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Calf Serum (calf + serum)
Kinds of Calf Serum Selected AbstractsHypertrophy and physiological death of equine chondrocytes in vitroEQUINE VETERINARY JOURNAL, Issue 6 2007Y. A. Ahmed Summary Reasons for performing study: Equine osteochondrosis results from a failure of endochondral ossification during skeletal growth. Endochondral ossification involves chondrocyte proliferation, hypertrophy and death. Until recently no culture system was available to study these processes in equine chondrocytes. Objective: To optimise an in vitro model in which equine chondrocytes can be induced to undergo hypertrophy and physiological death as seen in vivo. Methods: Chondrocytes isolated from fetal or older (neonatal, growing and mature) horses were cultured as pellets in 10% fetal calf serum (FCS) or 10% horse serum (HS). The pellets were examined by light and electron microscopy. Total RNA was extracted from the pellets, and quantitative PCR carried out to investigate changes in expression of a number of genes regulating endochondral ossification. Results: Chondrocytes from fetal foals, grown as pellets, underwent hypertrophy and died by a process morphologically similar to that seen in vivo. Chondrocytes from horses age >5 months did not undergo hypertrophy in pellet culture. They formed intramembranous inclusion bodies and the cultures included cells of osteoblastic appearance. Pellets from neonatal foals cultured in FCS resembled pellets from older horses, however pellets grown in HS underwent hypertrophy but contained inclusion bodies. Chondrocytes from fetal foals formed a typical cartilage-like tissue grossly and histologically, and expressed the cartilage markers collagen type II and aggrecan mRNA. Expression of Sox9, collagen type II, Runx2, matrix metalloproteinase-13 and connective tissue growth factor mRNA increased at different times in culture. Expression of fibroblast growth factor receptor-3 and vascular endothelial growth factor mRNA decreased with time in culture. Conclusions: Freshly isolated cells from fetal growth cartilage cultured as pellets provide optimal conditions for studying hypertrophy and death of equine chondrocytes. Potential relevance: This culture system should greatly assist laboratory studies aimed at elucidating the pathogenesis of osteochondrosis. [source] The antiapoptotic effects of blood constituents in patients with chronic lymphocytic leukemiaEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5 2003Yonit Bomstein Abstract: Objective: Clonal B-lymphocytes of chronic lymphocytic leukemia (B-CLL) are characterized by decreased sensitivity to programmed cell death and, therefore, they accumulate in vivo. However, these malignant cells die rapidly in vitro. In the current study we concentrated on the contribution of autologous serum (AS) and lymphocyte subsets to the survival of the malignant cells in vitro. Methods: Mononuclear cells from the peripheral blood of 26 CLL patients and 24 controls were incubated overnight in the presence or absence of AS and heat-inactivated AS (HI-AS) or fetal calf serum (FCS). Also, isolated B cells were incubated at different concentrations in the presence of AS and/or isolated T cells. The level of apoptosis of CD19+ cells was measured by flow cytometry. Results: Spontaneous apoptosis of unfractionated B-CLL cells incubated with AS, FCS or without serum was significantly lower than the rate of B-cell death in the control group, in similar culture conditions. AS had an antiapoptotic effect on unfractionated B-CLL cells when compared with FCS. The rate of apoptosis of B-CLL cells was directly associated with stage. HI of AS had a variable effect, which was related to the stage of the disease. High concentrations of B cells and the addition of autologous T cells reduced the rate of apoptosis when incubated without serum. The antiapoptotic effect of T cells was most prominent in progressive stages. Conclusions: B-CLL cells exhibit decreased spontaneous apoptosis, which is partially prevented by humoral (AS) and cellular (T cells and B-CLL cells) factors. The equilibrium between apoptotic and antiapoptotic factors changes with disease progression. [source] Effect of Silicate-Substitution on Attachment and Early Development of Human Osteoblast-Like Cells Seeded on Microporous Hydroxyapatite Discs,ADVANCED ENGINEERING MATERIALS, Issue 1-2 2010Katharina Guth Hydroxyapatite (HA) is a well-established graft material used in bone repair. Silicon-substituted hydroxyapatite (SA; 0.8,wt% Si) has shown greater bone ingrowth and bone coverage than phase pure HA. To assess the effect of microporosity on sensitivity of cell attachment to surface physiochemistry, microporous SA and HA discs, and control Thermanox (TMX) discs were incubated with osteoblast-like cells (5,×,104 HOS-TE85 cells) under differing tissue culture conditions. To investigate early cellular attachment, organization, and differentiation, cells were also stained for integrin,,5,1, actin, and runt-related transcription factor (RUNX-2), respectively, after incubation on HA, SA, and TMX discs for 3 days. No significant differences emerged between HA, SA, and TMX discs in mean numbers of cells attached in serum free medium (SFM) over 90,min incubation. In contrast, significantly more cells were attached to SA than HA after 180,min incubation in complete medium (C-MEM) containing fetal calf serum (p,<,0.05). Cell attachment to SA and HA discs pre-conditioned in SFM supplemented with fibronectin (FN) was lower than discs pre-conditioned in C-MEM, suggesting sensitivity of an active FN conformation to the presence of co-adsorbates. Confocal microscopy demonstrated significantly more co-localization of integrin ,5,1 and actin on SA than HA. Translocalization of RUNX-2 to the nucleus was stronger in cells incubated on SA. Microporosity did not diminish the effect of surface physiochemistry on cell adhesion, and enhanced cell attachment for SA appears to be mediated by differences in the quality of adsorbed protein rather than via direct effects of substrate chemistry. [source] Biocompatibility of various root canal filling materials ex vivoINTERNATIONAL ENDODONTIC JOURNAL, Issue 8 2008R. Scotti Abstract Aim, To evaluate the biocompatibility of a resin-based endodontic filler (RealSeal) using the indirect cytotoxicity test. Methodology, Human gingival fibroblasts were cultured ex vivo. Pellets of the materials to be tested were incubated for 24, 48, and 72 h at 37 °C under sterile conditions to obtain their eluates. The fibroblasts were exposed to either diluted (50%) or undiluted eluates for 24 h. A culture medium with foetal calf serum was added to the control wells. Cell viability was estimated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method. The data concerning cell viability were statistically analyzed using one-way anova test and Bonferroni multiple comparisons test. Results, Eluates obtained after 24 h of incubation with the resin filler did not reduce cellular viability. An increase in cellular viability, as compared with control cells, was observed in the gutta-percha group. The undiluted eluate from the polyether material was cytotoxic, causing an 82 ± 4% decrease in cellular viability. Eluates obtained after 48 h of incubation with the resin filler increased cellular viability, whereas the polyether significantly reduced viability. Gutta-percha did not cause any detectable change. After 72 h of incubation the eluate of the resin filler caused an increase in cellular viability, as did gutta-percha, whereas polyether caused a significant decrease. Conclusions, RealSeal resin filler was nontoxic in this laboratory model. Further investigations are necessary to verify its usefulness in clinical applications. [source] Influence of the growth phase and culture medium on the survival of Mannheimia haemolytica during storage at different temperaturesJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2004E. van Rensburg Abstract Aims:, To quantify the influence of the growth phase, storage temperature and nutritional quality of the plate count medium on the apparent viability of Mannheimia haemolytica during storage at different temperatures. Methods and Results:,Mannheimia haemolytica was grown in shake flasks and in aerobic continuous culture to investigate factors affecting cell viability during storage, which was determined using plate counts on different media and epifluorescence microscopy. The high specific death rates of cells harvested after cessation of exponential growth and stored at 22, 4, ,18 and ,75°C could be related to the rapid onset of exponential death in batch cultures. Yeast extract supplementation of the culture medium increased the viability of cells at most of the above-mentioned storage temperatures. Of the total cell count in continuous culture, only 48% could be recovered on brain,heart infusion agar, whereas supplementation of the agar medium with foetal calf serum increased the plate count to 71% of the total count. Conclusions:,Mannheimia haemolytica cells harvested from the exponential growth phase had the highest survival rate during storage at low temperatures. Plate count values also depended on the nutritional quality of the agar medium. Significance and Impact of the Study:, Results presented here impact on the procedures for culture preservation and plate count enumeration of this fastidious animal pathogen. [source] Genetically Manipulated Human Skeletal Myoblast Cells for Cardiac TransplantationJOURNAL OF CARDIAC SURGERY, Issue 6 2002Kh H Haider Aim: Considering the promise of skeletal myoblast cell transplantation to improve cardiac function in myocardial myopathies, we aim in the present study to investigate the potential of human skeletal myoblast cells (HSMC) as a carrier for therapeutic genes for the heart muscle. Methods: Skeletal muscle sample is obtained from rectus femoris of the donor and is processed in the tissue culture to generate HSMC by a patented process of Cell Therapy Inc. The HSMC are grown in large 225 mm2 tissue culture flasks coated with collagen for enhanced cell adherence, using patented Super Medium (Cell Therapy Inc., Singapore) containing 10% fetal calf serum, to 80% confluence. The HSMC are passaged at regular time intervals of 48-72 hours to prevent in vitro differentiation. The HSMC thus obtained are transduced three times with retroviral vector carrying Lac-Z reporter gene before transplantation. The Lac-Z transduced HSMC are harvested by trypsinization, washed and re-suspended in serum free Super Medium. Ischemic Porcine model is created by clamping ameroid ring around left circumflex coronary artery in Yorkshire swine, four weeks prior to cell transplantation. For cell transplantation, the animal is anaesthetized, ventilated and heart is exposed by left thoracotomy. Fifteen injections (0.25 ml each) containing 300 million cells are injected in to the left ventricle endocardially under direct vision. For control animal, only culture medium without cells is injected. The animal is euthanized at pre-determined time, heart is explanted and processed for histological examination. The cryosectioning of the tissue and subsequent staining for Lac-Z expression and Hematoxylin-Eosin staining is carried out by standard methods. Results: The skeletal muscle samples processed by the patented method of Cell Therapy yield 85-90% pure HSMC. The preliminary data shows that repeated transductions of myoblast cells with retrovirus carrying Lac-Z yield highly efficient 70-75% Lac-Z positive HSMC population (Figure 1). Dye exclusion test using Trypan blue reveals >95% cell viability at the time of injection. Gross sections of the cardiac tissue stained positive for Lac-Z expression (Figure 2). Histological examination showed the presence of grafted myoblast cells expressing Lac-Z gene in the cardiac tissue (Figure 3). Conclusion: In the light of our preliminary results, we conclude that HSMC may prove to be excellent carriers of transgene for cardiac muscle cells which otherwise are refractory to ordinary gene transfection methods. The use of HSMC mediated gene delivery to cardiac muscle is safer as compared to direct injection of viral vectors in to the heart muscle. Furthermore, the grafted myoblast cells will additionally serve to strengthen the weakened heart muscle. Figure 1.Human Skeletal myoblasts transduced with Lac-Z carrying retrovirus and stained with x-gal. Figure 2.Gross sections of heart muscle stained for Lac-Z expression. Figure 3.X-gal stained porcine heart muscle counter-stained with Eosin. The heart was explanted 6 weeks after transplantation of Lac-Z stained human myoblasts. The arrow shows Lac-Z expressing myoblast cells. [source] Heparin modulates the growth and adherence and augments the growth-inhibitory action of TNF-, on cultured human keratinocytesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2004Ilkka T. Harvima Abstract Previous works suggest the involvement of mast cells in the epithelialization of chronic wounds. Since heparin is a major mediator stored in the secretory granules of mast cells, the purpose of this work was to elucidate the function of heparin in epithelialization using in vitro culture models. For this, low- and high-calcium media in monolayer and epithelium cultures of keratinocytes were used. Also, an assay based on keratinocyte adherence onto plastic surface was used as well. Heparin (0.02,200 ,g/ml) inhibited keratinocyte growth in a non-cytotoxic and dose-dependent manner in low- and high-calcium media, Keratinocyte-SFM® and DMEM, in the absence of growth factors and serum. Also, heparin inhibited the growth of keratinocyte epithelium in the presence of 10% fetal calf serum and DMEM. Instead, in the presence of Keratinocyte-SFM and growth factors, heparin at 2 ,g/ml inhibited the growth by 18% but at higher heparin concentrations the inhibition was reversed to baseline. TNF-, is another preformed mediator in mast cell granules and it inhibited keratinocyte growth in monolayer and epithelium cultures. Interestingly, heparin at 2,20 ,g/ml augmented or even potentiated this growth-inhibitory effect of TNF-,. The association of TNF-, with heparin was shown by demonstrating that TNF-, bound tightly to heparin-Sepharose chromatographic material. However, heparin could not augment TNF-,-induced cell cycle arrest at G0/G1 phase or intercellular adhesion molecule-1 expression in keratinocytes. In the cell adherence assay, heparin at 2 ,g/ml inhibited significantly by 12,13% or 33% the adherence of keratinocytes onto the plastic surface coated with fibronectin or collagen, respectively, but this inhibition was reversed back to baseline at 20 or 200 ,g/ml heparin. Also, heparin affected the cell membrane rather than the protein coat on the plastic surface. In conclusion, heparin not only inhibits or modulates keratinocyte growth and adherence but it also binds and potentiates the growth-inhibitory function of TNF-,. © 2004 Wiley-Liss, Inc. [source] Induction of oxidative stress by homocyst(e)ine impairs endothelial function,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2001Vibhas S. Mujumdar Abstract Previous studies have demonstrated a relationship between hyperhomocysteinemia and endothelial dysfunction, reduced bioavailability of nitric oxide, elastinolysis and, vascular muscle cell proliferation. In vivo decreased nitric oxide production is associated with increased matrix metalloproteinase (MMP) activity and formation of nitrotyrosine. To test the hypothesis that homocysteine neutralizes vascular endothelial nitric oxide, activates metalloproteinase, causes elastinolysis and vascular hypertrophy, we isolated aortas from normotensive Wistar rats and cultured them in medium containing homocysteine, and calf serum for 14 days. Homocysteine-mediated impairment of endothelial-dependent vasodilatation was reversed by co-incubation of homocysteine with nicotinamide (an inhibitor of peroxinitrite and nitrotyrosine), suggesting a role of homocysteine in redox-mediating endothelial dysfunction and nitrotyrosine formation. The Western blot analysis, using anti-nitrotyrosine antibody, on aortic tissue homogeneates demonstrated decreased nitrotyrosine in hyperhomocysteinemic vessels treated with nicotinamide. Zymographic analysis revealed increased elastinolytic gelatinase A and B (MMP-2, -9) in homocysteine treated vessels and the treatment with nicotinamide decreases the homocysteine-induced MMP activation. Morphometric analyses revealed significant medial hypertrophic thickening (1.4,±,0.2-fold of control, P,=,0.03) and elastin disruption in homocysteine-treated vessels as compared to control. To determine whether homocysteine causes endothelial cell injury, cross-sections of aortas were analyzed for caspase activity by incubating with Ac-YVAD-AMC (substrate for apoptotic enzyme, caspase). The endothelium of homocysteine treated vessels, and endothelial cells treated with homocysteine, showed marked labeling for caspase. The length-tension relationship of homocysteine treated aortas was shifted to the left as compared to untreated aortas, indicating reduced vascular elastic compliance in homocysteine-treated vessels. Co-incubation of homocysteine and inhibitors of MMP, tissue inhibitor of metalloproteinase-4 (TIMP-4), and caspase, YVAD-CHO, improved vascular function. The results suggest that alteration in vascular elastin/collagen ratio and activation of MMP-2 are associated with decreased NO production in hyperhomocysteinemia. J. Cell. Biochem. 82:491,500, 2001. © 2001 Wiley-Liss, Inc. [source] Role of phospholipases A2 in growth-dependent changes in prostaglandin release from 3T6 fibroblastsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2001Teresa Sánchez Previously, we reported a growth-dependent change in prostaglandin production as a consequence of a marked growth-dependent alteration in arachidonic acid (AA) mobilization from phospholipids. Our present results show that fetal calf serum (FCS) and 4,-phorbol-12-myristate acetate (PMA) caused an enhancement of phospholipase A2 (PLA2) activity in the membrane fraction of non-confluent cells allowing PLA2 access to its substrate and the release of AA. Western blot analysis has shown that FCS and PMA increased secreted PLA2 (sPLA2) expression in non-confluent 3T6 fibroblast cultures. Moreover, FCS and PMA induced dithiothreitol-sensitive and bromoenol lactone-sensitive PLA2 activities in cytosol and membrane fraction. However, these stimuli did not modify significantly the PLA2 activity in both fractions when 3T6 fibroblasts reached a high cell density. This could be associated with the impairment of AA mobilization in these cell culture conditions. On the other hand, we observed that FCS and PMA induced the same prostaglandin H synthase-2 induction in non-confluent and confluent culture conditions. Moreover, the prostaglandin E2 levels reached in cell culture supernatants were independent of the degree of confluence when AA was added exogenously. These results suggest that the changes of intracellular distribution of PLA2 activity of sPLA2 and iPLA2 stimulated by exogenous stimuli may be controlled by cell density conditions which constitute an important mechanism in the regulation of prostaglandin release.© 2001 Wiley-Liss, Inc. [source] Ex vivo expansion of apheresis-derived peripheral blood hematopoietic progenitorsJOURNAL OF CLINICAL APHERESIS, Issue 1 2002Zeev Estrov Because the administration of hematopoietic growth factors and the use of stem cell support often fails to alleviate the neutropenic phase induced by cytotoxic drugs, several investigators have attempted to expand ex vivo hematopoietic progenitors for clinical use. These attempts have clearly shown that the cultured cells are functional and can be safely administered to patients, but that the in vivo performance is disappointing and the concept as a whole is not yet clinically useful. The major reasons for these unsuccessful attempts are thought to be cumbersome cell fractionation techniques, contamination, prolonged incubation, and the use of less than ideal cytokine combinations. In response, we have developed a simple procedure for ex vivo expansion of myeloid progenitor cells. In this assay, unfractionated mononuclear cells from apheresis donors are incubated in nonpyrogenic plastic bags for 7 days in the presence of culture medium either containing fetal calf serum or human plasma, granulocyte colony-stimulating factor, and stem cell factor. We have demonstrated that under these conditions the number of colony-forming units (CFU) granulocyte-macrophage (CFU-GM) and of CFU-granulocyte-macrophage-erythroid-megakaryocyte (CFU-GEMM) increased 7- and 9-fold, respectively, by day 7 and the number of burst-forming units-erythroid (BFU-E) increased 2.7-fold by day 5 of culture. Significant increases in the numbers of cells expressing CD34+, CD34+/CD38+, CD34+/CD33+, CD34+/CD15+, and CD34+/CD90+ and significant declines in the numbers of cells expressing CD34+/CD38- and CD19 surface antigens were also observed. The relative numbers of cells expressing T-cell markers and CD56 surface antigen did not change. By using different concentrations of various hematopoietic growth factor combinations, we can increase the number of mature and immature cells of different hematopoietic lineages. J. Clin. Apheresis 17:7,16, 2002. © 2002 Wiley-Liss, Inc. [source] In vivo magnetic resonance imaging of iron oxide,labeled, arterially-injected mesenchymal stem cells in kidneys of rats with acute ischemic kidney injury: Detection and monitoring at 3TJOURNAL OF MAGNETIC RESONANCE IMAGING, Issue 6 2007Harald Ittrich MD Abstract Purpose To evaluate MRI for a qualitative and quantitative in vivo tracking of intraaortal injected iron oxide,labeled mesenchymal stem cells (MSC) into rats with acute kidney injury (AKI). Materials and Methods In vitro MRI and R2* measurement of nonlabeled and superparamagnetic iron oxide (SPIO)-labeled MSC (MSCSPIO) was performed in correlation to cellular iron content and cytological examination (Prussian blue, electron microscopy). In vivo MRI and R2* evaluation were performed before and after ischemic/reperfusion AKI (N = 14) and intraaortal injection of 1.5 × 106 MSCSPIO (N = 7), fetal calf serum (FCS) (medium, N = 6), and SPIO alone (N = 1) up to 14 days using a clinical 3T scanner. Signal to noise ratios (SNR), R2* of kidneys, liver, spleen, and bone marrow, renal function (creatinine [CREA], blood urea nitrogen [BUN]), and kidney volume were measured and tested for statistical significance (Student's t -test, P < 0.05) in comparison histology (hematoxylin and eosin [H&E], Prussian blue, periodic acid-Schiff [PAS], CD68). Results In vitro, MSCSPIO showed a reduction of SNR and T2* with R2* , number of MSCSPIO (R2 = 0.98). In vivo MSCSPIO administration resulted in a SNR decrease (35 ± 15%) and R2* increase (101 ± 18.3%) in renal cortex caused by MSCSPIO accumulation in contrast to control animals (P < 0.01). Liver, spleen, and bone marrow (MSCSPIO) showed a delayed SNR decline/R2* increase (P < 0.05) resulting from MSCSPIO migration. The increase of kidney volume and the decrease in renal function (P < 0.05) was reduced in MSC-treated animals. Conclusion Qualitative and quantitative in vivo cell-tracking and monitoring of organ distribution of intraaortal injected MSCSPIO in AKI is feasible in MRI at 3T. J. Magn. Reson. Imaging 2007;25:1179,1191. © 2007 Wiley-Liss, Inc. [source] Characterization by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry of the major photoproducts of temoporfin (m -THPC) and bacteriochlorin (m -THPBC)JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 7 2001Marc Angotti Abstract The photobleaching of 5,10,15,20-tetrakis(m -hydroxyphenyl)chlorin (temoporfin, m -THPC) and 5,10,15,20-tetrakis(m -hydroxyphenyl)bacteriochlorin (bacteriochlorin, m -THPBC) was studied in ethanol,water (1 : 99, v/v) and in physiological medium (phosphate-buffered saline, PBS) with or without fetal calf serum (FCS). m -THPC solution was irradiated with the laser radiation of 650 nm, whereas m -THPBC solution underwent two consecutive irradiations at 532 and 650 nm. The photoproducts were characterized by UV,visible absorption spectrophotometry and by matrix-assisted laser desorption/ionization (MALDI) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). Independent of the solvent used, the phototransformation of either photosensitizer yielded the formation of 5,10,15,20-tetrakis (m -hydroxyphenyl)porphyrin (m -THPP) through a major dehydrogenation process. Copyright © 2001 John Wiley & Sons, Ltd. [source] Homeostasis of neuroactive amino acids in cultured cerebellar and neocortical neurons is influenced by environmental cuesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1-2 2005Helle Waagepetersen Abstract Neuronal function is highly influenced by the extracellular environment. To study the effect of the milieu on neurons from cerebellum and neocortex, cells from these brain areas were cultured under different conditions. Two sets of cultures, one neocortical and one cerebellar neurons, were maintained in media containing [U- 13C]glucose for 8 days at initial concentrations of 12 and 28 mM glucose, respectively. Other sets of cultures (8 days in vitro) maintained in a medium containing initially 12 mM glucose were incubated subsequently for 4 hr either by addition of [U- 13C]glucose to the culture medium (final concentration 3 mM) or by changing to fresh medium containing [U- 13C]glucose (3 mM) but without glutamine and fetal calf serum. 13C Nuclear magnetic resonance (NMR) spectra revealed extensive ,-aminobutyric acid (GABA) synthesis in both cultured neocortical and cerebellar neurons after maintenance in medium containing [U- 13C]glucose for 8 days, whereas no aspartate labeling was observed in these spectra. Mass spectrometry analysis, however, revealed high labeling intensity of aspartate, which was equal in the two types of neurons. Addition of [U- 13C]glucose (4 hr) on Day 8 in culture led to a similar extent of labeling of GABA in neocortical and in cerebellar cultures, but the cellular content of GABA was considerably higher in the neocortical neurons. The cellular content of alanine was similar regardless of culture type. Comparing the amount of labeling, however, cerebellar neurons exhibited a higher capacity for alanine synthesis. This is compatible with the fact that cerebellar neurons could ameliorate a low alanine content after culturing in low glucose (12 mM) by a 4-hr incubation in medium containing 3 mM glucose. A low glucose concentration during the culture period and a subsequent medium change were associated with decreases in glutathione and taurine contents. Moreover, glutamate and GABA contents were reduced in cerebellar cultures under either of these conditions. In neocortical neurons, the GABA content was decreased by simultaneous exposure to low glucose and change of medium. These conditions also led to an increase in the aspartate content in both types of cultures, although most pronounced in the neocortical neurons. Further experiments are needed to elucidate these phenomena that underline the impact of extracellular environment on amino acid homeostasis. © 2004 Wiley-Liss, Inc. [source] Carrier proteins determine local pharmacokinetics and arterial distribution of paclitaxelJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 9 2001Mark A. Lovich Abstract The growing use of local drug delivery to vascular tissues has increased interest in hydrophobic compounds. The binding of these drugs to serum proteins raises their levels in solution, but hinders their distribution through tissues. Inside the arterial interstitium, viscous and steric forces and binding interactions impede drug motion. As such, this might be the ideal scenario for increasing the amount of drug delivered to, and residence time within, arterial tissues. We quantified carrier-mediated transport for paclitaxel, a model hydrophobic agent with potential use in proliferative vascular diseases, by determining, in the presence or absence of carrier proteins, the maximum concentration of drug in aqueous solution, the diffusivity in free solution, and the diffusivity in arterial tissues. Whereas solubility of paclitaxel was raised 8.1-, 21-, and 57-fold by physiologic levels of ,1 -acid glycoproteins, bovine serum albumin, and calf serum over that in protein-free solution, diffusivity of paclitaxel in free solution was reduced by 41, 49, and 74%, respectively. When paclitaxel mixed in these solutions was applied to arteries both in vitro and in vivo, drug was more abundant at the tissue interface, but protein carriers tended to retain drug in the lumen. Once within the tissue, these proteins did not affect the rate at which drug traverses the tissue because this hydrophobic drug interacted with the abundant fixed proteins and binding sites. The protein binding properties of hydrophobic compounds allow for beneficial effects on transvascular transport, deposition, and distribution, and may enable prolonged effect and rationally guide local and systemic strategies for their administration. © 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1324,1335, 2001 [source] Pharmacokinetics and pharmacokinetic/pharmacodynamic integration of marbofloxacin in calf serum, exudate and transudateJOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 3 2002F. SHOJAEE ALIABADI Aliabadi, F. S., Lees, P. Pharmacokinetics and pharmacokinetic/pharmacodynamic integration of marbofloxacin in calf serum, exudate and transudate. J. vet. Pharmacol. Therap.25, 161,174. Marbofloxacin is a fluoroquinolone antimicrobial drug used in cattle for the treatment of respiratory infections. In this investigation the pharmacokinetics (PK) of marbofloxacin were determined after intravenous and intramuscular dosing at a dosage of 2 mg/kg. In addition the ex vivo pharmacodynamics (PD) of the drug were determined in serum and three types of tissue cage fluid (transudate, inflammatory exudate generated by carrageenan and exudate generated by lipopolysaccharide). Marbofloxacin PK was characterized by a high volume of distribution after dosing by both routes (1.28 L/kg intravenous and 1.25 L/kg intramuscular). Corresponding area under the concentration,time curve (AUC) and elimination half-life (t˝el) values were 9.99 and 10.11 ,g h/mL and 4.23 and 4.33 h, respectively. Values of AUC for carrageenan-induced exudate, lipopolysaccharide-induced exudate and transudate were, respectively, 8.28, 7.83 and 7.75 ,g h/mL after intravenous and 8.84, 8.53 and 8.52 ,g h/mL after intramuscular dosing. Maximum concentration (Cmax) values were similar for the three tissue cage fluids after intravenous and intramuscular dosing. For in vivo PK data values of AUC: minimum inhibitory concentration (MIC) (AUIC) ratio for serum were 250 and 253, respectively, after intravenous and intramuscular dosing of marbofloxacin against a pathogenic strain of Mannheimia haemolytica (MIC=0.04 ,g/mL). For all tissue cage fluids AUIC values were >194 and >213 after intravenous and intramuscular dosing, and Cmax/MIC ratios were 9 or greater, indicating a likely high level of effectiveness in clinical infections caused by M. haemolytica of MIC 0.04 ,g/mL or less. This was confirmed by both in vitro (serum) and ex vivo (serum, exudate and transudate) measurements, which demonstrated a concentration-dependent killing profile for marbofloxacin against M. haemolytica. Ex vivo, after 24-h incubation, virtually all bacteria were killed (<10 cfu/mL) in all samples collected up to 9 h (serum), 24 h (carrageenan-induced exudate and transudate) and 36 h (lipopolysaccharide-induced exudate). Application of the sigmoid Emax equation to the ex vivo antibacterial data provided, for serum, AUIC24 h values of 37.1 for bacteriostasis, 46.3 for bactericidal activity and 119.6 for elimination of bacteria. These data may be used as a rational basis for setting dosing schedules which optimize clinical efficacy and minimize the opportunities for emergence of resistant organisms. [source] Comparison by restriction fragment differential display RT‐PCR of gene expression pattern in bovine oocytes matured in the presence or absence of fetal calf serumMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 1 2001S. Jacek Rzucidlo Abstract A novel restriction fragment differential display (RFDD) RT‐PCR has been used to compare patterns of mRNA expression in bovine oocytes matured in vitro in the presence (10%) or absence of fetal calf serum (FCS). Total RNA extracted from matured and denuded oocytes was processed using display Profile kit (Display System Biotech). RFDD RT‐PCR products were separated on 6% polyacrylamide gel and analyzed using a Storm 860 scanner. Selected bands representing potentially differentially expressed fragments were excised from the gel and re‐amplified. Re‐amplified fragments with size matched to the original fragment were cloned into the TA vector and sequenced. Initially, 10 and 15 differentially expressed fragments were isolated from oocytes matured in the presence and absence of FCS, respectively. Eight out of 10 and 10 out of 15 fragments were re‐amplified successfully as evidenced bysize similarity to the original fragments. Finally, the size of six inserts sequenced from each group matched the size of corresponding original as well as re‐amplified fragments. Sequence comparison search revealed similarity of some isolated fragments to 18s ribosomal RNA, bovine apolipoprotein A‐I, bovine mitochondrion DNA, human CGI‐79 mRNA, human Ab1‐interactor protein, and bovine satellite DNA. The other sequenced fragments may represent novel genes. We showed that RFDD RT‐PCR can be effectively applied to contrast gene expression pattern in bovine oocytes and that presence or absence of FCS during maturation interval affects gene expression pattern in matured bovine oocytes. Mol. Reprod. Dev. 59:90–96, 2001. © 2001 Wiley‐Liss, Inc. [source] Effects of intravenous anesthetics on interleukin (IL)-6 and IL-10 production by lipopolysaccharide-stimulated mononuclear cells from healthy volunteersACTA ANAESTHESIOLOGICA SCANDINAVICA, Issue 2 2002M. Takaono Background: Surgical trauma has been shown to augment the plasma concentrations of proinflammatory cytokines, which are important mediators of host defense mechanisms and the systemic inflammatory response syndrome (SIRS). Recently, it has been shown that certain kinds of surgery provoke not only a proinflammatory response (SIRS) but also a concurrent anti-inflammatory response. The aim of this study was therefore to examine the effects of intravenous anesthetics on the synthesis of interleukin (IL)-6 (a proinflammatory cytokine) and IL-10 (an anti-inflammatory cytokine) by lipopolysaccharide (LPS)-stimulated mononuclear cells from healthy volunteers. Methods: Peripheral blood mononuclear cells (PBMCs) from 17 healthy volunteers, separated by centrifugation on a Ficoll-Hypaque gradient, were washed and suspended in RPMI containing 10% heat-inactivated fetal calf serum (FCS). After adding RPMI-FCS containing various concentrations of intravenous anesthetics (propofol, thiopental, ketamine and midazolam), the PBMCs were incubated overnight in the presence of a submaximal concentration of LPS. The supernatants were collected and their IL-6 and IL-10 contents were assayed using enzyme-linked immunosorbent assay kits. Results: Propofol inhibited both IL-6 and IL-10 production at 0.5 µg/mL, 5 µg/mL and 50 µg/mL. Conversely, thiopental induced IL-10 production at 2 µg/mL and 20 µg/mL. Conclusion: Propofol appears to inhibit both IL-6 and IL-10 production by LPS-stimulated PBMCs in vitro. Further study is required to clarify the mechanism of the suppressive effect of propofol. [source] Establishment and Characterization of a Normal Melanocyte Cell Line Derived from Pig SkinPIGMENT CELL & MELANOMA RESEARCH, Issue 4 2003Sophia Julé Several minipig strains develop spontaneous malignant melanoma. As a first step toward the analysis of genes involved in the tumoral progression of melanoma in these animal models, we developed culture conditions for pig melanocytes whereby melanocytes from normal epidermis can be isolated directly onto mitotically inactivated keratinocytes in Eagle's minimal essential medium supplemented with fetal calf serum, tetradecanoyl phorbol acetate (TPA) and cholera toxin. We also derived an immortal line of pigmented melanocytes from the epidermis of a healthy Meishan pig. This cell line, designated PigMel, retains differentiation function in culture, dependence on TPA and cholera toxin and a diploid chromosome number. PigMel melanocytes exhibit morphological and molecular characteristics common to normal mammalian skin melanocytes. [source] Analysis of pyridoquinoline derivatives by liquid chromatography/atmospheric pressure chemical ionization mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 11 2001Y. Picó A method using liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) has been developed for the characterization and determination of pyridoquinoline derivatives 4,6-bis(dimethylaminoethylamino)-2,8,10-trimethylpyrido[3,2-g]quinoline, 4,6-bis(dimethylaminoethoxy)-2,8,10-trimethylpyrido[3,2-g]quinoline and 4,6-bis[(dimethylaminoethyl)thio]-2,8,10-trimethylpyrido[3,2-g] quinoline, all with potential antitumor properties. LC separation was performed on a conventional C18 column using a binary mobile phase composed of acetonitrile and 50,mM aqueous ammonium formate at pH 3. The APCI mass spectra obtained showed that proton addition giving [M,+,H]+ was the common mode of ionization to the amino- and thiopyridoquinolines, whereas the alkoxypyridoquinoline was identified by the main formation of the [M,,,(C2H3)N(CH3)2,+,H]+, followed by the [M,+,H]+ ion. The LC separation conditions and MS detection parameters were optimized for the determination. The analytical method was also applied to the determination of these pyridoquinoline derivatives in fetal calf serum using liquid-liquid extraction with dichloromethane. Acceptable recovery values were obtained, ranging between 45 and 98%. Copyright © 2001 John Wiley & Sons, Ltd. [source] Use of Fetuin Before and During Vitrification of Bovine OocytesREPRODUCTION IN DOMESTIC ANIMALS, Issue 3 2008G Horvath Contents After vitrification of oocytes, fertilization rates and subsequent development are unsatisfactory, possibly due in part to zona hardening. Foetal calf serum (FCS) can prevent zona hardening because of its fetuin content, but FCS composition varies among batches, and may contain viruses. In this study, we therefore compared media supplemented with different sources of macromolecules, 2% bovine serum albumin (BSA), 2% BSA + 1 mg/ml fetuin and 20% FCS, for handling oocytes for 10,30 min prior to vitrification. None of the treatments resulted in developmental rates comparable with the non-vitrified controls, but FCS inclusion in pre-vitrification handling medium resulted in higher blastocyst production per oocyte (p < 0.05) (10.8%) on day 9 of culture than BSA (5.3%) or BSA + fetuin (6.4%). Blastocysts developing from oocytes from all vitrification treatments were somewhat retarded relative to those developed from non-vitrified oocytes. We also tested the use of fetuin during vitrification as well as two different exposure times with cryoprotectants, 180 and 30 s. There was no significant effect of fetuin or exposure time on rates of subsequent blastocyst production. [source] Comparative IFN- , Secretion after Hatching by Bovine Blastocysts Derived Ex Vivo and Completely Produced In VitroREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2007JA Neira Contents The interferon-tau (IFN- ,) secretion levels after hatching by bovine blastocysts derived from in vitro maturated oocytes (Group A) and from in vivo (Group B) were investigated considering embryo quality. Only very homogeneous blastocysts of excellent or good quality were considered from day 7 of culture (Group A) and day 7 after artificial insemination with frozen-thawed from the same bull used for in vitro fertilization (Group B). All embryos were individually cultured into a 50 ,l droplet of synthetic oviduct fluid medium with 10% fetal calf serum. After 24-h culture both Group A (n =44) and B (n = 40) secreted <54 pm IFN- ,. After 48-, 72-, 96- and 120-h culture, Group A daily secreted 143 ± 24 pm IFN- , (n = 19) vs 85 ± 12 pm IFN- , (n = 21) for Group B (p < 0.01), 491 ± 128 pm IFN- , (n = 29) vs 216 ± 37 pm IFN- , (n = 23) (NS), 499 ± 135 pm IFN- , (n = 26) vs 353 ± 93 pm IFN- , (n = 21) (NS), 559 ± 136 pm IFN- , (n = 22) vs 333 ± 75 pm IFN- , (n = 20) (NS), respectively. Taken all together during 5 days, Group A produced per embryo 1690 ± 290 pm IFN- , (n = 22) vs 982 ± 182 pm IFN- , (n = 20) for Group B (p < 0.05). For all culture time there were sizable percentages of embryos that did not produce concentrations of IFN- , above a certain cut-off level, and as such were not used to compute the means. In respect of the embryo quality whatever the groups after days 7,12 of culture, IFN- , secretions were 1815 ± 453 pm (n = 10) for the embryos of excellent quality vs 1356 ± 200 pm (n = 28) for those of good quality (NS) and 360 ± 188 pm (n = 4) (p < 0.05) for embryos of fair quality. A positive relationship between IFN- , production and in vitro development of quality I embryos was observed, whatever the embryos origins and, the embryos completely produced in vitro secreted more IFN- , than the embryos produced in vivo. [source] Isolation of a porcine UDP-GalNAc transferase cDNA mapping to the region of the blood group EAA locus on pig chromosome 1ANIMAL GENETICS, Issue 3 2001E. Meijerink In our studies of the genes constituting the porcine A0 blood group system, we have characterized a cDNA, encoding an ,(1,3)N-acetylgalactosaminyltransferase, that putatively represents the blood group A transferase gene. The cDNA has a 1095-bp open reading frame and shares 76.9% nucleotide and 66.7% amino acid identity with the human ABO gene. Using a somatic cell hybrid panel, the cDNA was assigned to the q arm of pig chromosome 1, in the region of the erythrocyte antigen A locus (EAA), which represents the porcine blood group A transferase gene. The RNA corresponding to our cDNA was expressed in the small intestinal mucosae of pigs possessing EAA activity, whereas expression was absent in animals lacking this blood group antigen. The UDP-N-acetylgalactosamine (UDP-GalNAc) transferase activity of the gene product, expressed in Chinese hamster ovary (CHO) cells, was specific for the acceptor fucosyl- ,(1,2)galactopyranoside; the enzyme did not use phenyl- , - D -galactopyranoside (phenyl- , -D-Gal) as an acceptor. Because the ,(1,3)GalNAc transferase gene product requires an ,(1,2)fucosylated acceptor for UDP-GalNAc transferase activity, the ,(1,2)fucosyltransferase gene product is necessary for the functioning of the ,(1,3)GalNAc transferase gene product. This mechanism underlies the epistatic effect of the porcine S locus on expression of the blood group A antigen. Abbreviations: CDS: coding sequence; CHO: Chinese Hamster Ovary; EAA: erythrocyte antigen A; FCS: foetal calf serum; Fuc,(1,2)Gal: fucosyl- ,(1,2)galactopyranoside; Gal: galactopyranoside; GGTA1: Gal,(1,3)Gal transferase; PCR: polymerase chain reaction; phenyl- , -D-Gal: phenyl- , - D -galactopyranoside; R: Gal,1-4Glc,1-1Cer; UDP-GalNAc: uridine diphosphate N-acetylgalactosamine [source] A cellular model system of differentiated human myotubes,APMIS, Issue 11 2001M. GASTER The aim of this study was to select an effective and stable protocol for the differentiation of human satellite cells (Sc) and to identify the optimal time period for the experimental use of differentiated human Sc-cultures. In order to identify the differentiation conditions which give a good survival of myotubes and a high grade of differentiation, Sc-cultures were induced to differentiate in media supplemented with either 2% fetal calf serum (FCS) 2% horse serum (HS) or 10% HS. Based on higher CK-activities in cultures differentiating in FCS-supplemented media compared to horse sera, fetal calf serum was chosen to induce differentiation. The ATP, DNA and protein content increased during the first 4 days after induction of differentiation and was followed by a period with minor changes. The maximal differences of ATP, DNA and protein between days 4,10 were evaluated and the differences in the three components were found to be less than 20% of the average value with a certainity of more than 0.9. Day 8-myotubes were investigated morphologically and were found immunoreactive for fast myosin, and expressed areas with clear cross striation. We recommend the use of differentiated Sc-cultures in the period from day 4 to 8 after induction of differentiation as only minor differentation-related changes will take place in the cells during this period of time. [source] Dose-dependent influences of dietary ,-1,3-glucan on innate immunity and disease resistance of hybrid striped bass Morone chrysops×Morone saxatilisAQUACULTURE RESEARCH, Issue 14 2009Peng Li Abstract To investigate potential use of dietary ,-1,3-glucan for health management of hybrid striped bass, juvenile fish were fed diets supplemented with yeast glucan (MacroGuard®) at 0.05%, 0.1% or 0.2% of diet for 4 weeks, followed by immune response assays and a bath challenge with Streptococcus iniae. Dietary glucan significantly (P<0.05) enhanced neutrophil oxidative radical production, and fish fed 0.1% glucan had a significant (P<0.05) reduction in mortality (10%) after bacterial challenge compared with fish fed the control diet (46.7%). However, accumulative mortality of fish fed 0.2% glucan was not significantly different from that of fish fed the control diet. To further elucidate this observation, macrophages from sub-adult hybrid striped bass were isolated and cultured in L-15 medium with 10% foetal calf serum and penicillin/streptomycin supplemented with 0.2, 0.5, 1, 2, 5, 20 and 100 ,g soluble glucan (MacroGuard®) mL,1 for 24 and 48 h. Intracellular superoxide anion production was significantly (P<0.001) increased by 0.5 ,g glucan mL,1, but significantly (P<0.001) suppressed by doses >5 ,g glucan mL,1. It is concluded that dietary yeast glucan has potential for use in diet formulations of hybrid striped bass to limit the adverse effects of S. iniae, but dosage should be an important consideration in administration. [source] Nuclear transfer in loach (Paramisgurnus dabryanus Sauvage) by cell-to-cell electrofusionAQUACULTURE RESEARCH, Issue 4 2001F Hongtuo Abstract To study nuclear transfer in the loach (Paramisgurnus dabryanus Sauvage), blastula and gastrula cells were fused with UV-inactivated oocytes by cell-to-cell electrofusion. To facilitate nuclear transfer, blastula and gastrula cells were cultured or incubated at 4 °C in different solutions. TC-199 medium supplemented with 20% calf serum was the best culture solution, and effectively retained the totipotence of blastula or gastrula cells for up to 10 days. It was found that gastrula cells incubated at 4 °C had the same totipotence as blastula cells. The optimal UV dosage for inactivation of the oocyte chromatin was 180,240 mJ cm,2. Electrofusion was carried out in a cone-shaped fusion chamber, which permitted the recipient oocyte and the donor blastula cell to contact one another. The electrofusion procedure resulted in a 10% success rate of normal-appearing fish. Genetic analysis indicated that the nuclear material originated from the donor cell (blastomere) and the oocyte pronucleus did not take part in development. [source] 0.2 T magnetic field inhibits angiogenesis in chick embryo chorioallantoic membraneBIOELECTROMAGNETICS, Issue 5 2004Marco Ruggiero Abstract Inhibition of angiogenesis is a major target in the fight against cancer and other diseases. Although the effects of static magnetic fields on cancer development and cell growth have been investigated, effects on angiogenesis have received no attention so far. In this study we report the effects on angiogenesis of exposure to 0.2 T static magnetic field. Angiogenesis was analyzed using the chick embryo chorioallantoic membrane assay. Exposure to 0.2 T static magnetic field was achieved by placing the eggs for 3 hr in the isocentre of the magnet of a sectorial magnetic resonance tomograph used in clinical practice. In sham exposed specimens treated with phosphate buffered saline (negative control), no significant vascular reaction was detectable; 3 hr exposure to 0.2 T static magnetic field did not affect the basal pattern of vascularization or chick embryo viability. Prostaglandin E1 and fetal calf serum elicited a strong angiogenic response in sham exposed eggs. This angiogenic response was significantly inhibited by 3 hr exposure to 0.2 T static magnetic field. These findings point to possible use of static magnetic field in inhibiting angiogenesis; this effect could be exploited for treatment of cancer and other diseases where excessive angiogenesis is involved. Bioelectromagnetics 25:390,396, 2004. © 2004 Wiley-Liss, Inc. [source] Oestrogen deficiency causes DNA damage in uterine leiomyoma cells: a possible mechanism for shrinkage of fibroids by GnRH agonistsBJOG : AN INTERNATIONAL JOURNAL OF OBSTETRICS & GYNAECOLOGY, Issue 1 2001Ya-Min Cheng 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 , -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. [source] Folate affects apoptosis in human trophoblastic cellsBJOG : AN INTERNATIONAL JOURNAL OF OBSTETRICS & GYNAECOLOGY, Issue 12 2000R. P. M. Steegers-Theunissen Clinical Epidemiologist Effects of folate deficiency on the rate of apoptosis in human cytotrophoblastic cells has been investigated. Apoptosis was determined in cytotrophoblastic cells after culture in 1. control medium, 2. folate-free medium and 3. folate-free medium plus 10% fetal calf serum. Apoptosis rates in cells cultured in mediums 2 and 3 were significantly higher than those cultured in the control medium (P < 0.02 and P < 0.03, respectively). In conclusion, human cytotrophoblastic cells show a significantly increased rate of apoptosis in vitro after culture in a folate-free medium. Possible explanations for the association between folate deficiency and pregnancy complications are suggested. [source] Induction of proliferation and differentiation of cultured urothelial cells on acellular biomaterialsBJU INTERNATIONAL, Issue 6 2004Gouya Ram-Liebig OBJECTIVE To determine the optimum conditions for the proliferation of urothelial cells, leading to the confluent coverage of large surfaces of biocompatible membranes, and for their terminal differentiation. MATERIALS AND METHODS Porcine and human urothelial cells were cultured on different matrices under different growth conditions. Proliferative activity and the viability of cells were evaluated using fluorescent markers for nuclei and cytoplasm. Growth and differentiation were assessed by histological, histochemical and immunohistochemical methods. RESULTS Under fibroblastic induction and supplementation of 5% fetal calf serum (FCS), urothelial cells showed more proliferation than in other conditions tested. Terminal differentiation of superficial cells was achieved by lowering the concentration of FCS to 1% at the air,liquid interface. CONCLUSIONS The mitogenic effects of the extracellular matrix content of biological membranes and fibroblastic inductive factors are synergistic with each other, and can compensate for a low FCS concentration and the absence of other additives. Lowering the FCS concentration to 1% inhibits the proliferation of urothelial cells and permits their terminal differentiation. [source] Reduction of vascular smooth muscle cell proliferation by immunomodulationBRITISH JOURNAL OF SURGERY (NOW INCLUDES EUROPEAN JOURNAL OF SURGERY), Issue 4 2001Y. C. Chan Background: Immunological factors may play an important role in mediating the progression of atherosclerosis and myointimal hyperplasia, with heat shock proteins being implicated as possible autoantigens. The authors have shown previously that immunomodulation can reduce vascular smooth muscle cell (vSMC) proliferation following balloon injury to rat carotid arteries. The aim of the present study was to examine the effects of immunomodulatory agents on the proliferation of rat aortic vSMCs remote from the area of balloon injury. The agents used were SRL172 (heat-killed Mycobacterium vaccae) and heat shock protein 65 kDa (HSP65) in Freund's incomplete adjuvant. Both these agents are known to influence T-cell responses. Methods: Male Sprague,Dawley rats were used. All immunizations were given subcutaneously. Four groups were studied (ten animals in each group): group 1 animals were immunized with normal saline, group 2 received SRL172, group 3 SRL172 and HSP65,Freund's, and group 4 HSP65,Freund's. Three immunizations were performed as well as carotid balloon injury. Three animals died, leaving 37 for analysis. Some 5 weeks later the animals were killed and the aorta was harvested. Standard explant techniques were applied to grow aortic vSMCs until confluency, passaged three times, quiesced, and fetal calf serum (FCS) of varying concentrations (0·4,10 per cent) was then added, incubated for another 48 h and cell counts carried out. Results: The proliferation rate of aortic vSMCs in the control group was significantly greater than that in the other study groups (Fig.). While all the treatment groups had significantly less proliferation compared with the control group (*P < 0·05, ,P < 0·01, Mann,Whitney U test), no statistically significant differences existed between any of the study groups. Conclusion: Immunomodulation may result in a reduction of vSMC proliferation. Although the precise mechanisms involved are unclear, these results are in concordance with previous findings that T-cell immunomodulation decreases the development of myointimal hyperplasia after injury, and suggest that a fundamental phenotypic shift has been produced by these immunizations. [source] | |||||||||||||||||||