Bovine endometrium-derived cultured cells are suitable for lipofection

Bovine-derived cultured cells, including Madin-Darby bovine kidney cells, are used worldwide; however, lipofection tend to result in low transfection efficiency, which has impeded the progress of veterinary research. We performed experiments to confirm the lipofection efficiency of bovine-derived cultured cells, to identify cells that suitable for lipofection. Several bovine tissues (endometrium, testis, ear tissue and foetal muscle) were collected, and primary cultured cells were prepared. Lipofection assay showed that only bovine endometrium (BE)-derived cells could be transfected efficiently (50‒70%). BE cells can be divided into at least two types of cell populations (BE-1 and BE-2). The BE-1 cells, which were suitable for lipofection, were obtained by passages at short intervals and were negative for cytokeratin- and positive for vimentin-expression; the BE-2 cells did not have these characteristics and were not suitable for lipofection. Furthermore, the BE-1 cells and artificially immortalised cells of BE-1, iBE-1 cells, were utilised in a reporter assay requiring the introduction of multiple DNAs. Endometrial tissues can be collected from living cows, and BE-1 cells can be obtained easily by controlling passaging timing. The production of BE-1 cells and sharing the methods required to prepare them will contribute to the development of veterinary research.

Potencial antiproliferativo do óleo essencial de Origanum majorana Linn. em células de melanoma e seus efeitos intracelulares em células neoplásicas e não-neoplásicas

Os objetivos desse estudo foram avaliar a atividade intracelular do óleo essencial de Origanum majorana (MARJ) nas linhagens Madin Darby Bovine Kidney (MDBK) e de melanoma metastático murino (B16F10) e elucidar a sua composição química. Através da citometria de fluxo foram analisados os parâmetros fluidez de membrana (F), espécies reativas de oxigênio (ERO), índice de fragmentação de DNA (IFD), potencial de membrana mitocondrial (PMM), ciclo celular, lipoperoxidação da membrana plasmática (LPO), viabilidade celular, necrose e apoptose das linhagens celulares tratadas com MARJ nas concentrações de 0,25, 0,5 e 1 mg/mL por 24 horas. A composição do MARJ foi determinada através de cromatografia gasosa, sendo seus componentes majoritários os terpenos: cis-sabineno hidratado, 4-terpineol e gama-terpineno. Os resultados mostraram que todas as concentrações de MARJ reduziram ERO, LPO e as células em fase S da linhagem MDBK. Nas células B16F10, os tratamentos com MARJ reduziram ERO e as células em fase G2, aumentando as células na fase S. As concentrações de 0,5 e 1 mg/mL causaram redução na F e 0,5 mg/mL aumentou o PMM na linhagem MDBK, enquanto LPO foi reduzida na concentração de 0,5 mg/mL nas células B16F10. Os resultados das avaliações da produção de ERO, da integridade da membrana plasmática e de LPO indicaram atividade antioxidante nas linhagens estudadas. Através da análise do ciclo celular foi possível observar que MARJ exerceu efeito antiproliferativo sobre as células de melanoma, sendo capaz de interromper a multiplicação desse tipo celular.

Transcriptome and Proteomic Analysis Reveals Up-Regulation of Innate Immunity-Related Genes Expression in Caprine Herpesvirus 1 Infected Madin Darby Bovine Kidney Cells

Caprine herpesvirus 1 (CpHV-1) is a member of the alpha subfamily of herpesviruses, which is responsible for genital lesions and latent infections in goat populations worldwide. In this study, for the first time, the transcriptome and proteomics of CpHV-1 infected Madin Darby bovine kidney (MDBK) cells were explored using RNA-Sequencing (RNA-Seq) and isobaric tags for relative and absolute quantitation-liquid chromatography tandem mass spectrometry (iTRAQ-LC-MS/MS) technology, respectively. RNA-Seq analysis revealed 81 up-regulated and 19 down-regulated differentially expressed genes (DEGs) between infected and mock-infected MDBK cells. Bioinformatics analysis revealed that most of these DEGs were mainly involved in the innate immune response, especially the interferon stimulated genes (ISGs). Gene Ontology (GO) enrichment analysis results indicated that the identified DEGs were significantly mainly enriched for response to virus, defense response to virus, response to biotic stimulus and regulation of innate immune response. Viral carcinogenesis, the RIG-I-like receptor signaling pathway, the cytosolic DNA-sensing pathway and pathways associated with several viral infections were found to be significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Eleven selected DEGs (Mx1, RSAD2, IFIT1, IFIT2, IFIT5, IFIH1, IFITM3, IRF7, IRF9, OAS1X and OAS1Y) associated with immune responses were selected, and they exhibited a concordant direction both in RNA-Seq and quantitative real-time RT-PCR analysis. Proteomic analysis also showed significant up-regulation of innate immunity-related proteins. GO analysis showed that the differentially expressed proteins were mostly enriched in defense response and response to virus, and the pathways associated with viral infection were enriched under KEGG analysis. Protein-protein interaction network analysis indicated most of the DEGs related to innate immune responses, as DDX58(RIG-I), IFIH1(MDA5), IRF7, Mx1, RSAD2, OAS1 and IFIT1, were located in the core of the network and highly connected with other DGEs. Our findings support the notion that CpHV-1 infection induced the transcription and protein expression alterations of a series of genes related to host innate immune response, which helps to elucidate the resistance of host cells to viral infection and to clarify the pathogenesis of CpHV-1.

A Comparative Study of the Cytotoxic Effects and Oxidative Stress of Gossypol on Bovine Kidney and HeLa Cell Lines

Background: Cotton seed is one of the main sources of protein in animal feeds, containing gossypol, which has been shown to have toxic effects. Results reported by various studies also indicate the anti-cancer effects of gossypol on various cell types. However, its toxic effects on human and animal cells have not been fully established. This study was planned to investigate, for the first time, the cytotoxic effects and oxidative stress induced by gossypol on normal Bovine Kidney (BK) and HeLa cell lines, representing typical healthy and cancer cells, respectively. Methods: The BK and HeLa cell lines were treated for 24, 48 or 72 hours with 5, 10 or 20 ppm of gossypol (+/-). The cellular bio-availability and cytotoxicity were measured by MTT assay. The catalase and Malondialdehyde (MDA) levels were also measured to represent the oxidative stress parameters. Results: The percentages of cytotoxicity in BK and HeLa cell lines were calculated at a gossypol concentration of 5, 10 and 20 ppm over 24, 48 or 72 hours of incubation, respectively. The Lethal Concentration 50 (lC50) values were also determined for the two cell lines. No changes in the catalase and lipid peroxidase activities were observed in either cell line. Conclusion: The percentage of the gossypol cytotoxicity was concentration-dependent. By comparing the IC50 in both cell lines using one-way Analysis of Variance (ANOVA) analysis, a significant difference was observed, suggesting that Hela cells were less sensitive to gossypol than the BK cells. Lack of changes in the oxidative stress, as tested by catalase and MDA assays, demonstrated that gossypol did not induce oxidative stress in either cell line.

In vitro infection of Madin-Darby bovine kidney (MDBK) cells with Eimeria acervulina sporozoites: quantitative analysis of parasite cellular invasion and replication using real-time polymerase chain reaction (PCR)

Poultry coccidiosis causes considerable economical losses to the livestock industry. Eimeria parasites are responsible for this disease. On a global scale, E. acervulina and E. tenella are amongst the most common Eimeria spp. infecting broilers. E. tenella is commonly used as infection model in in vivo and in vitro studies. On the other hand, E. acervulina has barely been studied under in vitro conditions. A well established and widely used in vitro model for E. tenella infection is the Madin-Darby bovine kidney cell line (MDBK); however, little is known regarding suitability of MDBK cells as host cells for E. acervulina. We infected MDBK monolayers with two different doses, 5 × 104 and 2 × 105, of E. acervulina sporozoites and evaluated cultures at 24 and 96 h post infection (hpi). For comparison, we ran an identical infection assay using E. tenella sporozoites. To assess parasite reproduction, the number of DNA copies of E. acervulina SCAR marker and E. tenella ITS-1 gene was quantified using real-time quantitative PCR. We found that the number of E. acervulina copies increased significantly at 24 hpi in comparison to E. tenella (p < 0.05). After 96 hpi, E. acervulina gene copies were considerably reduced while E. tenella continued to multiply (p < 0.05). Our results show that MDBK monolayers could be used for in vitro research aimed to study E. acervulina sporozoite cell invasion. Nevertheless, modifications of in vitro cultivation appear necessary to allow qualitative and quantitative studies over longer periods of parasite reproduction.

Creation of an ex-vivo bovine kidney flow model for testing embolic agents: work in progress

Objectives To develop an ex- vivo perfusion flow model using a bovine kidney for future testing of embolic agents in an inexpensive and easy way. Methods Six bovine adult kidneys were used for this study. Kidneys were cannulated and perfused via a roller pump. Three embolic agents, coils, Gelfoam, and a glue mixture of Histoacryl + Lipiodol, were deployed by targeting three secondary segmental arteries per kidney via a 5Fr catheter under fluoroscopic guidance. Cannulation time, success rate of segmental artery selection and embolic agent deployment, total operational time, and fluoroscopy dose were recorded. Results Average kidney weight was 0.752 +/− 0.094 kg. All six bovine kidneys were successfully cannulated in 21.6 min +/− 3.0 min. Deployment of coils and glue was achieved in every case (12/12); however, Gelfoam injection was not successful in one instance (5/6, 83%). Coil deployment demonstrated no embolic effect while Gelfoam and glue injections demonstrated decreased distal contrast filling post-embolization. Mean dose area product was 12.9 ± 1.8 Gy·cm2, fluoroscopy time was 10 ± 4 min and operational time was 27 ± 8 min. Conclusions We describe the creation of an ex vivo bovine kidney flow model for the preclinical evaluation of different embolic materials. The flow model can be modified to provide extensive bench testing and it is a promising tool for hands -on training in basic and advanced embolization techniques .

DDIT3 targets innate immunity via the DDIT3-OTUD1-MAVS pathway to promote BVDV replication

DNA damage inducible transcript 3 (DDIT3) plays important roles in ER stress-induced apoptosis and autophagy, but its role in innate immunity is not clear. Here, we report that DDIT3 inhibits the antiviral immune response during bovine viral diarrhea virus (BVDV) infection by targeting mitochondrial antiviral signaling (MAVS) in Madin-Darby bovine kidney (MDBK) cells and in mice. BVDV infection induced high DDIT3 mRNA and protein expression. DDIT3 overexpression inhibited type I interferon (IFN-I) and IFN-stimulated gene production, thereby promoting BVDV replication, while DDIT3 knockdown promoted the antiviral innate immune response to suppress viral replication. DDIT3 promoted NF-κB-dependent OTU deubiquitinase 1 (OTUD1) expression. Furthermore, OTUD1 induced upregulation of the E3 ubiquitin ligase Smurf1 by deubiquitinating Smurf1, and Smurf1 degraded MAVS in MDBK cells in a ubiquitination-dependent manner, ultimately inhibiting IFN-I production. Moreover, knocking out DDIT3 promoted the antiviral innate immune response to reduce BVDV replication and pathological changes in mice. These findings provide direct insights into the molecular mechanisms by which DDIT3 inhibits IFN-I production by regulating MAVS degradation. IMPORTANCE Extensive studies have demonstrated roles of DDIT3 in apoptosis and autophagy during viral infection. However, the role of DDIT3 in innate immunity remains largely unknown. Here, we show that DDIT3 was positively regulated in bovine viral diarrhea virus (BVDV)-infected Madin-Darby bovine kidney (MDBK) cells and could significantly enhance BVDV replication. Importantly, DDIT3 induced OTU deubiquitinase 1 (OTUD1) expression by activating the NF-κB signaling pathway, thus increasing intracellular Smurf1 protein levels to degrade MAVS and inhibit IFN-I production during BVDV infection. Together, these results indicate that DDIT3 plays critical roles in host innate immunity repression and viral infection facilitation.

IN VITRO ASSESSMENT OF THE BIOLOGICAL ACTIVITY OF A NEW REGENERATIVE AGENT PREPARED FROM THE CONCENTRATE OF DEPROTEINIZED DERMAL LAYER OF PORCINE SKIN

Background. Presently, a prospective direction for the development of regenerative medicine in the world is the search for regulatory molecules and the identification of molecular targets to stimulate the body's endogenous regenerative potential. The concentrate of the deproteinized dermal layer of porcine skin (СDDLPS) is a new therapeutic agent with restorative properties, the action of which is directed on the induction of the self resources of cells. Aim. The assessment of the effect of СDDLPS on the proliferative activity of mammalian cells of different histogenesis in vitro. Materials and Methods. To determine the amino acid composition of the СDDLPS liquid chromatography and biochemical methods were used. The biological effects and mechanisms of action of the drug were investigated by cell culture and molecular biological methods. The research was carried out using stable cell lines: human keratinocytes (HaCaT cell line), porcine endothelial cells (PAE cell line), bovine kidney cells (MDBK cell line) and mouse fibroblasts (3T3A31 cell line). Results. The cells of the bovine kidney MDBK cell line were the most sensitive to the effect of the CDDLPS. Also, the obtained results suggest that, depending on the concentration, the drug not only stimulates cell proliferation by 10–30 %, but also significantly enhances biosynthetic processes in cells, in particular, protein synthesis by 20–40 %. Conclusions. CDDLPS is an effective and affordable therapeutic agent with restorative properties, the biological activity of which manifests itself in the activation of cell biosynthetic and proliferative potentials and is comparable to effects of some growth factors, in particular epidermal growth factor