Infectious bronchitis virus replication in the chicken embryo related cell line

The susceptibility of the chicken embryo related (CER) cell line to infectious bronchitis virus (IBV M41) was characterized after five consecutive passages in CER cells. Virus replication was monitored by cytopathic effect observation, electron microscopy, indirect immunofluorescence, and reverse transcription polymerase chain reaction (RT-PCR). At 96 h post-infection (p.i.), the cytopathic effect was graded 75% by cell fusion, rounding up of cells and monolayer detachment, and the electron microscopy image characterized by coronavirus morphology. Cytoplasmic fluorescence was readily observed by from 24 h p.i. onwards, and at all times the respective viral RNA from IBV-infected monolayers was demonstrated by RT-PCR. Extra-cellular virus was measured by virus titration performed on chicken kidney cells and embryonated chicken eggs, and respective titres ranged from 4.0 to 6.0 log(10) EID50/ml on embryonated chicken eggs, and from 2.0 to 6.0 log(10) TCID50/ml on both CER cells and chicken kidney cells studied from 24 to 120 h p.i. These results confirmed that the M41 strain replicated well in the CER cell line.

Chicken embryo related (CER) cell line for quantification of rabies neutralizing antibody by fluorescent focus inhibition test

Levels of rabies virus neutralization antibody in sera from vaccinated dogs and cattle were either measured by mouse neutralization test (MNT) or by rapid fluorescent focus inhibition test (RFFIT), performed on CER monolayers. The two tests were compared for their ability to detect the 0.5 International Units/ml (I.U.) recommended by the World Health Organization (WHO) as the minimum response for proof of rabies immunization. A significant correlation was found between the two tests (n = 211; r = 0.9949 in dogs and 0.9307 in cows, p < 0.001), good sensitivity (87.5%), specificity (94.7%) and agreement (96.6%) as well. RFFIT method standardized on CER cell system for neutralizing antibodies detection turns the diagnosis easier and less expensive, specially when a great number of samples must be tested from endemic areas as commonly found in Brazil. (c) 2005 the International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

Rabies neutralizing antibody detection by indirect immunperoxidase serum neutralization assay performed on chicken embryo related cell line

The aim of this study was to evaluate the indirect immunoperoxidase virus neutralization (IPVN) and mouse neutralization test (MNT) to detect antibodies against rabies virus from vaccinated dogs and cattle. The IPVN was set up for the ability to measure 0.5 International Units/ml (IU) of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. IPVN was developed and standardized in chicken embryo related (CER) cell line when 141 dog and 110 cattle sera were applied by serial five-fold dilutions (1:5, 1:25, 1:125) as well as the positive and negative reference controls, all added in four adjacent wells, of 96-well microplates. A 50 µl amount of CVS32 strain dilution containing 50-200 TCID50/ml was mixed to each serum dilution, and after 90 min 50 µl of 3 x 10(5) cells/mlcell suspension added to each well. After five days of incubation, the monolayers were fixed and the IPVN test performed. The correlation coefficient between the MNT and IPVN performed in CER cells was r = 0.9949 for dog sera (n = 100) and r = 0.9307 for cattle sera (n = 99), as well as good specificity (94.7%), sensitivity (87.5%), and agreement (96.6%) were also obtained. IPVN technique can adequately identify vaccinated and unvaccinated animals, even from low-responding vaccinated animals, with the advantage of low cost and faster then MNT standard test.

Susceptibility of mammalian cell line for isolation of IBDV from clinical samples

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genotoxic, mutagenic and cytotoxic effects of the commercial dye CI Disperse Blue 291 in the human hepatic cell line HepG2

Textile dyes are discarded into the aquatic ecosystem via industrial effluents and potentially expose humans and local biota to adverse effects. The commercial dye CI Disperse Blue 291 which contains the aminoazobenzene 2-[(2-bromo-4,6-dinitrophenyl)azo]-5(diethylamino)-4-methoxyacetanilide (CAS registry no. 56548-64-2), was tested for genotoxicity and cytotoxicity in the human hepatoma cell line HepG2, using the comet assay, micronucleus (MN) test and a cell viability test. Five different concentrations of the test compound were examined: 200 mu g/ml, 400 mu g/ml, 600 mu g/ml, 800 mu g/ml and 1000 mu g/ml. An increase in comet tail length and in the frequency of MN was detected with exposure of cells to concentrations of the commercial dye from 400 pg/ml. Furthermore, the dye was found to decrease cell viability. The results of this study demonstrate for the first time the genotoxic and mutagenic effects of the dye CI Disperse Blue 291 in mammalian cells, thus stressing the need to develop non-mutagenic dyes and to invest in improving the treatment of effluents. These measures will help to prevent harmful effects that these compounds can have on humans and aquatic organisms that come in contact with them. (C) 2007 Elsevier Ltd. All rights reserved.

Effects of light-curing time on the cytotoxicity of a restorative resin composite applied to an immortalized odontoblast-cell line

This in vitro study evaluated the cytotoxic effects of a restorative resin composite applied to an immortalized odontoblast-cell line (MDPC-23). Seventy-two round resin discs (2-mm thick and 4 mm in diameter) were light-cured for 20 or 40 seconds and rinsed, or not, with PBS and culture medium. The resin discs were divided into four experimental groups: Group 1: Z-100/20 seconds; Group 2: Z-100/20 seconds/rinsed; Group 3: Z100/40 seconds; Group 4: Z-100/40 seconds/rinsed. Circular filter paper was used as a control material (Group 5). The round resin discs and filter papers were placed in the bottom of wells of four 24-well dishes (18 wells for each experimental and control group). MDPC-23 cells (30,000 cells/cm(2)) were plated in the wells and allowed to incubate for 72 hours. The zone of inhibition around the resin discs was measured under inverted light microscopy; the MTT assay was carried out for mitochondrial respiration and cell morphology was measured under SEM. The scores obtained from inhibition zone and MTT assay were analyzed with the Kruskal-Wallis followed by Dunnett tests. In Groups 1, 2, 3 and 4, the thickness of the inhibition zone was 1,593 +/- 12.82 mum, 403 +/- 15.49 mum, 1,516 +/- 9.81 mum and 313 +/- 13.56 mum, respectively. There was statistically significant difference among the experimental and control groups at the 0.05 level of significance. The MTT assay demonstrated that the resin discs of the experimental groups 1, 2, 3 and 4 reduced the cell metabolism by 83%, 40.1%, 75.5% and 24.5%. Only between the Groups 2 and 4 was there no statistically significant difference for mitochondrial respiration. Close to the resin discs, the MDPC-23 cells exhibited rounded shapes, with only a few cellular processes keeping the cells attached to the substrate or, even disruption of plasma membrane. Adjacent to the inhibition zone, the cultured cells exhibited multiple fine cellular processes on the cytoplasmic membrane organized in epithelioid nodules, similar to the morphology observed to the control group. Based on the results, the authors may conclude that the Z-100 resin composite light cured for 20 seconds was more cytopathic to MDPC-23 cells than Z-100 light cured for 40 seconds. The cytotoxic effects of the resin discs decreased after rinsing them with PBS and culture medium. This was confirmed by MTT assay and upon evaluation of the inhibition zone, which was narrower following rinsing of the resin discs.

Cytotoxic effects of current dental adhesive systems on immortalized odontoblast cell line MDPC-23

Objectives: Evaluate the cytotoxic effect of the three dental adhesive systems. Methods: The immortalized mouse odontoblast cell line (MDPC-23) was plated (30,000 cell/cm 2) in 24 well dishes, allowed to grow for 72 h, and counted under inverted light microscopy. Uncured fresh adhesives were added to culture medium to simulate effects of unset adhesive. Three adhesives systems were applied for 120 min to cells in six wells for each group: Group 1) Single Bond (3M), Group 2) Prime & Bond 2.1 (Dentsply), and Group 3) Syntac Sprint (Vivadent). In the control group, PBS was added to fresh medium. The cell number was counted again and the cell morphology was assessed under SEM. In addition, the adhesive systems were applied to circles of filter paper, light-cured for 20 s, and placed in the bottom of 24 wells (six wells for each experimental materials and control group). MDPC-23 cells were plated (30,000 cell/cm 2) in the wells and allowed to incubate for 72 h. The zone of inhibition around the filter papers was measured under inverted light microscopy; cell morphology was evaluated under SEM; and the MTT assay was performed for mitochondrial respiration. Results: The fresh adhesives exhibited more toxic (cytopathic effects) to MDPC-23 cells than polymerized adhesives on filter papers, and as compared to the control group. The cytopathic effect of the adhesive systems occurred in the inhibition zone around the filter papers, which was confirmed by the MTT assay and statistical analysis (ANOVA) combined with Fisher's PLSD test. In the control group, MDPC-23 cells were dense on the plastic substrate and were in contact with the filter paper. In the experimental groups, when acid in the adhesive systems was removed by changing the culture medium, or when the adhesives were light-cured, some cells grew in the wells in spite of the persistent cytotoxic effect. Significance: All dentin adhesive systems were cytotoxic odontoblast-like cells. Both acidity and non-acidic components of these systems were responsible for the high cytopathic effect of those dental materials. © 1999 Academy of Dental Materials. Published by Elsevier Science Ltd. All rights reserved.

Evaluation of serum- and animal protein-free media for the production of infectious bronchitis virus (M41) strain in a continuous cell line

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effect of curing regime on the cytotoxicity of resin-modified glass-ionomer lining cements applied to an odontoblast-cell line

Objective: The aim of this in vitro study was to evaluate the cytotoxicity of resin-modified glass-ionomer lining cements submitted to different curing regimes and applied to an immortalized odontoblast-cell line (MDPC-23). Methods: Forty round-shaped specimens of each experimental material (Fuji Lining LC and Vitrebond) were prepared. They were light-cured for the manufacturers' recommended time (MRT = 30 s), under-cured (0.5 MRT = 15 s), over-cured (1.5 MRT = 45 s) or allowed to dark cure (0 MRT). Sterilized filter papers soaked with either 5 μL of PBS or HEMA were used as negative and positive control, respectively. After placing the specimens individually in wells of 24-well dishes, odontoblast-like cells MDPC-23 (30,000 cells/cm2) were plated in each well and incubated for 72 h in a humidified incubator at 37 °C with 5% CO2 and 95% air. The cytotoxicity was evaluated by the cell metabolism (MTT assay) and cell morphology (SEM). Results: Fuji Lining LC was less cytotoxic than Vitrebond (p < 0.05) in all the experimental conditions. However, the cytotoxicity of Fuji Lining LC was noticeably increased in the absence of light-curing while the same was not observed for Vitrebond. The length of light-curing (15, 30 or 45 s) did not influence the toxicity of both lining materials when they were applied on the odontoblast-cell line MDPC-23. Significance: The light-activation plays an important role in reducing the cytotoxicity of Fuji Lining LC. Following the manufacturer' recommendation regarding the light-curing regime may prevent toxic effect to the pulp cells. © 2005 Academy of Dental Materials.

Cytotoxic effects of hard-setting cements applied on the odontoblast cell line MDPC-23

Objective: The present study evaluated the cytotoxic effects of hard setting applied on the odontoblastlike cells MDPC-23. Study design: Eighty round-shaped samples were prepared with the following experimental materials: calcium hydroxide, Vitrebond, RelyX Luting, and RelyX Unicem. The samples were placed in serum-free culture medium and incubated for 24 hours or 7 days at 37°C with 5% CO 2 and 95% air. The odontoblast cells were plated in the wells and incubated for 72 hours. After this period, the complete culture medium was replaced by the extracts obtained from every sample, and the methyltetrazolium assay was carried out to evaluate the cell metabolism. Results: For the 24-hour period, the experimental materials calcium hydroxide, Vitrebond, RelyX Luting, and RelyX Unicem decreased the cell metabolic activity by 91.52%, 81.14%, 78.17%, and 2.64%, respectively. For the 7-day period, calcium hydroxide, Vitrebond, RelyX Luting, and RelyX Unicem decreased the metabolic activity of the MDPC-23 cells by 91.13%, 87.27%, 79.04%, and 10.51%, respectively. Conclusion: RelyX Unicem presented the lowest cytopathic effects to the cultured odontoblast cell line. © 2007 Mosby, Inc. All rights reserved.

Effects of (-)-cubebin (Piper cubeba) on cytotoxicity, mutagenicity and expression of p38 MAP kinase and GSTa2 in a hepatoma cell line

(-)-Cubebin is a lignan extracted from the seeds of the pepper Piper cubeba, a commonly eaten spice with beneficial properties, including trypanocidal, anti-inflammatory, analgesic, anti-proliferative and leishmanicidal activities. Because of its therapeutic potential, we investigated the effects of (-)-cubebin on the cytotoxicity, cell proliferation kinetics, mutagenicity and expression of p38 MAP kinase and glutathione S-transferase a2 (GSTa2) using real-time RT-PCR in Rattus norvegicus hepatoma cells. We found that 280 μM (-)-cubebin was cytotoxic after 24, 48 and 72. h of exposure, but not mutagenic at 0.28 μM, 2.8 μM and 28 μM after 26. h. Similarly, exposure to 0.28 μM, 2.8 μM and 28 μM (-)-cubebin for 24, 48, 72 and 96. h did not alter the cell proliferation kinetics. Cells exposed to 28 μM (-)-cubebin for 24. h did not exhibit changes in p38 MAP kinase and GSTa2 expression, indicating that cellular changes were not induced by extracellular stimuli and that (-)-cubebin is likely not metabolized via this pathway. Our results suggest that high levels of (-)-cubebin should be consumed with caution due to the cytotoxic effect observed at the highest concentration. However, at lower concentrations, no cytotoxic, mutagenic or proliferative effects were observed, providing further evidence of the safety of consuming (-)-cubebin. © 2013 Elsevier Inc.

Drifter technique: a new method to obtain metaphases in Hep-2 cell line cultures

A linhagem celular Hep-2 é formada por células de carcinoma da laringe e é muito utilizada em modelos de carcinogênese e mutagenêse. Para avaliar o potencial proliferativo desta linhagem, desenvolvemos uma metodologia citogenética (técnica do sobrenadante) para obtenção de metáfases a partir de células que, ao entrarem em mitose, perdem adesão celular, ficando em suspensão no meio de cultura. Através deste procedimento, foram contadas 2000 células, correspondendo a um índice mitótico (IM) de 22.2% . Apesar de o IM obtido por esta técnica não ter sido estatisticamente diferente do IM obtido por outras metodologias citogenéticas clássicas, a técnica do sobrenadante é vantajosa porque elimina o uso de alguns reagentes utilizados na obtenção de metáfases e também diminui o consumo de reagentes de manutenção desta linhagem.

Conventional cytogenetic characterization of a new cell line, ACP01, established from a primary human gastric tumor

Gastric cancer is the second most frequent type of neoplasia and also the second most important cause of death in the world. Virtually all the established cell lines of gastric neoplasia were developed in Asian countries, and western countries have contributed very little to this area. In the present study we describe the establishment of the cell line ACP01 and characterize it cytogenetically by means of in vitro immortalization. Cells were transformed from an intestinal-type gastric adenocarcinoma (T4N2M0) originating from a 48-year-old male patient.This is the first gastric denocarcinoma cell line established in Brazil. The most powerful application of the cell line ACP01 is in the assessment of cytotoxicity. Solid tumor cell lines from different origins have been treated with several conventional and investigational anticancer drugs. The ACP01 cell line is triploid, grows as a single, non-organized layer, similar to fibroblasts, with focus formation,heterogeneous division, and a cell cycle of approximately 40 h. Chromosome 8 trisomy, present in 60% of the cells, was the most frequent cytogenetic alteration. These data lead us to propose a multifactorial triggering of gastric cancer which evolves over multiple stages involving progressive genetic changes and clonal expansion.

Cytogenetic characterization and evaluation of c-MYC gene amplification in PG100, a new Brazilian gastric cancer cell line

Gastric cancer is the fourth most frequent type of cancer and the second cause of cancer mortality worldwide. The genetic alterations described so far for gastric carcinomas include amplifications and mutations of the c-ERBB2, KRAS, MET, TP53, and c-MYC genes. Chromosomal instability described for gastric cancer includes gains and losses of whole chromosomes or parts of them and these events might lead to oncogene overexpression, showing the need for a better understanding of the cytogenetic aspects of this neoplasia. Very few gastric carcinoma cell lines have been isolated. The establishment and characterization of the biological properties of gastric cancer cell lines is a powerful tool to gather information about the evolution of this malignancy, and also to test new therapeutic approaches. The present study characterized cytogenetically PG100, the first commercially available gastric cancer cell line derived from a Brazilian patient who had a gastric adenocarcinoma, using GTG banding and fluorescent in situ hybridization to determine MYC amplification. Twenty metaphases were karyotyped; 19 (95%) of them presented chromosome 8 trisomy, where the MYC gene is located, and 17 (85%) presented a deletion in the 17p region, where the TP53 is located. These are common findings for gastric carcinomas, validating PG100 as an experimental model for this neoplasia. Eighty-six percent of 200 cells analyzed by fluorescent in situ hybridization presented MYC overexpression. Less frequent findings, such as 5p deletions and trisomy 16, open new perspectives for the study of this tumor.