Concentrations Of Fetal Calf Serum Research Articles

A role for Noggin in the development of oligodendrocyte precursor cells

Oligodendrocyte precursor cells (OPCs) can be differentiated in culture into either oligodendrocytes or type-2 astrocytes (2As), depending on the culture conditions. Whereas oligodendrocyte development can occur in the absence of inducing signals, 2A development apparently cannot. Fetal calf serum (FCS) and bone morphogenetic proteins (BMPs) are powerful inducers of 2A development in culture, but there is no compelling evidence that OPCs develop into astrocytes in vivo. We show here that BMPs are made by glial cells in the developing rat optic nerve, raising the question of why 2As do not normally develop in the optic nerve. We demonstrate that the BMP antagonist Noggin is strongly expressed by both OPCs and type-1 astrocytes in the developing optic nerve. We also show that depletion of Noggin by a small interference RNA inhibits OPC proliferation and induces 2A differentiation in the presence of a low, non-2A-inducing concentration of FCS. By contrast, enforced expression of Noggin in OPCs blocks FCS-induced 2A differentiation. These findings suggest that BMPs in FCS are largely responsible for the 2A-inducing activity of FCS and that Noggin may normally inhibit the formation of 2As in the developing CNS.

A model for 3-dimensional growth of bladder cancers to investigate cell-matrix interactions

Elucidating the mechanisms by which the phenotype of cancer cells is modulated by the extracellular matrix (ECM) potentially identifies mechanisms that could be exploited for cancer control. Three readily available bladder cancer cell lines of different aggressiveness were grown on a bioscaffold material (Small Intestine Submucosa: SIS) under a variety of media and nutrient schedules to determine the influence of epigenetic factors on phenotype. The aggressive TCCSUP and J82 lines displayed an invasive phenotype when fed twice weekly with medium containing 10% fetal calf serum (FCS), but grew as a layered, noninvasive structure when fed daily with the same nutrient. The papillary RT4 cells grew as a nearly normal appearing layered phenotype when fed with medium containing 10% FCS, regardless of the feeding schedule. The papillary phenotype appeared only when the fetal calf serum concentration was reduced to 1% and the cells were fed twice weekly. Immunohistochemical staining showed E-cadherin was detected in the nucleus of the TCCSUP line rather than on the cell periphery, thus, demonstrating that normalization of the TCCSUP line into a layered phenotype did not alter the fundamental dysregulation of the cadherin-B-catenin-cytoskeleton complex. In contrast, in the RT4 cells the biomarker was distributed discretely around the cell periphery in 10% fetal calf serum but in the nucleus in 1% fetal calf serum and twice weekly feeding. Modulating the phenotype of cancer cells from invasive to noninvasive through manipulation of matrix and nutrient components presents a model system for identifying the key factors involved in invasiveness and may identify new therapeutic targets and markers.

Increasing the yield of human mononuclear cells and low serum conditions for in vitro generation of macrophages with M-CSF

The yield of mononuclear cells extracted from peripheral blood using standard protocols is frequently inadequate when working with material of limited availability. In addition, the regular usage of autologous and fetal calf serum (FCS) to generate human macrophages in vitro may complicate antigen uptake, processing and presentation on HLA molecules. We optimized the yield of mononuclear cells from 34±3% to 65±5% by collecting both the interface and more than half of the overlayering supernatant, followed by three washes at 4 °C. Monocytes from 12 individuals were cultured 1–4 days with 0–100 ng/ml macrophage colony-stimulating factor (M-CSF) at either 1% (low) or 5% (v/v) FCS. Regardless of number of days in culture, maximal (50–100 ng/ml) M-CSF stimulation and low FCS induced 65±5% esterase-positive cells in all individuals compared to 52±7% without M-CSF ( p<0.001). M-CSF increased the mean proportion of esterase-positive cells in both 1% and 5% FCS with a negative interaction found between 5% FCS and M-CSF ( p<0.05). All cells were positive for CD14 and HLA class II but cell number did not increase. The generation of human macrophages by M-CSF at low FCS should prove useful in studies where higher FCS concentrations may interfere with the assay.

Proliferation and differentiation of bovine type A spermatogonia during long-term culture.

The present study was aimed at developing a method for long-term culture of bovine type A spermatogonia. Testes from 5-mo-old calves were used, and pure populations of type A spermatogonia were isolated. Cells were cultured in minimal essential medium (MEM) or KSOM (potassium-rich medium prepared according to the simplex optimization method) and different concentrations of fetal calf serum (FCS) for 2-4 wk at 32 degrees C or 37 degrees C. Culture in MEM resulted in more viable cells and more proliferation than culture in KSOM, and better results were obtained at 37 degrees C than at 32 degrees C. After 1 wk of culture in the absence of serum, only 20% of the cells were alive. However, in the presence of 2.5% FCS, approximately 80% of cells were alive and proliferating. Higher concentrations of FCS only enhanced numbers of somatic cells. In long-term culture, spermatogonia continued to proliferate, and eventually, type A spermatogonial colonies were formed. The majority of colonies consisted mostly of groups of cells connected by intercellular bridges. Most of the cells in these colonies underwent differentiation because they were c-kit positive, and ultimately, cells with morphological and molecular characteristics of spermatocytes and spermatids were formed. Occasionally, large round colonies consisting of single, c-kit-negative, type A spermatogonia (presumably spermatogonial stem cells) were observed. For the first time to our knowledge, a method has been developed to allow proliferation and differentiation of highly purified type A spermatogonia, including spermatogonial stem cells during long-term culture.

Effect of increasing selenite concentrations, vitamin E supplementation and different fetal calf serum content on GPx1 activity in primary cultured rabbit hepatocytes

Primary rabbit hepatocytes from 6 week old female New Zealand White rabbits (3.0 × 10 6 viable hepatocytes per treatment) were incubated for 24 h or 48 h with two basic variants of the selenium and vitamin E free DMEM/F12-HAM nutrition medium containing 2.5% or 10% fetal calf serum (FCS). Selenium and vitamin E concentrations of the media were varied by the addition of 0, 10, 50 and 100 ng Se/mL medium as sodium selenite and 100 μg α-tocopheryl acetate/mL. Lactic dehydrogenase (LDH) leakage of the hepatocytes was not influenced by the various selenium concentrations of the media, whereas vitamin E addition significantly inhibited LDH release. The activity of cellular glutathione peroxidase (GPx1) was markedly induced by increasing the selenium supplementation of the culture media. Vitamin E supply further enhanced GPx1 induction. In hepatocytes cultivated at the lower serum concentration (2.5% FCS), increasing the selenite concentration of the media raised GPx1 and reduced the intracellular levels of the reduced tripeptide glutathione (GSH). No vectored relation between the selenium concentration of the media and the activity of superoxide dismutase (SOD) could be observed. After both incubation periods (24 h and 48 h) SOD activity was significantly higher in the cytosol of hepatocytes grown in media containing 10% FCS as compared to cells incubated at the 2.5% FCS level. Furthermore, SOD activity was reduced by the addition of vitamin E to the media. In conclusion the results indicate an effective metabolism of rabbit hepatocytes for selenite even in amounts as low as nanograms. A general cytoprotective role for vitamin E can be shown by its ability to decrease LDH leakage and by the reduction of SOD activity.

Establishment and characterization of a new epithelial cell line, KC-1, from koala (Phascolarctos cinereus) conjunctiva.

A novel, untransformed koala cell line (KC-1) was established by culturing koala conjunctival tissue in growth medium, which has permitted the study of the cell biology of this unique system. After the establishment of the KC-1 cell line, the cells were characterized by light microscopy, doubling time, and Western blot analysis. Light microscopy revealed that the cells have an epithelial morphology. Doubling times were significantly different (P < 0.015) depending on fetal calf serum (FCS) concentration (16.5 h in 10% FCS and 26.5 h in 2% FCS). Cells constricted while in suspension but were shown to attach to the coverslip (or flask) and flatten rapidly, less than 1 h after seeding. To confirm the epithelial nature of the cells, protein was extracted and Western blot analysis was performed. Subsequent probing with primary and secondary antibodies (monoclonal anticytokeratin clone C-11 IgG1 and anti-mouse IgG) revealed two bands at 45 and 52 kDa (compared against a protein molecular weight marker) that correspond to primary type I keratin and major type II keratin, respectively, expressed in simple epithelial cells. The koala cell line was adapted to grow continuously in Dulbecco modified Eagle medium containing 10% FCS for at least 30 passages. This unique cell line is an ideal tool for further investigation on koala cell biology and cytogenetics and for exploration of the pathophysiological mechanism of eye infections caused by different pathogens in koalas.

Cell kinetics of human nasal septal chondrocytes in vitro: importance for cartilage grafting in otolaryngology.

In the field of reconstructive plastic surgery, grafts of autologous cartilage are sometimes used to replace damaged or pathologic tissues, particularly in the nose, ear, and trachea. However, this procedure is difficult to apply, especially because of the scarcity of donor sites. In this study, we have cultured and characterized human chondrocytes from human nasal septal cartilage biopsies. The proliferative activity was evaluated by incorporation of 3H-thymidine into deoxyribonucleic acid (DNA) both under normal culture conditions and with different growth factors and serum concentrations. Identification of chondrocytes in culture was performed with immunohistochemistry and production of matrix with specific histochemical indicators. We observed a significant increase of cell kinetics of differentiated chondrocytes, embedded with intense metachromatic matrix, in the presence of transforming growth factor beta, and low concentrations of fetal calf serum. Therefore, in suitable conditions, human chondrocytes obtained even from small specimens can produce in vitro considerable quantities of pure autologous cartilaginous tissue within a few days. This newly formed cartilage can be used as a grafting material in reconstructive surgery, particularly in otolaryngology.

ISOLATION AND CHARACTERIZATION OF CANINE SATELLITE CELLS

Satellite cells were isolated from biopsies of the biceps femoris of adult dogs. Virtually all cells expressed muscle-specific proteins. Proliferation of satellite cells increased as the concentration of fetal calf serum (FCS) was increased from 1 to 10% of the basal medium. The addition of mitogenic growth factors resulted in greater proliferation than that of cells cultured in basal medium alone. Maximum proliferation was obtained when fibroblast growth factor-basic (FGF2) was added to the medium, but differences existed between sources or types. Proliferation did not plateau when the concentration of recombinant human FGF2 was 75 ng/ml but reached maximum levels when 50 ng/ml of bovine FGF2 or 10 ng/ml of growth hormone or insulin-like growth factor-1 were added to the medium. Proliferation of satellite cells decreased when more than 5 ng/ml of transforming growth factor-alpha was included in the medium. Exposure of canine satellite cells to chemically defined media induced greater fusion of total nuclei (ODM-34%; 4F, ITT-CF, and SFG-23%) than exposure to other treatments, such as basal medium plus 2 mg/ml of 1-beta-d-arabinofuranosylcytosine, 5% chick embryo extract, 1% horse serum (average 9% fused nuclei), or 1% FCS (2% fused nuclei). Actin, myosin, desmin, neural cell adhesion molecule, MyoD1, and myogenin were expressed by canine satellite cells, but expression of major histocompatibility complex class II antigen was not detected. Reverse transcriptase-polymerase chain reaction detected expression of messenger ribonucleic acid for interleukin-6 (IL-6), IL-15, and leukemia inhibitory factor by canine satellite cells. Collectively, these data suggest that isolated canine satellite cells display properties of other types of myogenic cells and may be useful for further study of the regulation of postnatal myogenesis.

In vitro lead-induced cell toxicity and cytoprotective activity of fetal calf serum in human fibroblasts.

The underlying mechanisms by which lead ions produce their deleterious effects prior to the onset of clinical symptoms are incompletely understood. This study aimed to assess lead-induced cell toxicity mechanisms by focusing on the effects of the metal on cell growth, DNA synthesis, cellular ATP, intracellular hexosaminidase activity and lysosomal function, and examine the possible cytoprotective role of fetal calf serum (FCS). Several human dermal cultured fibroblast lines were exposed to Pb (400 microM) for 1-6 days with 2, 5, and 10% FCS. The earliest toxic effect of Pb was significant inhibition of DNA synthesis after 24 h direct exposure; this harmful effect was not progressive during the first 3 days, but worsened clearly on the 4th day regardless of the FCS concentration. Atime-dependent depletion of intracellularATP content was also caused by ionic lead, thereby compromising the cell energy charge which precedes cell death. Fibroblast growth was progressively and significantly inhibited from day 2 onwards; the greatest noxious effect was observed in the presence of 2% FCS: 49% reduction in cell proliferation after 5 days. Lead salts produced loss of cell adhesion to the culture dish which worsened from the 2nd day and was more pronounced when FCS in growth medium was decreased. Toxic actions on lysosomal membrane integrity provoked a decrease in neutral red uptake (NRU) which was exposure time-dependent and more marked with 2% FCS. In contrast, increased relative NRU (to 20% at 4 days), suggestive of endocytosis-induced lysosome enlargement, was observed in Pb-exposed cells. Intracellular hexosaminidase activity was not negatively affected until 5 days after exposure to Pb salts. FCS had a significant cytoprotective effect on Pb-induced toxicity.

Mechanisms underlying chlorhexidine-induced cytotoxicity

Chlorhexidine (CLX) is the most widely used antiseptic for wound and skin disinfection. Despite its potent bactericidal action, skin irritation is observed when it is used topically. This study aimed to evaluate the mechanisms underlying CLX-induced toxicity on human dermal fibroblasts with special emphasis on factors that may mediate or counteract its undesirable effects. Cells were exposed to CLX concentrations of 0.00005–0.025% for 3, 6, 8 or 24 h in the absence or presence of different concentrations of foetal calf serum (FCS) (2, 5 and 10%). Depletion of cell ATP occurred, in a time- and concentration-dependent manner, in all experimental conditions at [CLX] >0.001%. At 24 h of CLX exposure time, the decrease in intracellular ATP was produced from a 10-times lower CLX concentration (0.0001%). Concentrations ⩾0.02% produced total loss of ATP. However, cell survival was maintained after CLX treatment for 3 and 8 h and CLX concentrations ⩾0.005% were required to produce total cell death. CLX exerted an inhibitory concentration-dependent effect on DNA synthesis from concentrations as low as 0.0001%. Only FCS at 10% appeared to have a cytoprotective action against CLX-induced cytotoxicity.

Corneal Cryopreservation with Dextran

Different methods of corneal cryopreservation have been introduced, those employing intracellular cryoprotectants such as Me2SO or glycerol being the most widely favored. We investigated the influence of several freeze-thaw trauma variables on the survival of porcine endothelial monolayers when employing the extracellular cryoprotective agent dextran. We first examined the effects of various dextran concentrations and then, having ascertained the optimal concentration, further investigated the influence of fetal calf serum (FCS) concentration in the cryopreservation medium, the cooling rate, the thawing temperature, and the length of the preincubation in the freezing medium prior to cryopreservation. The numerical densities of endothelial cells were determined at dissection in hypoosmotic balanced salt solution and after organ culture by staining with alizarin red S and trypan blue. Morphological evaluation was not performed directly after thawing but after a subsequent organ culture at 37°C to detect latent cell damage after freeze-thaw trauma. Our data revealed that corneas cryopreserved in minimal essential medium containing 10% dextran but lacking FCS, preincubated for 3 h, frozen at a cooling rate of 1°C/min, and thawed at 37°C incurred the lowest cell losses (22.4%, SD ± 3.8). We conclude that dextran is an effective cryoprotectant for freezing of porcine corneas. However, variations between species in the results of cryopreservation require further investigation of an in vivo animal model and studies with human corneas before its clinical use can be recommended.

Stimulation of the growth of "normal" thyrocytes by lymphocytes infiltrating the tissue of diffuse toxic goiter

The capacity of lymphocytes isolated from diffuse toxic goiter (DTG) tissue to stimulate the proliferation of normal thyrocytes isolated from paranodular tissue adjacent to nodular euthyroid goiter was studied. Normal and DTG thyrocytes were incubated for 24 h with fetal calf serum (FCS) in various concentrations, DTG orparanodular lymphocytes, or both FCS and lymphocytes. The proliferation rate of normal thyrocytes in the presence of FCS alone increased with increase of the serum concentration, while the proliferation rate of DTG thyrocytes did not depend on the concentration of FCS. DTG lymphocytes, but not paranodular ones, stimulated the proliferation of normal thyrocytes. The growth-stimulating effect of DTG lymphocytes was paralleled by loss of normal thyrocytes sensitivity to growth factors (FCS). These data indicate a probable role of intrathyroid lymphocytes in increase of thyrocyte count characteristic of DTG and confirm a previous hypothesis on decrease in expression of surface antigens (receptors) on DTG cells under the effect of intrathyroid lymphocytes.

Low serum conditions for in vitro generation of human macrophages with macrophage colony stimulating factor

Animal serum is often used to generate human macrophages in vitro. Since fetal calf serum (FCS) may complicate antigen uptake, processing and presentation on HLA molecules, we tested the ability of M-CSF to generate macrophages at low fetal calf serum conditions. Peripheral blood monocytes from 12 individuals were cultured 1–4 days with 0–100 ng/ml macrophage colony stimulating factor (M-CSF) at either 1 (low) or 5% (v/v) FCS. Regardless of number of days in culture, maximal (50–100 ng/ml) M-CSF stimulation and low FCS induced 65±5% esterase positive cells in all individuals compared to 52±7% without M-CSF ( P<0.001). M-CSF increased the mean proportion of esterase positive cells after 24 or 96 h by 13% ( P<0.005) and 13% ( P<0.005), respectively, in 1% FCS, and 8% ( P<0.05) and 2% (NS), respectively, in 5% FCS, indicating a slight negative interaction between 5% FCS and M-CSF ( P<0.05). All cells were positive for CD14 and HLA class II, but cell number did not increase, confirming that M-CSF promote macrophage differentiation also at low FCS. M-CSF increased the average cell size after 24 or 96 h by 5.9±1.0 ( P<0.05) and 8.6±0.5 ( P<0.001) μm, respectively, without an increase in 5% FCS, further demonstrating the efficiency of M-CSF to promote macrophage generation at low FCS. The culture supernatants were negative for IL-1β and TNF-α, which demonstrates that M-CSF did not activate the macrophages. The generation of human macrophages by M-CSF at low FCS should prove useful in studies where higher FCS concentrations may interfere with the assay.

Optimised Conditions for the Production of Hepatitis B Virus from Cell Culture

In chronically infected patients, hepatitis B virus (HBV) particles reach numbers as large as >10⁹ genome equivalents (GE)/ml of serum. However, expression of infectious HBV particles in cell culture only yields 10⁵–10⁶ GE/ml, which is insufficient for many studies. HBV transcription and possibly replication is dependent on hepatocyte-specific differentiation. Thus, we tested several cell culture parameters that have been reported to enhance the expression of hepatocyte-specific markers, such as growth on different extracellular matrices, different cell culture media, low concentrations of fetal calf serum (FCS) and the addition of dimethyl sulfoxide (DMSO) to the medium. Lower concentrations of FCS, growth on collagen and inclusion of DMSO in the medium only moderately enhanced HBV production in vitro when applied individually. However, combinations of these parameters optimised cell culture conditions and reproducibly increased the release of HBV particles about 100-fold to titres >10⁸ GE/ml of culture medium.

Oestrogen deficiency causes DNA damage in uterine leiomyoma cells: a possible mechanism for shrinkage of fibroids by GnRH agonists

Objective To examine whether gonadotrophin-releasing hormone agonist or oestradiol can directly affect DNA in leiomyoma cells. Design In vitro explant culture of leiomyoma cells. Setting University research group. Sample Leiomyoma cells were cultured from the specimens of four premenopausal women at myomectomy. Methods The presence of gonadotrophin-releasing hormone receptor in leiomyoma cells was determined by reverse transcriptase–olymerase chain reaction. Leiomyoma cells were treated with gonadotrophin-releasing hormone agonist or cultured in different concentrations of oestrogen, progesterone or fetal calf serum for one, four or seven days. Main outcome measures Cell number, expression of proliferating cell nuclear antigen, and DNA damage after one, four or seven days of treatment. Results Gonadotrophin-releasing hormone receptor messenger ribonucleic acid was detected on cultured leiomyoma cells. Leiomyoma cell growth was not affected by the addition of gonadotrophin-releasing hormone agonist or progesterone, but increased with oestrogen or fetal calf serum supplementation. Overexpression of proliferating cell nuclear antigen was prevented in cultures added with oestrogen or fetal calf serum, but not related to gonadotrophin-releasing hormone agonist treatment. Significant decreases in DNA damage as indicated by decreased comet number were found in the leiomyoma cultures treated with oestrogen or fetal calf serum for four and seven days but not with gonadotrophin-releasing hormone agonist or progesterone. Furthermore, 5% fetal calf serum supplementation was more growth supporting and more significantly reduced the comet number than 250 pM 17 β-oestradiol. Conclusion Cell growth, proliferating cell nuclear antigen expression and DNA damage are dependent on oestrogen or fetal calf serum, but independent of gonadotrophin-releasing hormone agonist or progesterone. Our findings suggest that gonadotrophin-releasing hormone agonist-induced leiomyoma shrinkage may be due in part to a mechanism involving DNA damage, and support the hypothesis that gonadotrophin-releasing hormone agonist exerts its action indirectly through oestrogen action on the tumour level.

Oestrogen deficiency causes DNA damage in uterine leiomyoma cells: a possible mechanism for shrinkage of fibroids by GnRH agonists.

To examine whether gonadotrophin-releasing hormone agonist or oestradiol can directly affect DNA in leiomyoma cells. In vitro explant culture of leiomyoma cells. University research group. Leiomyoma cells were cultured from the specimens of four premenopausal women at myomectomy. The presence of gonadotrophin-releasing hormone receptor in leiomyoma cells was determined by reverse transcriptase-olymerase chain reaction. Leiomyoma cells were treated with gonadotrophin-releasing hormone agonist or cultured in different concentrations of oestrogen, progesterone or fetal calf serum for one, four or seven days. Cell number, expression of proliferating cell nuclear antigen, and DNA damage after one, four or seven days of treatment. Gonadotrophin-releasing hormone receptor messenger ribonucleic acid was detected on cultured leiomyoma cells. Leiomyoma cell growth was not affected by the addition of gonadotrophin-releasing hormone agonist or progesterone, but increased with oestrogen or fetal calf serum supplementation. Overexpression of proliferating cell nuclear antigen was prevented in cultures added with oestrogen or fetal calf serum, but not related to gonadotrophin-releasing hormone agonist treatment. Significant decreases in DNA damage as indicated by decreased comet number were found in the leiomyoma cultures treated with oestrogen or fetal calf serum for four and seven days but not with gonadotrophin-releasing hormone agonist or progesterone. Furthermore, 5% fetal calf serum supplementation was more growth supporting and more significantly reduced the comet number than 250 pM 17 beta-oestradiol. Cell growth, proliferating cell nuclear antigen expression and DNA damage are dependent on oestrogen or fetal calf serum, but independent of gonadotrophin-releasing hormone agonist or progesterone. Our findings suggest that gonadotrophin-releasing hormone agonist-induced leiomyoma shrinkage may be due in part to a mechanism involving DNA damage, and support the hypothesis that gonadotrophin-releasing hormone agonist exerts its action indirectly through oestrogen action on the tumour level.

Growth-altering effects of sodium hypochlorite in cultured human dermal fibroblasts

Sodium hypochlorite, the most widely used antimicrobial active chlorine compound in chemical disinfection, is little used as an antiseptic in clinical practice. This study aimed to assess the capacity of hypochlorite to alter human dermal fibroblast growth in vitro in relation to the concentration and exposure time. Effects of decreasing concentrations of hypochlorite (0.5%–0.00025%) on fibroblast adherence capacity and proliferation, according to varying exposure times and fetal calf serum (FCS) concentrations were investigated combining XTT assay, which provides cytochemical quantification of metabolically-active cell number, and total cell protein content, an indirect method for assessing substrate-adhered cell number. Initial cytotoxicity was produced at 0.0075% hypochlorite within contact time of two hours, provoking concentration-dependent cell detachment. From 0.1% upwards, NaOCl exerted a profound cytotoxic effect on fibroblasts. At later stages (4 h) and concentrations ≥0.01% hypochlorite produced dose-dependent mitochondrial dysfunction: cell survival progressively diminished from 71% to 10%. Cytotoxic effects were not significantly affected by exposure-time periods, probably because maximum chlorine is released within the first four hours. Hypochlorite concentrations from 0.005% to 0.00025% were found to have no inhibitory effects on cell growth; in fact, they appear to exhibit the opposite effect. Increments in protein content found after 24 h exposure ranged from 30% to 120% above control values. Hypochlorite is highly cytotoxic for fibroblasts at concentrations ≥0.01% provoking concentration-dependent loss of cell adherence capacity and mitochondrial dysfunction. In contrast, a mitogenic effect was observed with concentrations ≤0.005% which supports NaOCl as a source growth-promoting activity in cultured human fibroblasts. Hypochlorite proved to be a highly reactive molecule which inhibits or stimulates cell division according to the concentration.

Leishmania infantum: Stage-Specific Activity of Pentavalent Antimony Related with the Assay Conditions

Carrió, J., Colmenares, M. de, Riera, C., Gállego, M., Arboix, M., and Portús, M. 2000. Leishmania infantum: Stage-specific activity of pentavalent antimony related with the assay conditions. Experimental Parasitology95, 209–214. The determination of the intrinsic sensitivity of Leishmania strains to pentavalent antimonials in clinical trials, before treatment is begun, is essential in order to avoid failures and to allow alternative drugs to be chosen. A comparative study of SbV activity on promastigotes, axenic amastigote-like cells, and intracellular amastigotes of Leishmania infantum, when administered in the form of meglumine antimoniate and free, in hydrochloric solution, was performed. Results indicate that the conditions under which the promastigotes were cultured affect the IC50 obtained, although results were homogeneous when the products were assayed on axenic-like and intracellular amastigotes. The IC50 obtained for SbV in the form of meglumine antimoniate or in hydrochloric solution on promastigotes cultured in Schneider's medium depends on the growth rate of the culture and therefore could be regulated by modifying the fetal calf serum concentration in the medium. The pH of the culture medium strongly affected the activity of meglumine antimoniate but not that of the SbV hydrochloric solution on promastigotes cultured in Schneider's medium. This influence of pH was observed to a much lesser extent when promastigotes were cultured on M199 or RPMI media. In homogeneous culture conditions, which included the regulation of the promastigote growth rate through the heat-inactivated fetal calf serum concentration in the medium and the dilution of the meglumine antimoniate with Schneider's medium at pH 6.5, the activity of SbV, free or in the form of meglumine antimoniate, was the same in promastigotes, intracellular amastigotes, and axenic amastigote-like cells.

Accumulation of protoporphyrin-IX (PpIX) in leukemic cell lines following induction by 5-aminolevulinic acid (ALA).

We investigated the amounts of protoporphyrin IX (PpIX) accumulated in noninduced cells and following 5-aminolevulinic acid (ALA)-induction. Following ALA administration PpIX increased in all leukemic cell lines under investigation (HEL 26-fold, HL60 6-fold, Jurkat 3-fold, ML2 2-fold) but not in lymphocytes. Compared to other cell lines studied, HEL cells showed the lowest basal level of PpIX and the largest relative increase in PpIX. Despite a high increase following ALA treatment, the PpIX level in HEL cells is almost as low as in lymphocytes. It is in agreement with their relatively low sensitivities of ALA-induced photodynamic therapy (ALA-PDT) shown previously [(Grebenová, D., Cajthamlová, H., Bartosová, J., Marinov, J., Klamová, H., Fuchs, O., Hrkal, Z., 1998. Selective destruction of leukemic cells by photo-activation of 5-aminolevulinic acid induced protoporphyrin IX. J. Photochem. Photobiol. B: Biol. 47, 74-81)]. The ferrochelatase activities in the individual cell lines are in good inverse correlation with PpIX amounts accumulated in the ALA-induced cells, but not with the relative increase (ratio) of PpIX levels from basal to ALA-induced ones. This is most apparent in HEL cells and lymphocytes. There is probably different regulation of heme biosynthesis in erythroid cells, which are therefore not suitable for the studies of ALA-PDT mechanism. PpIX was accumulated more extensively in absence of fetal calf serum than in its presence. The amounts of PpIX accumulated in cells decreased exponentially with increasing fetal calf serum concentration.

Ionizing radiation‐induced death in bovine lens epithelial cells: Mechanisms and influence of irradiation dose rate

We recently reported, in a series of patients receiving total body irradiation before transplant, an influence of dose rate (DR) on cataract formation. The aim of our present in vitro study was to investigate the influence of DR and the mechanisms of lens cell death in a bovine model. After a single fraction of 10 Gy, delivered using low (0.05 Gy/min) or high (2 Gy/min) DR (LDR and HDR, respectively), cells were incubated in media supplemented with two different fetal calf serum (FCS) concentrations (1% and 10%). Cell proliferation was evaluated using Hoechst 33342 (HO) probe and cell viability, with neutral red probe. These fluorimetric assays used a cold light cytofluorimeter. After HO assay, stained cells were examined with fluorescence microscopy to evaluate the nuclear changes related to apoptosis. Global comparison of the mean HO fluorescent values observed with LDR/controls (c) vs. HDR/c revealed a significant difference only after 96 hr (P = 0.036). In 1% FCS conditions, the difference between HDR/c and LDR/c was also statistically significant at 96 hr (P = 0.04). Pairwise multiple comparison using values observed in 1% FCS conditions after 96 hr incubation showed significant difference between HDR vs. c (P = 0.001) and HDR vs. LDR (P = 0.007). This difference, in terms of fluorescence, was correlated to the proportion of cells with nuclear apoptotic morphology. In contrast, cell viability was not influenced by DR whatever the FCS concentration used, from 24 to 96 hr after irradiation. We conclude that our fluorimetric methodology is adapted to evaluate intracellular DNA modifications and cell viability after x-ray irradiation. We observed that a single fraction of 10 Gy induces in vitro lens epithelial cell apoptosis, which is influenced by DR. In humans, HDR is considered more cataractogenic than LDR. Thus, we speculate that lens cell apoptosis could be one of the major mechanisms of radiation-induced cataract. Further investigations are necessary to study the other possible mechanisms of cataractogenesis. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 138–144 (2000). © 2000 Wiley-Liss, Inc.