetase) activated in interferon-treated cell extracts or rabbit reticulocyte lysates by double-stranded RNA1–4. The major active species is the trimer, pppA2′p5′A2′p5′A. Cell-free protein synthesis is inhibited by subnanomolar concentrations of 2–5A and this inhibition seems to be mediated at least in part, by a nuclease which degrades mRNA5. However, 2–5A, the nuclease and the inhibition of protein synthesis are all unstable in cell-free systems. 2–5A is rapidly degraded in such systems prepared from control (or interferon-treated) cells, nuclease activity is transient and in the absence of a 2–5A regenerating system protein synthesis resumes if fresh mRNA is added6,7. Extracts derived from cells that have never been treated with interferon, therefore, have mechanisms for the synthesis of 2–5A (rabbit reticulocytes) and for responding to and degrading 2–5A. Thus, the 2–5A system, in addition to any role it has in the antiviral action of interferon, may also be involved in the regulation of normal cell growth and development. The extraordinary potency of 2–5A and its instability in cell-free systems makes the task of detecting 2–5A in intact cells very difficult. As an alternative we have studied the effect of exogenous 2–5A on protein synthesis in intact cells, using a recently described method for making animal cells reversibly permeable to small molecules8. Here we report that pppA2′p5′A2′p5′A and related 2′–5′ linked oligonucleotides inhibit protein synthesis in hypertonically treated BHK–21 cells.

Abstract: A population of neonatal mouse keratinocytes (epidermal basal cells) was obtained by gentle, short-term trypsin separation of the epidermal and dermal skin compartments and discontinuous Ficoll gradient purification of the resulting epidermal cells. Over 4--6 wk of culture growth at 32--33 degrees C, the primary cultures formed a complete monolayer that exhibited entire culture stratification and upper cell layer shedding. Transmission and scanning electron microscopy demonstrated that the keratinocyte cultures progressed from one to two cell layers through a series of stratification and specialization phenomena to a six to eight cell layer culture containing structures characteristic of epidermal cells and resembling in vivo epidermal development. The temporal development of primary epidermal cell culture specialization was confirmed by use of two histological techniques which differentially stain the specializing upper cell layers of neonatal mouse skin. No detectable dermal fibroblast co-cultivation was demonstrated by use of the leucine aminopeptidase histochemical technique and routine electron microscope surveillance of the cultures. Incorporation of [3H]thymidine ([3H]Tdr) was greater than 85% into DNA and was inhibited by both 20 micron cytosine arabinoside (Ara-C) and low temperature. Autoradiography and 90% inhibition of [3H]Tdr incorporation by 2 mM hydroxyurea indicated that keratinocyte culture DNA synthesis was scheduled (not a repair phenomenon). The primary keratinocytes showed an oscillating pattern of [3H]Tdr incorporation into DNA over the initial 23--25 days of growth. Autoradiography demonstrated that the cultures contained 10--30% proliferative stem cells from days 2-25 of culture. The reproducibility of both the proliferation and specialization patterns of the described primary epidermal cell culture system indicates that these cultures are a useful tool for investigations of functioning epidermal cell homeostatic control mechanisms.

Abstract: Spleen cells obtained from mice 5 to 40 days after infection with viable BCG organisms (BCG-spleens) were found to be unresponsive in vitro to both mitogenic and alloantigenic stimuli. Moreover, suppressor cells could be demonstrated in the spleens from these infected animals. When spleen cells from BCG-infected mice were added to either syngeneic or allogeneic normal spleen cells, the mixtures neither proliferated nor developed cytotoxic activity when cultured with alloantigen or with concanavalin A (Con A). The development of unresponsiveness post-infection paralleled the onset of suppressive activity. Spleen cells obtained from mice given heat-killed BCG were neither suppressive nor unresponsive. The suppressive activity of BCG-spleen cells was associated with an adherent, phagocytic cell that lacked membrane-associated Thy-1 antigen. Removal of this cell by passage through nylon wool columns resulted in a cell population that was no longer capable of suppression and that responded normally to alloantigen and to Con A. It would thus appear that BCG infection results in the development of a "suppressor" macrophage-like cell population within the spleen. The role of this cell type in regulation of the immune response in BCG-infected animals is as yet undefined.

Abstract: In Hodgkin's disease a possible mechanism for impaired cellular immunity is cell-mediated suppression, defined as the inhibitory interaction between suppressor cells and effector lymphocytes. To test for the presence of suppressor cells in peripheral blood, we have modified the standard, one-way mixed lymphocyte culture by adding mitomycin C-treated mononuclear cells from the responder. Suppression, expressed as a percent of the base-line mixed lymphocyte culture in which these extra cells are not present, results in a reduction of thymidine incorporated in the modified culture (i.e., 100% suppression = no net thymidine incorporation; 0% suppression = identical thymidine incorporation in both the modified and baseline culture). Suppression was found to be significantly increased in patients with both active Hodgkin's disease (78+/-4.6%) and remission Hodgkin's disease (58+/-9.3%) compared to normal individuals (21+/-6.9%) (mean+/-SE). The degree and frequency of suppression were not influenced by disease stage or prior therapy. Cell purification techniques revealed (in 10 patients studied) the suppressor cell to be a monocyte in 6, and a thymus-derived lymphocyte in 4. Possible genetic restriction of the suppressor cell interaction was indicated by a failure of suppressor cells to alter the response of lymphocytes from unrelated individuals, but suppression was obtained with lymphocytes from a histocompatible sibling. Although mononuclear cells from normal individuals suppress less frequently than cells from patients with Hodgkin's disease, normals may demonstrate suppression comparable to that observed in Hodgkin's patients. This finding suggests that suppression is a normal immunoregulatory mechanism which is altered in Hodgkin's disease.

Abstract: SUMMARY. The peripheral blood of 60 normal adults was separated into plasma, red cells, neutrophils, lymphocytes, monocytes and platelets. Lactoferrin concentrations were measured in the plasma and cell extracts and compared to those of lysozyme. The neutrophil lactoferrin content in males and post-menopausal females was found to be significantly higher than in pre-menopausal females. A small amount of lactoferrin was found in association with monocytes, but not with lymphocytes, erythrocytes and platelets. Neutrophil lysozyme concentrations did not exhibit any variation with sex and age; but the level in monocytes was higher than that in neutrophils. No correlation was observed between individual neutrophil lactoferrin values and the plasma level. Immunofluorescent studies showed neutrophils to have a lobulated pattern suggestive of nuclear staining. Monocytes did not show direct staining, but exhibited a peripheral pattern after prior exposure to lactoferrin-confirming the existence of a surface receptor. Gel chromatography indicated that neutrophil lactoferrin is in a polymerized or complexed form which elutes with the void volume on Sephadex G-zoo; serum lactoferrin consists of two forms, one of which also elutes with the void volume on Sephadex G-zoo.

Abstract: Streptococcus mutans strain B13 (serotype D) almost exclusively produced free glucosyltransferase (GTase) in the culture supernatant when grown in sucrose-free TTY broth medium, which was composed of Trypticase (Baltimore Biological Laboratory [BBL] Cockeysville, Md.), tryptose (Difco Laboratories, Detroit, Mich.), yeast extract (BBL), salts, and 1% glucose. Organisms grown in sucrose-free TTY broth retained very weak cell-associated GTase activity and did not adhere significantly to glass surfaces in the presence of exogenous sucrose. If sucrose was added to TTY broth, however, GTase was found on the cell surface where cell-bound, water-insoluble glucans were synthesized. Most commercially available products of Todd-Hewitt broth were found to contain trace amounts of sucrose, as did Trypticase soy broth (BBL), whereas brain heart infusion broth (Difco and BBL) was found to be essentially free of sucrose. Almost all detectable GTase activity was cell associated when S. mutans B13 was grown in Todd-Hewitt or trypticase soy broth. Heat-treated B13 cells grown in Todd-Hewitt broth and cell-free, water-insoluble glucans bound free GTase and produced marked adherence in the presence of sucrose. Experiments strongly suggest that the binding sites for free GTase are the surface glucans, and cell-associated and extracellular GTases are most likely alternate states of the same enzyme protein.

Abstract: Early passage mouse embryo fibroblasts, mouse 3T3 cell lines, and early passage diploid human fibroblasts grew to higher cell densities in tissue culture medium supplemented with serum than in medium supplemented with defibrinogenated platelet-poor plasma (PPP). Unlike the mouse cells, the human fibroblasts displayed this differential growth response only in the presence of hypophysiologic concentrations of calcium. The addition of heat-treated extracts of human platelets to PPP-supplemented medium stimulated the replication of both the normal mouse cells and early passage human embryo fibroblasts. Human or mouse fibroblasts transformed by either retroviruses or by SV40, including SV40 infected “serum revertants” and “flat transformants,” grew to equal cell densities in medium supplemented with either serum or PPP. Infection of Balb/c-3T3 cells with SV40 rapidly induced them to grow in PPP-supplemented medium demonstrating that the ability of SV40-transformed cell lines to proliferate in PPP-supplemented medium does not arise from the cell culture selection procedures usually employed to obtain stable virus-transformed cell lines. 3T3 cells infected but not transformed by retroviruses do not replicate in PPP-supplemented medium demonstrating that reduction of the growth requirement for the platelet growth factor(s) by retroviruses is a transformation-specific response. Cell cultures that did not proliferate well in PPP-supplemented medium did not form tumors when inoculated into athymic nude mice. Many, although not all, of the lines which grew well in PPP medium were tumorigenic in nude mice. Together, these findings indicate that: (1) normal fibroblast-like cells display a growth requirement for factor(s) present in serum but not found in PPP; (2) this serum specific growth factor is derived from platelets; (3) a primary response to viral transforming genes is a reduction in the growth requirement for these platelet-derived factors; and (4) cells that have a reduced requirement for the platelet-derived growth factor are often tumorigenic.

Abstract: Synchronized suspension cultures of Chinese hamster ovary cells (CHO) were labeled with various doses of 3H-thymidine or 125I-iododeoxyuridine to evaluate the cytocidal effects of intranuclear radionuclide decay. Damage produced by radionuclide decay outside the cell nucleus was studied on cells exposed to 125I labeled, monovalent concanavalin A. After labeling, the cells were resynchronized in G1-phase and incubated for 36 h at 4 degrees C to permit dose accumulation. Cell lethality was evaluated by the standard colony assay. Based on radionuclide incorporation data, cellular dimensions, and subcellular radionuclide distributions, the cumulative dose to whole cells, cell nuclei, and cellular cytoplasm was calculated from the known decay properties of 3H and 125I. As expected, DNA associated 125I (LD50: 60 decays/cell; 45 rad) was much more toxic to CHO cells than 3H (LD50: 1350 decays/cell; 380 rad) 380 rad) or external X-irradiation (LD50: 330 rad). In contrast, membrane associated 125I was surprisingly non-toxic (LD50: 19 600 decays/cell). At 19 600 decays/cell the dose to the cell membrane was approximately 52 krad and the overlap dose into the cytoplasm was about 2470 rad. Even at these high dose levels, membrane damage or cytoplasmic damage apparently did not contribute significantly to radiation induced cell death. With 19 600 decays on the plasma membrane the CHO nuclei received an overlap dose of about 410 rad. As can be seen from the LD50 data for 3H and X-rays, a nuclear dose of 410 rad should be sufficient to account for 50% cell death. These findings indicate that, although intranuclear decay by electron capture is extremely destructive, identical decay events in the plasma membrane cause only minimal cell damage. This parallels our earlier studies on 67Ga labeled leukemia cells which showed that electron capture decay in the cytoplasm is also highly ineffective in killing mammalian cells. It therefore appears that radiation-induced cell lethality in dividing mammalian cells results primarily from nuclear damage. Cytoplasmic or membrane contributions to radiation-induced cell death, if any, must be minimal. By implication, these findings refute the enzyme release hypothesis and similar theories designed to explain mitotic death in terms of cytoplasmic or membrane damage rather than nuclear damage.

Abstract: We have previously identified a molecule (named cell adhesion molecule [CAM]) that is involved in the in vitro aggregation of neural cells from chick embryos. In the present report, specific anti-CAM antibodies have been used to demonstrate that CAM is localized in neural tissues, and is associated with the plasma membrane of retinal cells and neurites. Furthermore, it has been shown by antibody absorption techniques that the decreased adhesiveness of cultured retinal cells obtained originally from older embryos is correlated with a decrease in the density or accessibility of cell adhesion molecules on the surface of these cells. The central role of CAM in neural cell aggregation has been established by the observation that anti-CAM Fab' fragments inhibit adhesion between neural cells in a variety of assays. To investigate the function of CAM and cell adhesion in developing tissues, aggregates of retinal cells that are capable of forming histotypic patterns in vitro were cultured in the presence and absence of anti-CAM Fab'. The Fab' was found to inhibit sorting out of cell bodies and neurites and to decrease the number of membrane-membrane contacts, suggesting that CAM is associated with cell- cell, cell-neurite, and neurite-neurite interactions.

Abstract: MURINE SARCOMA VIRUS (MSV)-transformed cells lack available receptors for epidermal growth factor (EGF)1,2. We have shown that this altered phenotype is the result of the endogenous production of growth factors by the MSV-trans-formed cells themselves. The major activity, isolated and purified from transformed cells, has been found in a 12,000-molecular weight peak and competed with EOF in an EGF-receptor-binding assay3. This growth factor, called sarcoma growth factor (SGF), stimulates cell division, causes normal cells to grow in soft agar and produces rapid, reversible morphological transformation of cells in monolayer culture3. There is no evidence that SGF acts as a complete carcinogen, producing permanent cell transformation; its properties resemble classical chemical promoters of carcinogenesis, like 12-O-tetradecanoylphorbol-13-acetate (TPA)4–6, the highly active component of croton oil. Whereas TPA is an exogenous plant derivative acting on an animal or a cell, SGF is an endogenous, virally induced growth promoter. In this respect it is interesting that retinoids (vitamin A and synthetic analogues)7 block the action in vivo of exogenous and endogenous promoters, preventing carcinogens from producing new tumours, but do not reverse the growth of many established tumours7–10. Retinoids prevent cancer of the lung7,11, skin9, bladder12 and mammary gland13 in experimental animals, block cell transformation induced by chemicals14 and radiation14,15 in culture, and reverse the anchorage-independent growth of transformed mouse fibroblasts16. If SGF is part of the natural tumour-promoting system and retinoids are part of the natural defence against that system, then one should be able to demonstrate a direct antagonism in cell culture. This, report establishes that this happens.

Abstract: Fibroblast cultures from patients with recessive dystrophic epidermolysis bullosa (RDEB) demonstrated an increased capacity to synthesize and secrete collagenase. This phenotypic trait appeared to distinguish RDEB from other genetically distinct forms of epidermolysis bullosa. The finding of increased collagenase may be a specific manifestation of these cells in that prototypic lysosomal and cytoplasmic enzymes were present in approximately normal concentrations. In addition, this trait persisted through many cell passages, suggesting that the property was genetically determined. The elevated concentrations of immunoreactive collagenase in fibroblast cultures of patients with RDEB reflected those previously observed in vivo (4) and support the concept of a pathogenetic role for the enzyme in the blistering phenomenon. In three of the cell lines, the increase in enzyme protein occurred in association with a structurally defective enzyme. The data suggest that this may be a characteristic of all RDEB cells.

Abstract: A cell fusion technique was used to produce hybridomas between the T lymphoma cell line, EL-4, derived from C57BL (H-2(b)), and an enriched population of human gamma globulin (HGG)-specific suppressor T cells prepared from the spleens of HGG-tolerant CBA mice (H-2(k)). Membrane fluorescence analysis of the hybridoma cells within 6 wk of cell fusion revealed expression of H-2(k) and I-J(k) gene products as well as H-2(b) antigens. Sonicates prepared from hybridomas which contained I-J(k) cells were tested for suppressive activity in vivo in irradiated mice given HGG-primed cells, dinitrophenyl (DNP)-primed cells, HGG-DNP, and horse erythrocytes. Among 18 such hybridoma lines, 6 showed specific suppressive activity, 5 nonspecific suppression, and 7 no suppression. Most lines progressively lost, with time, those properties derived from the normal parent cell. By about 3 mo after fusion few cells expressed CBA markers and only one cell line (number 77) retained some specific suppressive activity. In parallel with the losses was an alteration in chromosome number from near-tetraploid, soon after cell fusion, to near- diploid. Preliminary results with the T lymphoma-sensitive hypoxanthine aminopterin thymidine cell line, L5178, indicate retention of the expression of surface markers derived from the normal parent for 18 wk after hybidization. This suggests that T lymphoma cell lines may have to be screened for their capacity to produce hybridomas with stable properties.

Abstract: Cell systems as different as normal human blood lymphocytes and frog auricles release spontaneously a nucleoprotein complex in their culture medium. This release seems to be an active mechanism that is unrelated to cell death. The presence of RNA in this complex is demonstrated. The amount of extracellular RNA is regulated by the same homeostatic mechanism that has previously been shown to govern DNA release in the same cell systems. This extracellular RNA is linked by hydrogen bonds to the extracellular DNA and cannot be extracted by a usual phenol procedure, due perhaps to the presence of a glycoprotein. Further purifications by chloroform, sodium perchlorate, and hydroxyapatite are necessary to obtain an RNA molecule that is acid precipitable, RNase and KOH sensitive, and orcinol positive. The extracellular RNA sediments between 2.5 and 4S and is not a transfer RNA. It is more highly methylated than the 28S, 18S, and 4 to 5S cellular RNA. It activates DNA synthesis in vitro.

Abstract: Pretreatment of murine lymphoid cells with anti-Ia and C abrogated the proliferative response of these cells to Con A, but not to PHA. Reconstitution experiments demonstrated that T cell-enriched populations failed to restore Con A responsiveness and that T cell-depleted populations were more effective in restoring responsiveness to Con A. In particular, a population of 1000 R resistant, glass-adherent, non-T spleen cells was capable of completely restoring responsiveness to Con A when added in numbers as low as 4% of cultured cells. These splenic adherent cells were found to express Ia determinants encoded by at least two genes: one in I-A and the other in I-B, I-J, and/or I-E/C, and it was demonstrated that determinants encoded in these two regions were expressed on the same cell. These results demonstrate that non-T accessory cells may be the Ia+ cells entirely responsible for the anti-Ia and C-induced abrogation of T cell proliferative responses to Con A.

Abstract: By using cell-free systems prepared from uninfected and poliovirus-infected cells, we have been able to demonstrate that crude preparations of initiation factors from infected cells do not stimulate the initiation of translation by polyribosomes containing endogenous host cell mRNA. When tested with polysomes containing endogenous viral mRNA, however, they were able to stimulate initiation of translation nearly as well as uninfected cell initiation factors. The uninfected cell initiation factor preparations were able to stimulate initiation of translation of both cell and viral mRNA. The results indicate an mRNA-specific activity present in crude initiation factor preparations from infected cells. Furthermore, the ability of eIF2 from infected cells to form a ternary complex with GTP and formyl [35S]methionine-tRNAfmet, an mRNA-independent step in initiation, was found not to be deficient. Implications of these data for proposed mechanisms of poliovirus-induced host cell shutoff are discussed.

Abstract: Activation of the alternative complement pathway by human B cell lymphoma lines is correlated with the presence of Epstein Barr virus (EBV) in the cell genome. EBV-negative B cell lymphoma lines produce little activation of the alternative pathway as measured either by C3 deposition on the cell surface or C3 conversion and consumption of alternative pathway activity in the supernatant serum. By contrast, EBV-positive sublines derived by in vitro EBV conversion of EBV-negative parental lines produce considerable activation of the alternative pathway. This membrane-associated complement-activating mechanism reflects an EBV-induced membrane change in these cells and may provide a mechanism whereby EBV-transformed cells are controlled in vivo.

Abstract: We reported previously that, by mutagenesis of a malignant teratocarcinoma cell line, it is possible to obtain a number of variant clones that are incapable of forming progressive tumors. Each of these "tum-" variants is rejected in syngeneic mice and stimulates the production of immune memory cells (self-protection). We show here that four different tum- clones confer an immune protection against each other although this cross-protection is invariably weaker than the self-protection. Moreover, mice immunized with living tum- cells are partially protected against the original malignant teratocarcinoma cells, even though the latter cells are incapable of conferring any immune protection when injected after being killed by irradiation. These results indicate that each tum- variant carries at least one specific transplantation antigen that is absent from the original tumor cell line and from most other tum- variants. Other tumor-specific transplantation antigens are probably present on all the tum- variants and also on the malignant teratocarcinoma cell line.

Abstract: The infection cycle of Rickettsia tsutsugamushi in mouse peritoneal mesothelial cells, observed late in the course of an established infection, intimately involved the host cell plasma membrane. Organisms multiplied in the cytoplasm, moved to the cell periphery, and acquired a host-membrane coat as they budded from the cell surface. Rickettsiae enveloped by this membrane entered other mesothelial cells, apparently by a phagocytic mechanism. Organisms escaped from the phagocytic vacuole as the vacuole membrane and host membrane coat disintegrated. Free rickettsiae replicated by binary fission in the cell cytoplasm. Rickettsial infection of mesothelial cells induced conspicuous cellular hypertrophy with increased numbers of unaltered cytoplasmic organelles.