Lysozyme as a regulator of interleukin-2-activated lymphocyte proliferation

Summary Lysozyme at 1 to 100μg/ml of exposure levels augmented or inhibited proliferative response of human peripheral blood lymphocytes stimulated with interleukin-2 (IL-2). This contradictory effect of lysozyme depended on IL-2 concentration, activating state of lymphocytes, addition time of lysozyme, and serum existence. Lymphocytes increased their IL-2-mediated proliferating ability in response to lysozyme when stimulated with less than suboptimal concentration of IL-2. Lymphocyte activation with anti-CD3 antibody changed the augmented proliferative response into the inhibited response by lysozyme addition whereas elimination of MHC class II molecule-expressing cells augmented the response. Addition of lysozyme within 1 h after IL-2 exposure was most effective in promoting the proliferation whereas additions after 16 to 24 h were ineffective or inhibitory. Addition after longer than 24 h inversely restored the proliferative response. Serum seemed to retard lysozyme action because either sequential serum addition 1 h after exposure of IL-2 and lysozyme to cells or exposure of IL-2 and serum after pretreatment of cells with lysozyme changed the proliferative responsiveness from inhibition into augmentation. Thus lysozyme may regulate lymphocyte proliferation responding to a magnitude of antigenic stimuli and to the progression of cellular events that periodically occur.     

豆腐柴根提取物对小鼠T淋巴细胞增殖反应的影响

利用3H-TdR放射性来测定T淋巴细胞增殖强度的方法,研究了豆腐柴根提取物对小鼠T淋巴细胞增殖反应的影响.结果表明,豆腐柴根提取物在一定浓度范围内可促进刀豆蛋白(ConA)诱导T淋巴细胞发生增殖反应,其中2μg.mL-1提取物浓度效果最为明显.该研究为合理开发利用豆腐柴这一野生药物资源提供了科学的实验依据.     

Obtaining cell proliferation for chromosome preparation in gill tissue culture of the oyster Crassostrea gigas

The present results were obtained in the course of theadjustment to the oyster Crassostrea gigas of atissue culture technique recently developed for themussel Mytilus edulis. With respect to theprotocol originally described, the effects of twomodifications are reported: (1) replacement of chickembryo extract by chicken serum for medium enrichment,and (2) achievement of cultures in rotating tubes(roller drum) in place of stationary condition.Paradoxical results were obtained: whereas takenseparately, each modification exerted a negativeeffect which is statistically significant, combinated,they exerted a high positive effect representing athree-fold increase of the mean metaphase spreadnumber per slide (i.e. 71.5). Hypotheses are proposedto explain the mechanisms involved. It is suggestedthat the two additives work differently and thatcultures with chick embryo extract enriched mediumcould not withstand the condition generated by theroller drum. Conversely, cultures performed withchicken serum enriched medium would be in a betterphysiological state and the roller allow to obtain acell proliferation after only six days of incubation.     

Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European ‘Biorack’ provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the ‘Biorack’ facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatent in-flight), injection port, and supernatent collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatent, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground- based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities. J. Cell. Biochem. 70:252–267, 1998. © 1998 Wiley-Liss, Inc.     

Regulation of cell proliferation in a stratified culture system of epithelial cells from prostate tissue

Mechanisms controlling epithelial proliferation and differentiation in the prostate have been primarily investigated in mouse models. The regulation of proliferation and differentiation is poorly understood in human prostate epithelial cells. In vivo, the glandular prostate epithelium consists of a p63-positive proliferating basal cell layer and a post-mitotic p27-positive secretory cell layer. We have established an organized stratified culture system of human primary prostate epithelial cells to gain insight into mechanisms regulating proliferation and differentiation. In this system, expression of p63 is observed in the bottom layer. In addition, BrdU incorporation persists even though cells are confluent. In contrast, in the upper layer, p63 expression is greatly diminished, p27 is expressed, and the cells are growth arrested. Overexpression of cyclin D1 or knockdown of p27 does not increase proliferation. After inactivation of the nuclear phosphoprotein Rb, the cell layers remain organized and cell proliferation increases only in the bottom layer. Furthermore, the expression of p63 remains confined to the bottom layer after Rb inactivation. Altogether, this in vitro model recapitulates certain aspects of in vivo growth regulation and differentiation and suggests that the loss of Rb family proteins in human cells trigger hyperplasia but is not sufficient for transformation.This work was supported by the Departments of Pathology and Urology at Weill Medial College, by grants DAMD-17-02-1-0159, MEDC-GR-355, and P30 CA015704-30, and by grant RO1CA84069 to B.E.C.     

Cell culture density affects the proliferation activity of human adipose tissue stem cells

In this study, we investigated the effect of cell density on the proliferation activity of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT‐MSCs) over time in culture. Passage #4 (P4) and #12 (P12) AT‐MSCs from two donors were plated at a density of 200 (culture condition 1, CC1) or 5000 (culture condition 2, CC2) cells cm^?2. After 7 days of incubation, P4 and P12 AT‐MSCs cultured in CC1 were thin and spindle‐shaped, whereas those cultured in CC2 had extensive cell‐to‐cell contacts and an expanded cell volume. In addition, P4 and P12 AT‐MSCs in CC1 divided more than three times, while those in CC2 divided less than once on average. Flow cytometric analysis using 5(6)‐carboxyfluorescein diacetate N‐succinimidyl ester dye showed that the fluorescence intensity of AT‐MSCs was lower in CC1 than in CC2. Furthermore, expression of proliferation‐associated genes, such as CDC45L, CDC20A and KIF20A, in P4 AT‐MSCs was higher in CC1 than in CC2, and this difference was also observed in P12 AT‐MSCs. These data demonstrated that cell culture density affects the proliferation activity of MSCs, suggesting that it is feasible to design a strategy to prepare suitable MSCs using specific culture conditions. Copyright © 2016 John Wiley & Sons, Ltd.     

Suppression of lymphocyte proliferation by a nonspecific factor produced by the burkitt lymphoma-derived lymphoblast cell line

Summary The DAUDI lymphoblast cell line derived from a patient with Burkitt lymphoma was obtained from two different sources. One of these (DAUDI-I) produced a factor that inhibited lymphocyte proliferation in both human and mouse regardless of the stimulator, i.e. allogeneic lymphocytes or mitogens. Glutaraldehyde treatment eliminated production of the factor and demonstrated that DAUDI-I was capable of stimulating normal lymphocytes in MLR. A second DAUDI cell line (DAUDI-S) did not produce the inhibitory factor and was capable of MLR stimulation. Supported by the Children's Leukemia Foundation of Michigan, NIH Grants AI 11013 and AI 11335, and the Kidney Foundation of Michigan.     

GTSF-2: A new, versatile cell culture medium for diverse normal and transformed mammalian cells

Summary The aim of this study was to test the versatility of a new basal cell culture medium, GTSF-2. In addition to traditional growth-factors, GTSF-2 contains a blend of three sugars (glucose, galactose, and fructose) at their physiological levels. For these studies, we isolated normal endothelial cells from human, bovine, and rat large blood vessels and microvessels. In addition, GTSF-2 was also tested as a replacement for high-glucose-containing medium for PC12 pheochromocytoma cells and for other, transformed cell lines. The cell growth characteristics were assessed with a novel cell viability and proliferation assay, which is based on the bioreduction of the fluorescent dye, Alamar Blue. After appropriate calibration, the Alamar Blue assay was found to be equivalent to established cell proliferation assays. Alamar Blue offers the advantage that cell proliferation can be measured in the same wells over an extended period of time. For some of the cell types (e.g., endothelial cells isolated from the bovine aorta, the rat adrenal medulla, or the transformed cells), proliferation in unmodified GTSF-2 was equivalent to that in the original culture media. For others cell types (e.g., human umbilical vein endothelial cells and PC12 cells), GTSF-2 proved to be a superior growth medium, when supplemented with simple additives, such as endothelial cell growth supplement (bFGF) or horse serum. Our results suggest that GTSF-2 is a versatile basal medium that will be useful for studying organ-specific differentiation in heterotypic coculture studies.     

小鼠精原干细胞在三种培养基中的生长行为

目的：建立小鼠精原干细胞（SSCs）的体外长期培养体系。方法：用分别添加了等量的胶质细胞源神经营养因子（GDNF）、可溶性GFRα1和hFGF的DMEM／F12、KSR和StemPro-34 SFM三种无血清培养基和MEF饲养层分别培养经差异贴壁分选富集的小鼠SSCs,通过形态观察、标志基因的RT-PCR和免疫细胞化学分析检测其SSCs本原。结果：DMEM／F12与KSR可支持小鼠SSCs在体外存活6-7d,而StemPro-34 SFM能能维持SSCs体外增值一个月。结论：StemPro-34 SFM支持小鼠SSCs的体外增殖。     

Explants of porcine coronary artery in culture: A paradigm for studying the influence of heparin on vascular wall cell proliferation

Explant cultures of porcine coronary artery provided a coculture model, used as a paradigm of arterial wall in contact with vascular prosthesis which allowed the study of spatial and temporal changes in cell phenotype. First cells emerging from the explant had an endothelial phenotype monitored by cytoimmunostaining. Percentages of anti-smooth muscle α-actin labelled cells were assessed at early and late phase by flow cytofluorometric analysis to control the effect of heparin. At 100 μg ml^-1, no effect on α-actin labelled cell growth has been detected. This result contrasted with the inhibition of monolayer cell cultures. At 500 μg ml^-1, the proliferation of smooth muscle cells was reduced. This explant system should be useful for testing drugs susceptible to interfere with restenosis. This revised version was published online in August 2006 with corrections to the Cover Date.     

A tissue model for the study of cell proliferation in vitro

Summary A procedure for the cultivation of mesentery is described, in which the culture is fully representative of the tissue of origin. The intact mesenteric membrane—exposed to a minimum of trauma—was spread out over a hole in a filter paper strip in fluid medium and was cultivated free-hanging. Specimens from rats and guinea pigs were used. The organ culture model appears especially apt for cytochemical and proliferation studies. Proliferation variables based on Feulgen DNA analysis in individual, morphologically defined cells and on mitotic counting and radiochemical analysis were estimated. The tissue was fully viable in chemically defined growth medium and showed an almost unaltered light microscopical appearance after up to 52 hr in culture. Supported by the Swedish Cancer Society.     

The murine Kupffer cell. II. Accessory cell function in in vitro primary antibody responses, mitogen-induced proliferation, and stimulation of mixed lymphocyte responses

The ability of murine Kupffer cells to function in several in vitro immunologic systems was investigated. These cells have been shown previously to function as accessory cells in antigen-stimulated T cell proliferation in response to protein antigens. In the present study it has been demonstrated that murine Kupffer cells also are competent as accessory cells in in vitro primary antibody responses to TNP-KLH and for T cell proliferative responses to concanavalin A. In addition, murine Kupffer cells were found to be potent stimulators of mixed lymphocyte responses. These studies extend previous observations by demonstrating that Kupffer cells are competent accessory cells in several distinct in vitro correlates of in vivo immune responses. The role of Kupffer cells in in vivo immune responses, particularly those to enterically derived antigens, may require re-evaluation in the light of these findings.     

Factors controlling embryonic heart cell proliferation in serum-free synthetic media

Summary Embryonic chick cardiac cell cultures, plated on collagen-coated dishes, containing serum-free synthetic media proliferate actively. The basic medium contained Ham's F12 nutrient mixture, fetuin, ascorbic acid, and bovine serum albumin. This medium was supplemented with various combinations of factors; endothelial cell growth supplement (ECGS), epidermal growth factor (EGF), insulin (I), transferrin (T), selenium (S), hydrocortisone, and thyroxine or supplemented alone. Basic medium supplemented with ECGS alone contributes to the highest final cell density among all other factors used in various combinations or alone. The final cell density of the control culture with 2% fetal bovine serum was higher than those of all experimental cultures and an additional control culture grown in the basic medium. Combinations of factors without ECGS do not promote significant cell proliferation. Thyroxine is required to induce optimal differentiation and contractility of cardiac myocytes in vitro. Fibronectin and laminin did not show any more influence than collagen did on the growth and maintenance of cardiac myocytes in serum-free media. The proportion of cardiac muscle cells in ECGS-containing media was higher than those in other experimental media and control media with the exception of ECGS and ITS-containing medium that showed lower proportion of cardiac myocytes than that of serum-containing medium on Days 3 and 5. The profiles of incorporation of [³H]thymidine into DNA of heart cells in experimental and control cultures showed a peak in incorporation values within the first week of culture and subsequently declined. Autoradiography studies revealed that cardiac myocytes in culture supplemented with ECGS alone attained a peak in labeling index on Day 1 with approximately 62% labeled cells. Subsequently, the labeling indices declined. Cardiac myocytes grown in media without ECGS showed significantly lower labeling indices than those in ECGS-containing media. This study has demonstrated the influence of ECGS, EGF and ITS in promoting the growth of cardiac myocytes and also in contributing to the maintenance of contractile cardiac myocytes in serum-free, long-term culture. The influence of ECGS on heart cell proliferation is considered to be superior to that of EGF and ITS. This study was supported in part by a grant HL-25482 from the National Heart Lung and Blood Institute and a grant from the American Heart Association of Michigan.     

Development of improved media and culture conditions for clonal growth of normal diploid cells

Summary Multiplication of normal diploid cells in culture is controlled by a complex set of interacting extracellular variables. The amount of serum protein needed for colony formation by such cells is affected directly by many of the other variables, including the nature of the culture surface, the type of trypsinization procedure used, and the qualitative and quantitative composition of the culture medium. By a sequential process of adjusting all of these variables to optimum values for cellular multiplication with minimal amounts of serum protein, we have been able to obtain clonal growth of normal human and chicken cells with less than 500 μg per ml dialyzed serum protein. Precise quantitative adjustment of nutrient concentrations is particularly important. The multiplication-promoting functions of serum can be classified operationally as “replaceable” (those that can be replaced by modifying the medium or the culture conditions) and “nonreplaceable” (those that we have not yet been able to replace). Elimination of the requirement for replaceable functions of serum has improved greatly the specificity and sensitivity of the bioassay for the nonreplaceable functions. The nonreplaceable multiplication-promoting activity from fetal bovine serum for human diploid fibroblasts has been separated from fetuin and serum albumin and purified approximately 15-fold. Presented in the Opening Symposium on Nutritional Factors and Differentiation at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. This work was supported by Contract 223-74-1156 from the Bureau of Biologics, U.S. Food and Drug Adminsstration, Grant AG 00310 from the National Institute on Aging, and Grant CA 15305 from the National Cancer Institute.     

Complement-dependent induction of DNA synthesis and cell proliferation in human liver connective tissue cells in vitro

Summary Liver connective tissue cells (LCTC) isolated from patients with fibrotic livers have morphological and biochemical characteristics of myofibroblasts. We have examined the proliferation of LCTC derived from normal livers and from livers with fibrosis of different etiologies, as well as proliferation of skin fibroblasts. We have compared proliferation rates in the presence of fresh human serum and heat-inactivated serum. While skin fibroblast and LCTC from normal liver showed no difference, proliferation of LCTC from fibrotic livers was markedly decreased in the presence of heat-inactivated serum. We demonstrate that the native complement component C1 is a factor involved in the induction of DNA synthesis and proliferation of LCTC isolated from fibrotic livers. We propose that native C1, acting probably in cooperation with other growth factors, is involved in the expansion of connective tissue cells during the development of liver fibrosis.     

Paracrine control of tissue regeneration and cell proliferation by Caspase-3

Executioner caspases such as Caspase-3 and Caspase-7 have long been recognised as the key proteases involved in cell demolition during apoptosis. Caspase activation also modulates signal transduction inside cells, through activation or inactivation of kinases, phosphatases and other signalling molecules. Interestingly, a series of recent studies have demonstrated that caspase activation may also influence signal transduction and gene expression changes in neighbouring cells that themselves did not activate caspases. This review describes the physiological relevance of paracrine Caspase-3 signalling for developmental processes, tissue homeostasis and tissue regeneration, and discusses the role of soluble factors and microparticles in mediating these paracrine activities. While non-cell autonomous control of tissue regeneration by Caspase-3 may represent an important process for maintaining tissue homeostasis, it may limit the efficiency of current cancer therapy by promoting cell proliferation in those cancer cells resistant to radio- or chemotherapy. We discuss recent evidence in support of such a role for Caspase-3, and discuss its therapeutic implication.     

Cytokine production and lymphocyte proliferation in patients with Nocardia brasiliensis actinomycetoma

IFN-γ, TNF-α, IL-4, IL10 and IL-12 concentrations in the supernatant of peripheral blood mononuclear cell (PBMC) cultures and the in vitro proliferation of PBMC were studied in 25 patients with actinomycetoma caused by Nocardia brasiliensisand in 10 healthy controls from endemic zones. Cell cultures were stimulated by a N. brasiliensiscrude cytoplasmic antigen (NB) and five semi-purified protein fractions (NB2, NB4, NB6, NB8, and NB10) separated by isoelectric. Phytohemagglutinin (PHA) and purified protein derivative (PPD) of Mycobacterium tuberculosiswere used as control antigens. Skin tests were performed by injecting 0.1 ml of candidin and PPD intradermally (ID). Patients showed a poor response to tuberculin, while their response to candidin was more than two fold greater than that observed in the controls. Cell proliferation showed no statistically significant differences in either group. IFN-γ production was higher in the healthy controls than in the patients, whereas TNF-α secretion was slightly higher in the patients’ cultures. IL-4 was detected in the patients’ cultures but not in the controls. IL-10 and IL-12 were present at low concentrations in both groups. These results suggest that patients with actinomycetoma show normal antigen recognition, but with low IFN-γ production, and higher concentrations of IL-4, IL-10 and TNF-α in the patients’ PBMC cultures, indicating that they probably have a Th2 type of immune response.     

Epidermal cell proliferation and terminal differentiation in skin organ culture after topical exposure to sodium dodecyl sulphate

Summary Epidermal cell proliferation and differentiation were investigated in vitro after exposure to the anionic surfactant sodium dodecyl sulfate (SDS). Human skin organ cultures were exposed topically to various concentrations of SDS for 22 h, after which the irritant was removed. Cell proliferation was measured immunohistochemically by incorporation of bromodeoxyuridine (BrdU) into the DNA of cells during S-phase, while the expression of transglutaminase and involucrin were used as markers of differentiation. Cell proliferation was moderately increased at concentrations of SDS that did not affect the histomorphology (0.1% and 0.2% SDS). A marked increase of cell proliferation was observed 22 to 44 h after removal of SDS at a concentration (0.4%) that induced slight cellular damage. Exposure of human skin organ cultures to a toxic concentration of SDS (1.0%) led to decreased cell proliferation. Transglutaminase and involucrin were expressed in the more basal layers of the epidermis after exposure to 0.4% or 1.0% SDS. Moreover, intra-epidermal sweat gland ducts were positive for transglutaminase at these irritant concentrations. These in vitro data demonstrate that SDS-induced alterations of epidermal cell kinetics, as described in vivo are at least partly due to local mechanisms and do not require the influx of infiltrate cells. However, we were unable to relate the altered cell kinetics to the release of interleukin-1α or interleukin-6. Furthermore, supplementation of the culture medium with 12-hydroxyeicosantetraenoic acid did not affect epidermal cell proliferation. Rabbit skin cultures appeared more sensitive to SDS than human skin. At nontoxic doses, the irritant induced an increase of epidermal cell proliferation, similar to that observed in human skin discs.     

Novel cell culture device enabling three-dimensional cell growth and improved cell function

A better understanding of cell biology and cell-cell interactions requires three-dimensional (3-D) culture systems that more closely represent the natural structure and function of tissues in vivo. Here, we present a novel device that provides an environment for routine 3-D cell growth in vitro. We have developed a thin membrane of polystyrene scaffold with a well defined and uniform porous architecture and have adapted this material for cell culture applications. We have exemplified the application of this technology by growing HepG2 liver cells on 2- and 3-D substrates. The performance of HepG2 cells grown on scaffolds was significantly enhanced compared to functional activity of cells grown on 2-D plastic. The incorporation of thin membranes of porous polystyrene to create a novel device has been successfully demonstrated as a new 3-D cell growth technology for routine use in cell culture.