Eagle's MEM Research Articles

Adaptation of african swine fever virus (Asfarviridae: Asfivirus)to growth in the continuous culture PPK-66b cells by the method of accelerated passaging

African swine fever virus (ASF) is a large, enveloped virus with an icosahedral capsid morphology and a double-stranded DNA genome ranging in size from 170 to 190 kb. The replication cycle proceeds in two phases, the early phase lasting 4-6 hours and the late 8-20 hours after infection. The adaptation of the ASF virus to growth in continuous cell lines makes efficient and reliable genetic analysis and more accurate interpretation of its results. Adaptation of a new isolate of the ASF virus to growth in a continuous cell line by the method of accelerated passages and preliminary genetic analysis of the resulting strain. For virus isolation and passaging of the ASF virus, a porcine leukocyte cell culture (PL) and continuous cell cultures of porcine origin (ST, PK, PPK-66b) were used with Eagle MEM and HLA essential media with 10% porcine or fetal serum. The article presents data on the isolation and analysis of the changes in the reproductive properties of a new African swine fever (ASF) virus isolate in the process of adaptation to growth in a continuous piglet kidney cell culture clone b (PPK-66b). The current state of the problem of cultivation of the ASF virus, the features of its reproduction, and the basis of the genetic differentiation of its isolates are described in detail. Understanding the uniqueness of the nature of the ASF virus determined the approaches to the processes of its cultivation and adaptation. In this regard, the results of studies of cultural properties, and analysis of the nucleotide sequence of 6 genes of the new isolate, as well as phylogenetic analysis of these genes with already known strains and isolates of the ASF virus are presented. A new strain obtained in the process of cell adaptation of ASVF/Znaury/PPK-23 ASF virus by the accelerated passaging method reaches a high level of reproduction in 72 hours with an accumulation titer of 7.07 lg HAdE50/cm3. Primary genetic analysis allowed to establish the main phylogenetic relationships of the newly isolated strain with previously known variants of the current ASF panzootic.

The Potential of Cytotoxin and Antiviral in Sargassum polycystum and Sargassum ilicifolium’s Polysaccharides Extract

Marine algae known as one producers of bioactive compounds. This study aims to analyze the cytotoxicity and antiviral activity in Sargassum polycystum and Sargassum ilicifolium tested with Herpes Simplex Virus (HSV). The polysaccharides extract of algae was used in this study, as sulfated polysaccharides have been reported has bioactivity. Cytotoxicity either antiviral could be correlated with the sulfate content as well as nature and chemical composition of the polysaccharides. Cytotoxicity and antiviral analysis based upon cell viability. Using the Vero cell / HSV-1 model, cytotoxicity was evaluated by incubating cellular suspensions (3.5×105 cells.mL-1) with various dilutions (concentration from 1 to 500 µg.mL-1, four wells per concentration) of fractions in 96-well plates (72h, 37°C, 5% CO2) in Eagle's MEM containing 8% FCS. The cells were examined daily under a phase-contrast microscope to determine the minimum concentration of hydrolysate dry matter that induced alterations in cell morphology, including swelling, shrinkage, granularity and detachment. Algae S. illicifolium was found to have the highest cytotoxic content in each solution compared to S. polycystum. Algae S. illicifolium in KOH 4M (cellulose) reached 2,707 µg.ml-1, then HCl pH 2 (fucoidan) was 2,477 µg.ml-1, then CaCl2 2% (fucoidan) was 2,362 µg.ml-1, and in Na2CO3 3% (alginates) was 2,134 µg.ml-1. For antiviral, S. polycystum contained the highest antiviral compounds compared to S. illicifolium with KOH 4M (cellulose) solution was reached 67.02 µg.ml-1. Then in Na2CO3 3% (alginates) which was 33.25 µg.ml-1, then CaCl2 2% (fucoidan) which was 31.62 µg.ml-1,and HCl pH 2 (fucoidan) was 30.08 µg.ml-1. After all, the highest bioactivity compounds was found with KOH 4M (cellulose) for cytotoxicity in S. ilicifolium and antiviral activity in S. polycystum.

Collagen cross-linking treatment increases adhesion in mock corneal grafts.

We tested the hypothesis that collagen cross-linking (CXL) could be used to promote adhesion in mock corneal grafts. Donated human corneal tissue underwent epithelial debridement and was cut into sections measuring 4mm×3mm. Paired sections were sutured together with 10-0 vicryl, forming mock corneal grafts. Looped 6-0 sutures were placed at each end to facilitate tension measurement. Mock grafts underwent CXL before being cultured for 2days in Eagle's MEM culture medium. Control mock grafts did not undergo CXL treatment before culture. Tissue was obtained from 4 donors and a maximum of 2 controls and 2 treated grafts was obtained from each donor. Following the culture period, the 10-0 sutures were cut. The mock grafts were mounted on force transducers and were put under increasing tension until eventually the sections were pulled apart. The mean applied stress required to generate graft failure was calculated for all mock grafts±standard error of the mean. In the control group 0.236±0.09mPa of applied stress was required to cause graft failure, in comparison to 0.691±0.12mPa in the treated group. A paired t-test showed this result to be significant, (p=0.0087). The results of our study are consistent with our hypothesis that CXL treatment could be used to promote early adhesion between separate sections of corneal tissue.

Oxidative actions of hydrogen peroxide in human gingival and oral periosteal fibroblasts: Responses to glutathione and nicotine, relevant to healing in a redox environment

BackgroundThis study aims to validate pro-oxidant actions of nicotine (N), using hydrogen peroxide (H2O2) and the antioxidant glutathione (G) in an in vitro model of human gingival fibroblasts (HGF) and human oral periosteal fibroblasts (HPF); radiolabelled androgens are used as biomarkers of redox status. Oxidative stress is an important mediator of inflammatory repair. The androgen metabolite 5α-dihydrotestosterone (DHT) is an effective biomarker of oxidative stress and healing. Methods6 Cell-lines of HGF and HPF established in confluent monolayer culture were incubated in Eagle's MEM using 14C-testosterone and 14C-4-androstendione as substrate; in conjunction with effective concentrations of N, G and H2O2 established at N250, G3μg/ml and 3%H2O2 w/w, 0.5μl/ml. Combinations of H2O2G and H2O2GN were used in order to compare the oxidative effects of N/H2O2 and their responses to glutathione. At 24h, the medium was solvent extracted, evaporated to dryness and subjected to TLC in a benzene/acetone solvent system 4:1 v/v for the separation of metabolites. The separated metabolites were quantified using a radioisotope scanner. ResultsThe mean trends of 6 cell-lines for both substrates and each cell type demonstrated that the yield of the main metabolite DHT was significantly reduced by N and H2O2 alone (2-fold, n=6; p<0.01). The inhibition caused by H2O2 was overcome by the antioxidant glutathione in the combination H2O2G, to values similar to those of controls (n=6; p<0.01). It is relevant that when N was added to this neutralized combination, the decrease in yields of DHT triggered by N were comparable to those induced by H2O2; and retaining the positive effect of G. ConclusionOxidative stress mediated by H2O2 was overcome by glutathione and recurred when nicotine was added, suggestive of a pro- oxidant role for nicotine. Androgen biomarkers are a sensitive index of oxidative stress which affects wound healing.

Establishment and characterization of an embryonic cell line from Sarconesiopsis magellanica.

Sarconesiopsis magellanica (Le Guillou) (Diptera: Calliphoridae) is a necrophagous fly that is important in both human and veterinary medicines. This insect has been registered in Colombia as a biological indicator in estimating post-mortem interval. Insect cell cultures are an important biotechnological tool for basic and applied studies, and cell cultures derived from S. magellanica embryonic tissues are described in this study. S. magellanica embryonated eggs were taken for tissue explants. These were seeded in L-15, Grace/L-15, Eagle MEM, MM, VP12, MM/VP12, and Schneider culture media. The morphological, cytogenetic, biochemical, and molecular characteristics of the cell cultures were examined. Cell growth was achieved in the L15, Grace/L15, and Schneider culture media, and the confluent monolayers were obtained 8, 10, and 19 days after the embryonated eggs were explanted. However, the Schneider medium was the most efficient to develop the subcultures, and 21 passages have been maintained. The cell morphology of the primary cell cultures was initially heterogeneous, but in the confluent monolayer and in the subcultures there was greater cell morphology uniformity, fibroblastoid types being predominant. Cultured cells had a chromosomal number of 12, and the karyotypic complement consisted of five pairs of somatic chromosomes and one sexual pair. The cell culture isozyme patterns of S. magellanica coincided with adult samples from the same species. The molecular analysis, using RAPD-PCR, demonstrated the authentication of the cell cultures of this fly and their differentiation from other cultures derived from two sand flies species. This cell line is a new in vitro model that will be used in biomedical and biotechnological studies.

Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) Directly Regulates Osteogenesis

Bone formation requires synthesis, secretion, and mineralization of matrix. Deficiencies in these processes produce bone defects. The absence of the PDZ domain protein Na(+)/H(+) exchange regulatory factor 1 (NHERF1) in mice, or its mutation in humans, causes osteomalacia believed to reflect renal phosphate wasting. We show that NHERF1 is expressed by mineralizing osteoblasts and organizes Na(+)/H(+) exchangers (NHEs) and the PTH receptor. NHERF1-null mice display reduced bone formation and wide mineralizing fronts despite elimination of phosphate wasting by dietary supplementation. Bone mass was normal, reflecting coordinated reduction of bone resorption and formation. NHERF1-null bone had decreased strength, consistent with compromised matrix quality. Mesenchymal stem cells from NHERF1-null mice showed limited osteoblast differentiation but enhanced adipocyte differentiation. PTH signaling and Na(+)/H(+) exchange were dysregulated in these cells. Osteoclast differentiation from monocytes was unaffected. Thus, NHERF1 is required for normal osteoblast differentiation and matrix synthesis. In its absence, compensatory mechanisms maintain bone mass, but bone strength is reduced.

Osteoblastic Responses to LPS, Glucose-oxidised LDL and Minocycline: Therapeutic Targets for Periodontal and Cardiometabolic Diseases

To study redox responses of cultured osteoblasts, mediated by bacterial lipopolysaccharide (LPS), glucose (G), glucose-oxidised low density lipoprotein (GLDL) and minocycline (M) using radiolabelled steroid markers of redox status and wound healing. The clinical relevance of this concept in periodontitis patients with cardiometabolic risk markers is addressed. A well differentiated osteoblastic cell-line was cultured in Eagle's MEM in confluent monolayer, in 24 well multiwell plates. Radiolabelled testosterone was used as the steroid substrate. Experiments were set up with controls in the absence of agents, optimal concentrations (previously determined) of G, GLDL, LPS, M, GLDL+LPS and the latter combined with M (n = 8). At the end of a 24h incubation period, the reaction was terminated and the medium analysed for yields of the steroid metabolite 5α-dihydrotestosterone (DHT), the redox marker relevant to wound healing, the weaker androgen 4-androstenedione (4-A) and the diols. Analysis entailed thin layer chromatography and radioisotope scanning. The yields of DHT showed 1.4-fold and 2.3-fold decreases in response to GLDL and LPS respectively and a 1.3-fold reduction in response to the combination, when compared with controls in the absence of agents. Minocycline stimulated the yield of DHT by 1.4-fold, and when combined with GLDL+LPS, the decreased yield was overcome and raised to 2-fold above the combination in response to the addition of minocycline (n = 8; p < 0.001), when compared with controls. The trends in the yields of 4-A and diols were inversely related to each other with increases and decreases over controls respectively, in keeping with enzymic pathways. Decreased yields of the oxidative stress marker DHT in response to LPS, G and GLDL were overcome in the presence of minocycline, which demonstrates its potential role as an adjunctive therapeutic agent in an environment of oxidative stress. These applications could be extrapolated to periodontal disease and co-existing cardiometabolic risk markers, in the context of its antiinflammatory and antioxidant actions relevant to healing. In this paper, recent patents relevant to adjunctive therapeutic management of periodontal disease co-existing with cardiometabolic risk markers are addressed. There have been significant advances in therapeutic interventions for overcoming oxidative stress-inducing mechanisms that are common to these disease entities.

P4-S1.04 11C-Choline small animal PET in experimental Chlamydia muridarum infection

ObjectivesGenital tract infection of female mice with Chlamydia muridarum closely mimics C trachomatis acute genital tract infection in women. Aim of this study was to assess the predictivity of 11C-Choline...

1004 インプラント表面に生成する皮膜の電気化学的評価(OS13-(2)オーガナイズドセッション《生体医工学及びバイオマテリアル》)

Now, the people who substitute a joint, the tooth that a function deteriorated for an artificial joint, an artificial root increase rapidly. Therefore the characteristic found to artificial materials becomes higher very much. The metal materials are used as implant materials widely, and it is often that the fewness to show the toxicity is controlled by metal corrosion resistance. I was aimed for the chemical evaluation of metallic bio materials and I examined metallic bio materials based on the testing method for corrosion resistance of metallic bio materials by anodic polarization measurement (JIS T 0302) standardized by JIS. As a result, the oxidation film of stainless steel and Co-Cr-Mo alloy is appeared the peak of the current density in Eagle MEM. But the oxidation film of Zr-2.5Nb alloy is not

In Vitro Effects of Culture Medium and Serum on Germ Cells in Testis and Epididymis of Male Wistar Rats

Summary The present study was designed to examine the influence of culture media and serum on survival of in vitro rat sperms. The effects of different culture media with or without 10% fetal bovine serum (FBS) on spermatids and spermiums of Wistar rats were assessed in vitro between 2004 and 2005. Spermatozoa were cultured in Gamete-20, RPMI-1640, alphaMEM (AMEM), Dulbecco's modified Eagle's medium (DMEM) and Eagle's MEM (MEM). The number and morphology of cells was recorded at 4 and 24 h. Under all conditions the number and the viability of the cells decreased with time but parameters were positively affected by the presence of FBS. Viability rates of spermatids and spermatozoa reduced at 24 h of culturing. The survival and morphology were the best in RPMI with serum and worst in MEM without serum. These results indicate that different composition of culture media and FBS are important for maturation and survival of spermatozoa. Carefully selected culture media can play important roles in the generation of functional sperms as well as in the success of in-vitro fertilization (IVF) procedures.

Comparative Analysis of Reproduction of Influenza Virus Strains in Cell Lines Perspective for the Creation of Cultural Vaccines Grown on Nutrient Medium on the Basis of Rise Flour Protein Hydrolysate

We studied vaccine strains of influenza viruses A and B during their culturing in MDCK and Vero cells grown in Eagle's MEM medium and in a medium on the basis of enzyme hydrolysate of rise flour proteins with reduced (2%) content of fetal calf serum. Optimal conditions for cell culturing and reproduction of influenza virus strains in these cells were studied. Culturing of vaccine strains of influenza viruses in MDCK and Vero cells grown in nutrient media on the basis of rise flour protein hydrolysate yielded high infection titers, which suggests that this medium can be used for the development of cultural influenza vaccine.

Inhibition of the antigen-induced activation of RBL-2H3 cells by sinomenine

The present study was designed to investigate the effect of sinomenine (SIN), an alkaloid extracted from sinomenium acutum, on the antigen-induced activation of RBL-2H3. For this investigation, the RBL-2H3 cells were sensitized with dinitrophenyl (DNP)-specific IgE overnight in 1.0 ml of Eagle's MEM (EMEM), and varying doses of SIN were added to the culture medium for 30 min and challenged with dinitrophenyl-human serum albumin (DNP-HSA) to induce mast cell degranulation before supernatants were collected. The effects of SIN on antigen-induced release of β-hexosaminidase were measured by enzymatic assay, calcium influx by FACS, cytokines by ELISA, and signaling events by immunoblotting. The results showed that treatment with SIN was followed by a decrease in FcεRI-mediated mast cell release of β-hexosaminidase, production of IL-4 and TNF-α, phosphorylation of Gab2 (Scaffolding adapter Grb2-associated binder 2), Akt and p38 mitogen-activated protein kinase (MAPK). In addition, SIN had no effect on the phosphorylation of LAT and no significant difference on calcium mobilization was observed between control and SIN treated group. These results suggested that SIN might suppress the antigen-induced activation of RBL-2H3 cells via a Ca 2+ independent pathway.

Superoxide Dismutase and Catalase Activities in the Seminal Plasma of Normozoospermic and Asthenozoospermic Beagles

We measured the blood plasma testosterone (T) levels and superoxide dismutase (SOD) and catalase activities in the seminal plasma of the ejaculates of 5 normal (2-5 years old) and 5 asthenozoospermic (AZ-) (3-5 years old) Beagles. Sperm ejaculated by AZ-dogs was incubated for 3 hr in Eagle's MEM only (controls) or Eagle's MEM containing 100 units/ml of SOD or catalase. Sperm motility was examined during incubation. The mean (+/- SE) plasma T level of the AZ-dogs (1.2 +/- 0.2 ng/ml) was significantly lower than in the normal dogs (2.5 +/- 0.2 ng/ml) (P<0.005). The mean (+/- SE) seminal plasma SOD and catalase activities (18.8 +/- 1.9 and 0.5 +/- 0.1 unit/g protein, respectively) were significantly lower in the AZ-dogs than in the normal dog (43.3 +/- 2.5 and 2.2 +/- 0.4 unit/g protein, respectively) (P<0.001 and 0.01, respectively). The motility of sperm incubated in Eagle's MEM containing SOD or catalase was significantly higher than that of control sperm incubated in only Eagle's MEM after 2 or 3 hr of incubation (P<0.05). The results of this study indicate that poor T secretion by the testes and low antioxidant enzyme activities are related to AZ in the dog.

Fabrication and enzymatic degradation of fibronectin-based ultrathin films

Novel fibronectin (FN)-based ultrathin films were prepared by layer-by-layer (LbL) assembly. Among the various combinations of extracellular matrix (ECM) proteins and glycosaminoglycans (GAGs) such as FN, gelatin (G), α-elastin (E) and heparin (Hep), FN/Hep, FN/G and FN/E nanofilms were successfully fabricated in phosphate buffer solutions (pH 7.4). The film thickness of the nanofilms, in which each component can interact with each other by FN-specific interactions, was larger than that of other LbL films (E/Hep, G/E and G/Hep) prepared by electrostatic interactions. The FN/G film was rapidly decomposed by treatment with elastase, thus demonstrating, the enzymatic biodegradability of the nanofilm. We prepared the FN/heparinoid multilayers composed of FN and dextran sulfate (Dex), and its thickness was much larger than that of the FN/α-poly(L-lysine hydrochloride) (PLL) film prepared by LbL assembly using common electrostatic interactions. Furthermore, the FN/G and FN/Dex nanofilms prepared by FN-specific interaction were more stable in Eagle's MEM with 10% fetal bovine serum (FBS) than the electrostatic assembling films, FN/PLL and PLL/Dex. FN-based multilayers composed of FN and ECM components can be useful as artificial ECM films for tissue engineering and other biomedical applications.

Antioxidant Effects of Glutathione and IGF in a Hyperglycaemic Cell Culture Model of Fibroblasts: Some Actions of Advanced Glycaemic end Products (AGE) and Nicotine

The aim of this investigation was to establish potential oxidative effects of glucose, advanced glycation end products (AGE) and nicotine (N) in a fibroblast cell culture model using the anti-oxidants glutathione (G) and insulin like growth factor (IGF). Assays of androgen metabolites were used as biomarkers of healing in this context. Confluent monolayer cultures of human gingival fibroblasts were established in 24 well multiwell plates and incubated in Eagle's MEM for 24h using two radiolabelled androgen substrates 14C-testosterone/14C-4-androstenedione. The established effective concentrations of G1000, glutathione and AGE were used alone and in combination with nicotine and insulin-like growth factor. The medium was then solvent extracted for steroid metabolites, evaporated to dryness and subjected to thin layer chromatography in a benzene acetone solvent system 4:1 v/v for separation of formed metabolites. The metabolites were quantified, using a radioisotope scanner. Significant reduction in the yields of DHT in response to G1000, AGE and nicotine (n=6; p <0.003) were overcome by glutathione (n=6; p <0.002). The stimulatory effect of IGF when combined with AGE was further enhanced by the antioxidant effect of glutathione (n=6; p <0.003). Glucose, AGE and nicotine had a significant inhibitory effect on the yields of the androgen biomarker DHT, overcome by the antioxidant glutathione and IGF, suggestive of an oxidant role for the former agents and an anti-oxidant one for the latter. These agents affected yields of androgen metabolites, biomarkers of oxidative stress and repair, with potential implications on healing in uncontrolled diabetic smokers.

Effects of Hyaluronic Acid on Ca&lt;sup&gt;2+&lt;/sup&gt; Influx, Lactate Dehydrogenase Activity, and Cyclic AMP Synthesis in Canine Ejaculated Sperm during &lt;i&gt;In Vitro&lt;/i&gt; Capacitation

The effects of hyaluronic acid, which comprises the cumulus intercellular matrix, on Ca(2+) influx, lactate dehydrogenase (LDH) activity, and cyclic AMP synthesis in canine sperm during capacitation was investigated. Ejaculated sperm were collected from 10 Beagle dogs and the sperm were incubated for 4 hr in Eagle's MEM containing 10 microg/ml of hyaluronic acid. The percentages of actively motile sperm, hyperactivated sperm (HA-sperm), acrosome-reacted sperm (AR-sperm), and sperm labeled with fluoresceinated Ca(2+) indicator (Ca(2+)-labeled sperm) were evaluated to assess Ca(2+) influx into the sperm. LDH activity and cAMP concentration were measured in homogenized sperm. The mean percentages of motile sperm, HA-sperm, and Ca(2+)-labeled sperm in the MEM containing hyaluronic acid were higher than in the control medium (P<0.05, 0.05, and 0.01, respectively), but there was no difference between the percentages of AR-sperm. Mean LDH activity and mean cAMP concentration were also significantly higher than the control values (P<0.05). The percentages of HA-sperm correlated with those of Ca(2+)-labeled sperm (r(2)=0.810). The results indicate that hyaluronic acid increases Ca(2+) influx, LDH activity, and cAMP synthesis in canine ejaculated sperm during capacitation in vitro.

Effect of glutathione on arecanut treated normal human buccal fibroblast culture.

Experimental studies have shown arecanut to be a cytotoxic substance with mutagenic and carcinogenic potential. The present study was undertaken to evaluate the effect of glutathione on arecanut treated human buccal fibroblast culture and its potential as a chemopreventive agent. Fibroblast culture was done in Dulbecco's Modified Eagle's Medium MEM) supplemented with 10% Fetal Calf Serum (FCS) and antibiotic at 370C degrees in an atmosphere of 5% carbon di-oxide and 95% air. The fibroblast cells were subjected to different concentrations of aqueous extracts of raw and boiled arecanut. Fibroblasts were plated in two 24-well culture plates and in each plate, cells were dividt,ednto 2 groups; 600gg microml of reduced glutathione was added to the first group of cells; subsequently, aqueous extracts of raw and boiled arecanut at least and highest concentrations i.e., 20j. microml and 100lg microml were added to the first group of cells in the respective plates whereas the second group served as a control. The morphological alterations and cell survival were assayed at 24, 48, 72, and 96 hours. Results Morphologically, the initial (10 hours) attached fibroblast cells were converted from spheroidal shape towards hexagonal and finally to a fully extended spindle shaped configuration. The three morphological types of fibroblasts at 48 hours were F-I, F-II and F-III. Aqueous extract of raw arecanut exhibited significant cytotoxicity (p < .0 001) at all time periods studied, when compared against the control values of untreated fibroblasts. Addition of reduced glutathione to cultures showed a significant (p < 0. 001) reduction in cytotoxicity, as indicated by higher optical density values and morphological reversion to the spindle-shaped configuration. Co Addition of glutathione reduced the cytotoxic and morphological alterations of the fibroblasts treated with aqueous extracts of both raw and boiled arecanut.

Growth characteristics of canine pathogenic viruses in MDCK cells cultured in RPMI 1640 medium without animal protein

Madin Darby canine kidney (MDCK) cells were adapted to serum-free RPMI 1640 medium and used for cultivation of canine viruses. RPMI 1640 medium was supplemented with a soybean peptone, l-glutamine and antibiotics, so that the protein concentration was less than 5 μg/ml (RPMI/SP medium). The resulting adapted MDCK-SP cells showed steady growth after the twenty-eighth passage in RPMI/SP medium (MDCK-SP cell culture). Canine distemper virus, canine parvovirus, canine adenoviruses and canine parainfluenza virus, which are the principal components of canine combined virus vaccines, grew in the MDCK-SP cell culture as efficiently as the parental MDCK cells cultured in the conventional Eagle's MEM containing fetal bovine serum. Consequently, the use of MDCK-SP cell culture can make current canine vaccine products much safer, of higher quality and at lower cost.

Acquisition of anoikis resistance in human osteosarcoma cells does not alter sensitivity to chemotherapeutic agents

BackgroundChemotherapy-induced cell death can involve the induction of apoptosis. Thus, aberrant function of the pathways involved might result in chemoresistance. Since cell adhesion to the extracellular matrix acts as a survival factor that homeostatically maintains normal tissue architecture, it was tested whether acquisition of resistance to deadhesion-induced apoptosis (anoikis) in human osteosarcoma would result in resistance to chemotherapy.MethodsOsteosarcoma cell lines (SAOS-2 and TE-85) obtained from ATCC and were maintained in complete Eagle's MEM medium. Suspension culture was established by placing cells in tissue culture wells coated with poly-HEMA. Cell cytotoxicity was determined using a live/dead cytotoxicity assay. Cell cycle/apoptosis analyses were performed using propidium iodide (PI) staining with subsequent FACS analysis. Apoptosis was also assayed by Annexin-FITC/PI staining.ResultsEtoposide, adriamycin, vinblastine, cisplatin and paclitaxel were able to induce apoptosis in human osteosarcoma cells SAOS-2 regardless of their anoikis resistance phenotype or the culture conditions (adhered vs. suspended). Moreover, suspended anoikis resistant TE-85 cells (TE-85ar) retained their sensitivity to chemotherapy as well.ConclusionAcquisition of anoikis resistance in human osteosarcoma cells does not result in a generalized resistance to all apoptotic stimuli, including chemotherapy. Moreover, our results suggest that the pathways regulating anoikis resistance and chemotherapy resistance might involve the action of different mediators.

225 IN VITRO MATURATION OF LION OOCYTES

The potential for rescuing immature oocytes from the ovaries of females of rare species of felids that die or undergo ovariohysterectomy has been reported in only two studies (Johnston et al. 1991 Biol. Reprod. 45, 898-906; Jewgenow et al. 1997 J. Reprod. Fertil. Suppl. 51, 33-39), in which oocytes were maturated in complex media (Eagle's MEM, M199, respectively). The domestic cat is used as a research model for endangered species but there may be some differences that perturb the adaptation of in vitro maturation (IVM), fertilization, or culture methods of lion oocytes compared to cat oocytes. Therefore, in the present study we evaluated the in vitro response of lion oocytes to our system for in vitro maturation of domestic cat oocytes in a simple medium (Merlo et al. 2005 Theriogenology 63, 2032-2039). A 14-year-old lioness, referred to our clinic because of pyometra, underwent ovariohysterectomy. After premedication with acepromazine 0.1 mg/kg i.m. (Prequillan; ATI, Bologne, Italy) and ketamine 5 mg/kg i.m. (Ketavet 100; Intervet, Boxmeer, The Netherlands), anesthesia was induced with ketamine 0.05 mg/kg i.v. and diazepam 0.02 mg/kg i.v. (Diazepam; Intervet) and maintained with isoflurane (Forane; Abbott, Rome, Italy) after intubation. Ovaries were removed and stored in saline solution at room temperature until collection of oocytes (within 1 h). A total of 53 small follicles (2-4 mm), five corpora lutea and a 15-mm follicle were present on the ovaries. Oocytes were collected by aspirating visible follicles of each ovary with a 21-ga needle connected to a vacuum pump (K-MAR-5100; Cook Australia, Brisbane, Australia) at -75 mmHg; then CL were removed and the ovaries were minced with a scalpel blade in a 60-mm petri dish containing HEPES-SOF (H-SOF) for recovery of additional oocytes. A total of 45 oocytes were recovered, of which 19 were degenerate (42.2%); of the remaining 26, 12 were fully surrounded by cumulus cells, 9 had only corona radiata, and 5 were denuded. Degenerate oocytes were discarded and all other oocytes were washed twice in H-SOF and matured in a 35 mm petri dish containing SOFaaBSA 5 mg/mL plus 0.1 IU of porcine FSH-LH (Pluset; Laboratorios Calier, Barcelona, Spain), 25 �L/mL insulin-transferrin-selenium (ITS) (Sigma, Madrid, Spain), 1.2 mm l-cysteine (Sigma), and 25 ng/mL epidermal growth factor (EGF) (Sigma) for 24 h in a humidified atmosphere of 5% CO2 in air at 38.5�C. After maturation, cumulus cells were removed by pipetting oocytes into a 0.25% trypsin solution for 2 min. Then, denuded oocytes were washed once in H-SOF plus 10% FCS to inactive trypsin and twice in H-SOF before being stained with Hoechst 33342 (10 �g in 10 mL PBS) for 30 min at room temperature. After washing in PBS, oocytes were observed using fluorescence microscopy to determine maturation rate. Oocytes in telophase I or metaphase II were considered mature. Of 26 oocytes, 19 (73.1%) were mature and 7 (26.9%) were at the GV stage. These results demonstrated that lion oocytes can undergo successful IVM at a frequency that is similar to that of cat oocytes cultured in the same system (76.9%, P &gt; 0.05). Furthermore, the maturation rate obtained in a simple medium was higher or similar to those previously reported (mean: 22.9% and 69.7% respectively, by Johnston et al. 1991 and Jewgenow et al. 1997).