An improved method for elimination of mycoplasmas from cell cultures

Cell lines infected by different species of mycoplasma (Mycoplasma orale, Mycoplasma hominis) were decontaminated by co-culture with human blood monocyte (BM)-derived macrophages and pooled human immunoglobulin preparations. Co-cultures with BM-derived macrophages or murine peritoneal macrophages (PM) alone were not successful. The phenotype of infected cell lines did not differ from that of uninfected cell lines as revealed by morphological, enzymecytochemical, and immunocytochemical analysis.     

Use of polyethylene glycol-treated calf serum for cell cultures in virus and interferon studies.

Immunoglobulins and lipoproteins can be efficiently removed from calf serum by precipitation with polyethylene glycol (PEG) 6000 under sterile conditions. The PEG-treated serum is suitable for cell cultures used for virus growth and assays. Moreover, PEG was found to slow down the growth of L cells and to enhance the production and activity of mouse interferon.     

Effect of tuberculin on mitotic activity of cell cultures

Bulletin of Experimental Biology and Medicine -     

Antiviral activity of benzothiazole and benzothiazolinethione derivatives in cell cultures.

The virus inhibitory activity of benzothiazole, benzothiazolinethione and naphthothiazole derivatives was tested with vaccinia virus, Newcastle disease virus (NDV) and western equine encephalomyelitis (WEE) virus in the agar-diffusion plaque-inhibition test. Among 58 compounds examined, 5 showed medium activity and selectivity with vaccinia virus. The highest selective effect was found with 3-(2-ethylthio-6-benzothiazolylaminomethyl)-2-benzothiazolinethione. A much lower inhibitory effect was observed with several derivatives against WEE virus. One derivative (2-mercaptobenzothiazole) showed a low inhibitory effect against NDV.     

Interferon-gamma is an autocrine mediator for dendritic cell maturation

Maturation of dendritic cells (DC) is critical for efficient antigen presentation and initiation of an immune response. Interferon-gamma (IFN-gamma) is an important Th1 cytokine. In this study, we investigated the role of IFN-gamma in DC maturation using either IFN-gamma receptor deficient- or IFN-gamma overexpression-models. We showed that immature DC generated in vitro from bone marrow (BM) progenitor cells produced low level of IFN-gamma. After LPS stimulation, DC produced more IFN-gamma, and IFN-gamma productions were at comparable levels among C57BL/6 mice-derived DC (C57BL/6 DC), wild-type 129 mice-derived DC (129 DC) and IFN-gamma receptor deficient 129 mice-derived DC (IFN-gammaR-/-DC). We found that IFN-gammaR-/-DC exhibited decreased expression of CD54, CD86, reduced capacity to secrete IL-1beta and IL-12p70, and impaired capacity to stimulate alloreactive T cells and to drive Th1 differentiation. Transfection of IFN-gamma gene into DC promoted DC to express higher CD40, CD54, CD80, CD86, CCR7 and I-Ab, secrete more IL-1beta and IL-12p70, and more potently activate both CD4 and CD8 T cells. These data suggest that IFN-gamma signaling pathway is important for the maturation of DC in an autocrine fashion.     

Comparison of fortified calf serum, serum substitutes and fetal calf serum with or without extenders for propagation of cell cultures for virus plaque assays

Two studies comparing sewage-isolated and laboratory stock viruses were conducted to determine if alternative forms of serum or serum extenders could be used in place of fetal bovine serum without a significant loss of viral titer. In the first study, BGM cells were grown in standard MEM-L15 medium which was supplemented with Nuserum, Sigma serum replacement (CPSR-1), HyClone defined iron supplemented calf bovine serum, fetal bovine serum (FBS) or FBS supplemented with either SerXtend or Mito serum extenders. Comparison of virus titers showed that CPSR-1 gave the best overall results and was comparable to FBS. Of the serum extenders, only SerXtend improved virus recovery from sewage samples. In the second study, all sera were tested with and without SerXtend. In these experiments, SerXtend enhanced virus sensitivity of the BGM cell line grown in the HyClone serum but reduced the sensitivity of those cultured in Sigma serum. In both series, the growth of BGM cells was monitored for 12 weeks and all test products were shown to support long-term cell growth.     

Histamine-releasing activity of lymphocyte supernatants of guinea pig spleen cell cultures

We demonstrated the production of a histamine releasing factor (HRF) by 24-h cultures of guinea pig spleen cells which were stimulated or not with specific antigen (ovalbumin, OA) or mitogen (phytohemagglutinins or concanavalin A). HRF induced the release of histamine from homologous mesenteric mast cells in a dose-dependent fashion. The HRF-induced histamine release was not high compared to the release induced by calcium ionophore A23187, but higher than that induced by compound 48/80, polymyxin B and con canavalin A. The mast cells from sensitized guinea pigs released histamine when challenged with OA. We found that HRF-induced histamine release was additive to that induced by antigen, when both agents were added simultaneously to sensitized mast cells. The phenomenon was most significant when a suboptimal dose of antigen was used. Moreover, we did not observe any differences in the magnitude of HRF-induced histamine release between the mast cells from nonsensitized and sensitized guinea pigs. The time course of histamine release induced by HRF was significantly slower than that with specific antigen (10 min and 45 sec, respectively). Our results may suggest that HRF acts on mast cells through a different not immunological mechanism.     

TGF-betas synthesized by RPE cells have autocrine activity on mesenchymal transformation and cell proliferation.

The present study investigated the effects of transforming growth factor (TGF)-beta on retinal pigment epithelial (RPE) transformation in a simplified model and also whether or not TGF-beta exhibits similar proliferation effects on transformed RPE cells that it has on primary RPE cells. Furthermore, we examined the cell proliferation effects of RPE-conditioned medium (CM). A vertical wound measuring 2 mm in diameter was made on primary RPE monolayers. The expression of alpha-smooth muscle actin (SMA) by the cells located at the wound edges was observed using a confocal microscope under immunofluorescent staining. Cell proliferation was measured by incorporating 3H-thymidine into DNA. The presence of alpha-SMA was observed in the cells within the wound after treatment with TGF-beta2, while negative expression was observed in control cells. TGF-betas inhibited the proliferation of the primary cultures of RPE cells in a dose-dependent manner, but the spindle-shaped late-passaged RPE cells were not inhibited by these growth factors. The medium conditioned by RPE cells stimulated the proliferation of subconjunctival fibroblasts and inhibited the proliferation of primary RPE cells, in a manner similar to TGF-beta. These findings demonstrate that TGF-beta-stimulated RPE cells may evoke proliferative vitreoretinopathy through mesenchymal transformation and cell proliferation.     

Induction of DNase activity in monkey cell cultures infected by simian virus 40

Summary DNase activity following SV40 infection of CV1 cells has been followed. An increase in this activity late in infection is observed.     

Possible autocrine regulation of chromaffin cell activity in adrenal glands of the frog by endothelin-1-induced serotonin release

The aim of the present study was the demonstration of mechanisms of regulation of activity of chromaffin cells in the adrenal gland of Rana ridibunda (Anura-Amphibia). Previous studies have shown that endothelin-1 is an important factor for the maintenance of adrenal gland function. On the basis of these findings, frogs were injected with [Ala(1,3,11,15)]-endothelin-1 (0.025 mg/0.2 ml), which is a selective agonist of the endothelin B receptor, whereas control animals were injected with Ringer solution (0.2 ml). The adrenal glands were removed at 5, 20, and 60 min after injection and fixed, embedded in paraffin wax and stained by histological and immunohistochemical means, applied on adjacent 4-microm-thick sections. Sections were stained with hematoxylin and eosin for overall tissue analysis and, in parallel, serotonin was localized using the streptavidin-biotin complex technique while dopamine beta-hydroxylase and serotonin 2A receptors were shown by the peroxidase-antiperoxidase (PAP)-3,3'-diaminobenzidine tetrachloride (DAB) method. After injection of [Ala(1,3,11,15)]-endothelin-1, chromaffin cells secreted serotonin and synthesized dopamine beta-hydroxylase. In conclusion, these findings suggest that [Ala(1,3,11,15)]-endothelin-1 stimulates chromaffin cell activity in frog adrenal glands. Moreover, the presence of serotonin 2A receptors in chromaffin cells indicates that these cells are also targets for serotonin and that there is an autocrine signaling pathway in chromaffin cells. This is the first report providing data on the effects of endothelin-1 on chromaffin cells in frog adrenal glands.     

Production of interleukin 1 in glomerular cell cultures from rats with nephrotoxic serum nephritis.

To investigate the role of cytokines in the pathogenesis of experimental glomerulonephritis (GN), we examined interleukin 1 (IL-1) activity in isolated glomeruli of rats with an accelerated autologous form of nephrotoxic serum nephritis (NTSN). Glomeruli were isolated and explanted in tissue culture. The prominent feature of the glomerular outgrowth of the glomeruli in the NTSN was the presence of large numbers of type III cells (macrophages). In addition, there were significantly greater numbers of type II (mesangial) cells in culture from the NTSN rats as compared with glomeruli from normal rats, though the numbers of type I (epithelial) cells were the same. IL-1 bioactivity was significantly higher in culture supernatants generated by glomeruli isolated from NTSN animals versus normal animals early in the course of the disease. When glomerular culture supernatants and purified IL-1 were pre-incubated with an anti-IL-1 beta antibody, a parallel decrease of the biological activity was found, suggesting that the biologically active binding sites of the culture supernatants and monocyte-produced IL-1 share some common structure. The administration of a rabbit anti-rat macrophage serum (AMS) prevented the outgrowths of type III cells (macrophages) and type II (mesangial) cells and reduced the production of IL-1 activity in the NTSN rats. In this experimental model IL-1 synthesis, probably by multiple cell types, is present early in the disease process and perhaps may be an important mediator of glomerular immune injury.     

Cryopreservation technology for plant cell cultures

Summary Described in detail is a technique for the cryopreservation, long-term storage, thawing, recovery, and regrowth of embryogenic suspension cultures of maize (Zea mays L.) With this system we have successfully preserved many different culture lines, and recovered living material after more than 2 yr storage in liquid nitrogen. The recovered tissue is not apparently different from the starting material. With minor variations, the technique has also been used forDactylis glomerata and other suspension culture cells, as well as callus cultures. Approaches for the cryopreservation of other plant parts, such as seeds, embryos and meristems are discussed.     

A chemiluminescent assay for mycoplasmas in cell cultures

A chemiluminescent assay for the detection of mycoplasma contamination of cell cultures is described. Cells (and supernatant) derived from mycoplasma-contaminated cultures stimulate a burst of luminol-dependent chemiluminescence in cell suspensions containing phagocytic effector cell types. The assay conditions for spleen cells, human and bovine polymorphonuclear leucocytes as the responder or indicator cells have been optimized. The chemiluminescent assay can be utilized for both monolayer and suspension cell cultures and is more sensitive than colony formation on agar plates and electron microscopy. Results are obtained within 3-5 h including the time required for the preparation of the indicator cells. CL can be measured in the tritium window of standard liquid scintillation spectrometers after switching off the coincidence circuit.     

Proliferative activity and characteristics of immunocytochemically identified oligodendrocytes in embryonic mouse brain cell cultures

Dissociated brain cell cultures of 14-day-old mouse embryos (E 14) were used for studying, during development, the proliferative activity of oligodendrocytes which express myelin basic protein (MBP) and galactocerebroside (GC). This was done using a combination of 3H-Thymidine autoradiography and immunoperoxidase or immunofluorescence. Quantitative estimates of labeled cells were made using a Leitz Texture Analysis System (T.A.S.) coupled to a P.D.P. 11-34 minicomputer. Results showed that differentiated oligodendrocytes, which express both MBP and GC, are able to proliferate. According to the intensity of the immunostaining, strong MBP positive and weak MBP positive oligodendrocytes were observed. Only the weak MBP positive cells incorporated 3H-Thymidine. The highest percentage (22.5%) of 3H-Thymidine labeled oligodendrocytes was observed at day 6 in vitro, and was reduced by half at day 9 to 13. Oligodendrocytes which have undergone a first division are still able to proliferate.