Response Of Cultured Cells Research Articles

Real-time optoacoustic temperature determination on cell cultures during heat exposure: a feasibility study

Objective/Purpose: In order to study the effects of hyperthermia and other temperature-related effects on cells and tissues, determining the precise time/temperature course is crucial. Here we present a non-contact optoacoustic technique, which provides temperatures during heating of cultured cells with scalable temporal and spatial resolution.Methods: A thulium laser (1.94 µm) with a maximum power of 15 W quickly and efficiently heats cells in a culture dish because of low penetration depth (1/e penetration depths of 78 µm) of the radiation in water. A repetitively Q-switched holmium laser (2.1 µm) is used simultaneously to probe temperatures at different locations in the dish by using the photoacoustic effect. Due to thermoelastic expansion of water, pressure waves are emitted and measured with an ultrasonic hydrophone at the side of the dish. The amplitudes of the waves are temperature dependent and can be used to calculate the temperature/time course at any location of probing.Results: We measured temperatures of up to 55 °C with a heating power of 6 W after 10 s, and subsequent lateral temperature profiles over time. Within this profile, temperature fluctuations were found, likely owing to thermal convection and water circulation. By using cultured retinal pigment epithelial cells, it is shown that the probe laser pulses alone cause no biological damage, while immediate cell damage occurs when heating for 10 s at temperatures exceeding 45 °C.Conclusions: This method shows great potential not only as a noninvasive, non-contact method to determine temperature/time responses of cells in culture, but also for complex tissue and other materials.

Evaluation of the Effect of PEGylated Single-Walled Carbon Nanotubes on Viability and Proliferation of Jurkat Cells.

Among the numerous nanosized drug delivery systems currently under investigation, carbon nanotubes (CNTs), regardless of being single or multiple-walled, offer several advantages and are considered as promising candidates for drug targeting. Despite the valuable potentials of CNTs in drug delivery, their toxicity still remains an important issue. After the PEGylation of single-walled CNTs (SWCNTs) with phospholipid-PEG (Pl-PEG) conjugates to prepare water-dispersible nanostructures, the present study was designed to evaluate whether the functionalization with Pl-PEG derivatives could alter the cytotoxic response of cells in culture, affect their viability and proliferation. In-vitro cytotoxicity screens were performed on cultured Jurkat cells. The SWCNTs samples used in this exposure were pristine SWCNTs, Pl-PEG 2000/5000-SWCNTs at various concentrations. Jurkat cells were first incubated for 3 h at 37°C with test materials and seeded in 6-well culture plates at a given concentration. The plates were then incubated for 24, 48 and 72 h at 37°C in a 5% CO2 humidified incubator. Cell Viability and proliferation assay were performed using trypan blue exclusion test and the cell cycle kinetic status of Jurkat cells was analyzed by flow cytometry. Cell morphology was finally studied using double staining technique and a fluorescence microscope. We found that, regardless of the duration of exposure, functionalized SWCNTs were substantially less toxic, compared to pure SWCNTs and that the molecular weight of Pl-PEGs played an important role at higher concentrations. In conclusion, our noncovalent protocol seemed to be effective for increasing SWCNTs biocompatibility.

Low Dose of IL-12 Stimulates T Cell Response in Cultures of PBMCs Derived from Non Small Cell Lung Cancer Patients

Cancer induces tolerance by suppressing immune function, modulating the T helper activity and causing an imbalance of cytokines produced by T cells. IL-12 is an immune regulatory cytokine with potent anti-tumor activity and its signalling network leads to polarization of na?ve CD4+ T cells into Th1. In pre-clinical studies, administration of recombinant IL-12 by intravenous injection or IL-12 plasmid DNA by intra-tumoral injection showed some anti-tumor effects, measurable immunological responses, but also important dose-dependent side effects. We investigated the ability of low doses of IL-12 to modulate the T cell subpopulations in cultures of PBMCs derived from Non Small Lung Cancer (NSCLC) patients and to induce lysis of lung adenocarcinoma cells by T cells. PBMCs were stimulated with different doses of IL-12 and T cell phenotype was evaluated. IL-12 at 0.01 pg/ml significantly increased the number of CD4 and CD8 T cells, in particular of CD4/IFNγ producing cells. IL-12 did not stimulate T regulatory, but it increased the lysis of lung adenocarcinoma cells induced by T cells. Our results showed that low doses of IL-12 modulates T cell sub-populations in vitro and it increased their lytic activity on adenocarcinoma cells, thus we hypothesize the use of low dose of IL-12 as a therapeutic tool against pathologies characterized by a T cell imbalance, in order to promote an immuno-modulation.

The Fourth Dimension of Biochemical Pathways

The Fourth Dimension of Biochemical Pathways

Quantification of Ionizing Radiation-Induced Cell DeathIn Situin a Vertebrate Embryo

Quantitative studies of radiation cytotoxicity have been performed mostly in cells in culture. For a variety of reasons, however, the response of cells in culture may not reflect the response for cells in situ in a whole organism. We describe here an approach for quantification of radiation-induced cell death in vivo using the transparent embryo of the zebrafish, Danio rerio, as a model vertebrate system. Using this system, we show that the number of TUNEL-positive cells within a defined region increases approximately linearly with radiation dose up to 1 Gy. The results are consistent with predictions of a linear-quadratic model. The use of alternative models, accommodating a response threshold or low-dose hypersensitivity, did not significantly improve the fit to the observed data. Attenuation of the expression of the 80-kDa subunit of Ku, an essential protein for the nonhomologous end-joining pathway of repair, led to a dose reduction of 30- to 34-fold, possibly approaching the limit where each double-strand break causes a lethal hit. In both the Ku80-attenuated and the control embryos, apoptotic cells were distributed uniformly, consistent with a cell-autonomous mechanism of cell death. Together, these results illustrate the potential of the zebrafish for quantitative studies of radiation-induced cell death during embryogenesis and in vivo.

Functionalization density dependence of single-walled carbon nanotubes cytotoxicity in vitro

The cytotoxic response of cells in culture is dependant on the degree of functionalization of the single-walled carbon nanotube (SWNT). After characterizing a set of water-dispersible SWNTs, we performed in vitro cytotoxicity screens on cultured human dermal fibroblasts (HDF). The SWNT samples used in this exposure include SWNT-phenyl-SO 3H and SWNT-phenyl-SO 3Na (six samples with carbon/-phenyl-SO 3X ratios of 18, 41, and 80), SWNT-phenyl-(COOH) 2 (one sample with carbon/-phenyl-(COOH) 2 ratio of 23), and underivatized SWNT stabilized in 1% Pluronic F108. We have found that as the degree of sidewall functionalization increases, the SWNT sample becomes less cytotoxic. Further, sidewall functionalized SWNT samples are substantially less cytotoxic than surfactant stabilized SWNTs. Even though cell death did not exceed 50% for cells dosed with sidewall functionalized SWNTs, optical and atomic force microscopies show direct contact between cellular membranes and water-dispersible SWNTs; i.e. the SWNTs in aqueous suspension precipitate out and selectively deposit on the membrane.

Gallium arsenide exposure impairs processing of particulate antigen by macrophages: Modification of the antigen reverses the functional defect

Gallium arsenide (GaAs), a semiconductor used in the electronics industry, causes systemic immunosuppression in animals. The chemical's impact on macrophages to process the particulate antigen, sheep red blood cells (SRBC), for a T cell response in culture was examined after in vivo exposure of mice. GaAs-exposed splenic macrophages were defective in activating SRBC-primed lymph node T cells that could not be attributed to impaired phagocytosis. Modified forms of SRBC were generated to examine the compromised function of GaAs-exposed macrophages. SRBC were fixed to maintain their particulate nature and subsequently delipidated with detergent. Delipidation of intact SRBC was insufficient to restore normal antigen processing in GaAs-exposed macrophages. However, chemically exposed cells efficiently processed soluble sheep proteins. These findings suggest that the problem may lie in the release of sequestered sheep protein antigens, which then could be effectively cleaved to peptides. Furthermore, opsonization of SRBC with IgG compensated for the macrophage processing defect. The influence of signal transduction and phagocytosis via Fcγ receptors on improved antigen processing could be dissociated. Immobilized anti-Fcγ receptor antibody activated macrophages to secrete a chemokine, but did not enhance processing of unmodified SRBC by GaAs-exposed macrophages. Restoration of normal processing of particulate SRBC by chemically exposed macrophages involved phagocytosis through Fcγ receptors. Hence, initial immune responses may be very sensitive to GaAs exposure, and the chemical's immunosuppression may be averted by opsonized particulate antigens.

Short Chain Fatty Acids and Colon Cancer

The development of intestinal cancer involves complex genetic and epigenetic alterations in the intestinal mucosa. The principal signaling pathway responsible for the initiation of tumor formation, the APC-beta-catenin-TCF4 pathway, regulates both cell proliferation and colonic cell differentiation, but many other intrinsic and extrinsic signals also modulate these cell maturation pathways. The challenge is to understand how signaling and cell maturation are also modulated by nutritional agents. Through gene expression profiling, we have gained insight into the mechanisms by which short chain fatty acids regulate these pathways and the differences in response of gene programs, and of the specific regulation of the c-myc gene, to physiological regulators of intestinal cell maturation, such as butyrate, compared with pharmacological regulators such as the nonsteroidal antiinflammatory drug sulindac. Moreover, we used a combination of gene expression profiling of the response of cells in culture to sulindac and the response of the human mucosa in subjects treated with sulindac for 1 month, coupled with a mouse genetic model approach, to identify the cyclin dependent kinase inhibitor p21(WAF1/Cip1) as an important suppressor of Apc-initiated intestinal tumor formation and a necessary component for tumor inhibition by sulindac. Finally, the mucous barrier, secreted by intestinal goblet cells, is the interface between the luminal contents and the intestinal mucosa. We generated a mouse genetic model with a targeted inactivation of the Muc2 gene that encodes the major intestinal mucin. These mice have no recognizable goblet cells due to the failure of cells to synthesize and store mucin. This leads to perturbations in intestinal crypt architecture, increased cellular proliferation and rates of cell migration, decreased apoptosis and development of adenomas and adenocarcinomas in the small and large intestine and the rectum.

A Dipeptide Metalloendoprotease Substrate Completely Blocks the Response of Cells in Culture to Cholera Toxin

Prior exposure (15 min at 37 degrees C) of several cell types (Vero, SH-SY5Y neuroblastoma, human intestinal epithelial T84) to 3 mm N-benzoyloxycarbonyl-Gly-Phe-amide (Cbz-Gly-Phe-NH(2)), a competitive substrate for metalloendoproteases, completely suppressed cholera toxin (CT)-induced intracellular cAMP accumulation. The specificity of the inhibitory effect was demonstrated by the complete lack of effect of the dipeptide Cbz-Gly-Gly-NH(2), an inactive analogue of Cbz-Gly-Phe-NH(2). The effect was reversible and dose- (IC(50) as low as 0.2 mm depending on the cell type) and time-dependent. Adding Cbz-Gly-Phe-NH(2) during the lag phase caused a diminution of its inhibitory effect similar to that observed with brefeldin A (BFA). Whereas the dipeptide completely suppressed the CT-induced adenylate cyclase (AC) activity, a direct effect on AC is unlikely since the elevation of intracellular cAMP by forskolin was only slightly reduced. The A(1) peptide of CT and NAD(+) activated the AC to the same extent in membranes from control and Cbz-Gly-Phe-NH(2)-treated cells or when Cbz-Gly-Phe-NH(2) was added directly to the assay. The inhibitory effects of suboptimal amounts of Cbz-Gly-Phe-NH(2) and BFA were not additive pointing to a similar mode of action of the two substances. However, Madin-Darby canine kidney cells of which the Golgi structure is BFA-resistant were not resistant to the inhibitory action of Cbz-Gly-Phe-NH(2) on CT cytotoxicity. Several lines of evidence indicate that a perturbation of intracellular Ca(2+) homeostasis by Cbz-Gly-Phe-NH(2) is not responsible for the inhibitory effect of the dipeptide. The dipeptide had also no effect on the binding of (125)I-CT to cells and even increased its intracellular internalization. In contrast with BFA, Cbz-Gly-Phe-NH(2) did not completely suppress the formation of the catalytically active A(1) fragment from bound CT. The data are compatible with a role of metalloendoprotease activity in the intracellular trafficking and processing of CT, although other mechanisms of action of Cbz-Gly-Phe-NH(2) cannot be excluded.

Expression of a Cx43 Deletion Mutant in 3T3 A31 Fibroblasts Prevents PDGF-Induced Inhibition of Cell Communication and Suppresses Cell Growth

Communication through gap junctions was first suggested to have a role in the social control of cell growth over 30 years ago. However, despite extensive experimentation, the importance of gap junctions as a general mechanism of growth control remains to be established. A number of different studies have shown that a common early response of cells in culture to polypeptide growth factors such as PDGF is a rapid and transient inhibition of cell communication suggesting that a cell may have to lose communication with its neighbors before it can undergo cell division. Here we show that 3T3 A31 fibroblasts exposed to PDGF exhibit a 50% decrease in cell communication as measured by dye transfer in the absence of significant changes in the cellular content and distribution of Cx43. Likewise, PDGF inhibited cell communication in cells transfected either with a vector which did not contain a cDNA or with an expression vector encoding full-length Cx43 fused to a c-myc tag (Cx43-M). In contrast, 3T3 A31 fibroblasts transfected with an expression construct encoding a deletion mutant of Cx43 (Cx43-256M) consisting of amino acids 1–256 of Cx43 fused to a c-myc tag maintain high levels of gap junction activity following exposure to PDGF. These results suggest that sites which trigger loss of cell communication in response to PDGF are located within amino acids 257 to 382 of the Cx43 molecule. Cells transfected with an expression vector encoding full-length Cx43 fused to a c-myc tail exhibited a reduced basal growth rate compared to both parent cells and cells transfected with a control vector but maintained a strong mitogenic response to PDGF. In contrast, both the basal growth rate and the mitogenic response to PDGF was markedly reduced in cells which expressed Cx43-256M consistent with the hypothesis that loss of cell communication is required before a cell can respond to mitogenic stimuli.

The response of bovine granulosa cells to different gonadotrophins in culture.

Previous studies with bovine granulosa cells cultured in vitro indicated that follicle-stimulating hormone (FSH) stimulated differentiation and progesterone production of granulosa cells in a dose-dependent manner, this was due mainly to an increase in the number of differentiated cells. The objectives of the present study were to investigate (1) whether the response of bovine granulosa cells in culture to luteinising hormone (LH) and equine chorionic gonadotrophin (eCG) was similar to the response to FSH, and (2) whether granulosa cells derived from different cattle breeds responded similarly to gonadotrophin stimulation. Pairs of ovaries were recovered postmortem from Charolais (38) and Hereford (41) crossbred post-pubertal heifers, and granulosa cells were aspirated from 5-8 mm follicles. In two simultaneous experiments, granulosa cells (2-3 x 10(5) viable cells) were cultured with different gonadotrophins (oFSH or oLH in Experiment 1; oFSH or eCG in Experiment 2). Cell culture was for 4 days at 37 degrees C in a humidified atmosphere of 5% CO2 in air in 1 ml of serum-free culture medium. Progesterone production, total DNA and the protein content of granulosa cells on Day 4 of culture were determined. Log10 data were analyzed by analysis of variance and multiple linear regression. In Experiment 1, both FSH and LH stimulated progesterone production (ng microgram-1 DNA) and protein content (microgram microgram-1 DNA) of granulosa cells in a dose-dependent manner (P < 0.01). The relative potencies of FSH to LH (milli micron/milli micron) were found not to be different from unity. In Experiment 2, progesterone production and the protein content of granulosa cells were stimulated by both FSH and eCG in a dose-dependent manner (P < 0.001). The progesterone response curves (log/log) were linear up to 1-10 milli microns FSH and 1-10 iu eCG, and were Y = 1.67 + 0.093 FSH and Y = 1.60 + 0.091 eCG for progesterone production. Calculated on a milli micron/iu basis, FSH was found to be 5.8 times more potent than eCG (P < 0.05) in terms of stimulating progesterone production. Granulosa cells derived from Hereford crosses were more sensitive (P < 0.001) than those from Charolais crosses to gonadotrophin stimulation (31 and 42 times for FSH and eCG, respectively, in terms of progesterone production, and 4.8 and 3.1 times for FSH and eCG, respectively, in terms of protein content). The response curves for both FSH and eCG were similar within each breed. The slopes of the progesterone response curves, and the protein responses were similar for all the gonadotrophins. In conclusion, these results imply that FSH; LH and eCG have similar effects on the differentiation and progesterone production of bovine granulosa cells from 5-8 mm follicles cultured in vitro. Furthermore, granulosa cells from different breeds cultured in vitro had different sensitivities to gonadotrophin stimulation.

Cell culture from rat renal glomeruli

The primary culture of rat renal glomeruli was found to result in the ready outgrowth of two cells types. One type designated c-cells were cytokeratin positive and exhibited microvilli and cilia. The second type designated f-cells were vimentin positive and showed rugose surfaces. C-cells were polygonal in culture on plastic surfaces and were derived from cells of parietal epithelial origin. F-cells assumed a more extended form on plastic and were judged to be a sub-set of parietal epithelial cells. Neither cell type was derived from the visceral epithelium which was found to have been destroyed during isolation of the glomeruli. When cultured on isolated glomerular basement membrane both the c-cells and f-cells assumed a polygonal morphology but when grown on Matrigel the cells assumed the form of long strands interconnecting the outgrowths between the glomeruli. The appearance of the cells in the strands, judged from scanning electron microscopy, suggested that these were formed from f-cells but other cell types were entrained in the structures. Glomeruli subjected to vigorous proteinase digestion of the basement membrane allowed culture of a wider variety of cells. These included endothelial cells, judged by OX-43 antibody and anti-von Willebrand Factor staining, and mesangial cells. In cultures from glomeruli polygonal cells are often assumed to be visceral epithelial cells, the results from this study indicate that this assumption is unsound. The very different behaviour of cells grown on isolated basement membrane as compared with cells grown on Matrigel suggests that Matrigel may not faithfully mimic basement membrane with respect to cell response in culture.

Morphological and immunohistochemical differentiation patterns of rabbit uterine epithelium in vitro

We describe morphological and immunohistochemical changes of uterine epithelium from immature rabbits in vitro in response to hormonal treatments, using a matrix-coated semipermeable filter. These investigations were compared to in vivo studies of uterine epithelium from immature rabbits treated with estrogen and/or progesterone. In vitro, polarization of the epithelium seems to be best developed under progesterone dominance, and the pattern of cell organelles is similar to those seen in vivo. Two types of apical protrusions could be observed in cultures treated with progesterone, some shaped like domes, containing cell organelles, and some irregular in shape with small lucent vesicles. Both types of apical differentiation are typical for the in vivo situation. In vitro, estrogen leads to a more pseudostratified growth pattern of the cells. They develop apical protrusions with big vesicles probably containing mucin, as in vivo. Treatment with both steroid hormones leads to a heterogeneous response of the uterine epithelial cells in culture, some cells responding more to the estrogen, others to the progesterone whereas in vivo the progesterone-dominant features are obvious. Immunohistochemistry of uteroglobin in monensin-treated cultures gives evidence for uteroglobin secretion in all cultures, but to a lesser extent in the untreated, and this is strongly increased in cultures treated with estrogen and progesterone. These results correspond to observations made in vivo. This in vitro cell culture method seems therefore to provide a useful model for investigating the regulatory mechanisms of sexual steroid hormones and the cell biology of uterine receptivity.

Effects of hyperthermia on intracellular calcium concentration and responses of cancerous mammary cells in culture.

Effects of hyperthermia on the intracellular calcium concentration (Cai) of an established mouse breast cancer cell line, MMT060562, were studied using fura-2 fluorescence microscopy and the whole-cell clamp technique. A sudden change of temperature from 37 to 45 degrees C induced a transient increase in the fluorescence ratio permeability of the cell membrane and inward current. Deletion of extracellular calcium abolished the fluorescence ratio response to the rise in temperature. Cai of some cells increased after hyperthermia treatment at 44-48 degrees C for 20 min, but the average increase of Cai was negligible. After hyperthermia treatment, spontaneous oscillation of Cai, chemical responses to ATP and bradykinin and the mechanically-induced spreading response diminished. However, the mechanically induced increase of Cai within the stimulated cell remained even after hyperthermia treatment. Suppression of the ATP-induced Cai response recovered to about half the original level within 12 h. Blockage of protein synthesis with cycloheximide (100 microM) had no effect on the recovery. The D-myo-inositol 1,4,5-triphosphate (IP3)-dependent increase of Cai remained intact even after hyperthermia treatment. It is concluded that hyperthermia treatment increases both the permeability of the cell membrane and Cai, but decreases the sensitivity of cells to ATP and bradykinin, presumably due to modification of the signal transduction mechanism.

T cell regulation of human B cell proliferation and differentiation. Regulatory influences of CD45R+ and CD45R- T4 cell subsets.

The immunoregulatory functions of human T4 cell subpopulations defined by mAb to the CD45R molecule (2H4) were examined. Both CD45R- and CD45R+ T4 cells that had been treated with mitomycin C (CD45R- and CD45R+ T4-mito) provided help for the generation of Ig-secreting cells (ISC) in cultures stimulated by PWM or by immobilized mAb to CD3 (64.1). IL-2 enhanced the generation of ISC in PWM-stimulated cultures and in anti-CD3-stimulated cultures containing CD45R+ T4-mito. The generation of ISC was maximal in cultures containing anti-CD3-activated CD45R- T4-mito and was not increased by IL-2. By contrast, CD45R+ T4 cells that had not been treated with mitomycin C suppressed B cell responses in cultures stimulated with PWM or anti-CD3, whereas CD45R- T4 cells suppressed the generation of ISC only in cultures stimulated with anti-CD3. IL-2 enhanced suppression by anti-CD3, but not PWM, activated CD45R- T4 cells. Suppression by CD45R+ T4 cells was maximal and not increased by IL-2. CD45R+ T4-mito were more effective suppressor-inducers in PWM-stimulated cultures, promoting the differentiation of suppressor-effector cells from CD8+ T cells. However, both CD45R+ and CD45R- T4-mito exerted comparable suppressor-inducer function in anti-CD3-stimulated cultures. Moreover, in anti-CD3-stimulated cultures, T8 cells could function as both suppressor-effector cells and suppressor-inducer cells. One of the functions of suppressor-inducer cells in this system appeared to involve the production of IL-2. Thus, the addition of IL-2 facilitated the induction of suppressor-effector T8 cells by CD45R- T4-mito in PWM-stimulated cultures. Although IL-2 production by the T cell subsets varied widely depending on the nature of the stimulus, these differences could not entirely explain their capacity to function as helper cells, suppressor-effector cells or suppressor-inducer cells. These results indicate that both CD45R+ and CD45R- T4 cells can help or suppress B cell responses, as well as induce suppressor-effector T8 cells. Moreover, suppressor-inducer function of T cells is not limited to the T4 cell population, but rather can also be accomplished by T8 cells. The results indicate that both T4 cell subsets and T8 cells exert multiple regulatory effects on human B cell function, with the nature of the activating stimulus playing a major role in determining the functional capacity of various T cell subsets.

Mitogenic response of cells in culture to demineralized bone matrix

In this study the mitogenic effect of demineralized bone powder (DBP) on cells in tissue culture was evaluated. The cells studied have osteoblastic properties. At a DBP concentration of 100 μg/well, rat osteosarcoma cells (ROS) 17 2.8 cells, ROS 24 1 cells, and periosteal fibroblasts (POF) all showed a significant increase in proliferation in comparison to controls. The peak effect was on day two for ROS 17 2.8 (125% of control), on day three for ROS 24 1 (139% of control), and on day four for POF (145% of control). The results indicate that this in vitro system may be useful for evaluating the osteoinductive activity of DBP preparations.

Discussion: Mitogenic Response of Cells in Culture to Demineralized Bone Matrix

Discussion: Mitogenic Response of Cells in Culture to Demineralized Bone Matrix

In vitro response of leaf tissues from Lolium multiflorum ? a comparison with leaf segment position, leaf age and in vivo mitotic activity

Immature gramineous leaves provide a convenient system for comparing the response of cells in culture with their state of differentiation. Callusing frequency is compared with leaf segment position, leaf age and in vivo mitotic activity in Lolium multiflorum. (1) In a succession of one millimeter sections from the immature leaf base, callus was formed from the first and second sections but not the third or subsequent sections. The frequency of those explants callusing decreased with distance from the base of the leaf and with leaf age (or leaf extension growth). (2) In vivo, the proportion of cells in mitosis declined from around 10-14% at the base of young leaves to zero at 5 mm from the base and beyond. Mitotic activity also declined in leaves as they aged, and dividing cells were not observed in leaves 30 days from initiation or older. (3) A high frequency of callus formation was associated with a high mitotic index in the explant. But for corresponding mitotic indices, cells further away from the leaf base were less responsive in culture. (4) It is proposed that cells are becoming differentiated even in highly meristematically active regions of the leaf and concomitantly losing their ability to respond in culture.

The Clonogenic Response of Bovine Aortic Endothelial Cells in Culture to Radiation

The effect of ionizing radiation on the survival of bovine aortic endothelial (BAE) cells was determined by the in vitro colony formation method. The BAE cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum, antibiotics, and growth factors obtained from the culture of mouse S-180 cells. The cultured BAE cells were positive to the staining of antibodies against human factor VIII and formed clones in plastic culture flasks with a plating efficiency of about 11%. The survival curve of the BAE cells following an exposure to a single dose of X rays was characterized by D0 = 101 rad, Dq = 65 rad, and an extrapolation number (n) of 1.9. These parameters were not modified by the absence of growth factors at the time of irradiation. The response of BAE cells to radiation was dose-rate dependent. The split-dose studies demonstrated that the BAE cells were able to repair sublethal radiation damage within 1 h after irradiation.

Differential effect of interferon-γ and interleukin-2 on the induction of IgA and IgM anti-dextran responses

Supernatants from T-cell lines and T-cell hybridomas can substitute for T cells in the induction of the anti-α(1,3) Dextran B1355 plaque-forming cell response in culture. The present study sought to define the lymphokines required for the induction of IgA and IgM anti-α(1,3) dextran responses. Recombinant Interferon-γ (IFN-γ) supported the induction of low levels of IgA anti-α(1,3) dextran plaque-forming cells in splenic B-cell cultures. IgA responses were substantially increased when cultures containing IFN-γ were supplemented with an interleukin 2 (IL-2)-containing supernatant from the murine T-cell hybridoma BW.Mls, purified murine IL-2, or recombinant human IL-2. In striking contrast, IgM anti-α(1,3) dextran plaque-forming cells were not produced in cultures containing IFN-γ alone or in combination with purified or recombinant IL-2. However, substantial IgM responses could be produced in cultures containing IFN-γ and BW.Mls supernatant. This data indicates that there may be different lymphokine requirements for the induction of IgA and IgM anti-α(1,3) dextran B cells, or alternatively, such B cells may be in different stages of differentiation and therefore, not respond to the same lymphokines.