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Culture conditions for Equine bone marrow mesenchymal stem cells and expression of key transcription factors during their differentiation into osteoblasts

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Journal of Animal Science and Biotechnology 2014年第2期5卷 163-172页

作者： Elizabeth R A Glynn Alfredo Sanchez Londono Steven A Zinn Thomas A Hoagl Kristen E Govoni Department of Animal Science University of Connecticut3636 Horsebarn Road Ext.Unit 4040StorrsCT 06269-4040USA Department of Environmental and Population Health Tufts University North GraftonMA 01536USA

Background： The use of Equine bone marrow mesenchymal stem cells （BMSC） is a novel method to improve fracture healing in horses. However, additional research is needed to identify optimal culture conditions and to determine the mechanisms involved in regulating BMSC differentiation into osteoblasts. The objectives of the experiments were to determine： 1） if autologous or commercial serum is better for proliferation and differentiation of Equine BMSC into osteoblasts, and 2） the expression of key transcription factors during the differentiation of Equine BMSC into osteoblasts. Equine BMSC were isolated from the sterna of 3 horses, treated with purchased fetal bovine serum （FBS） or autologous horse serum （HS）, and cell proliferation determined. To induce osteoblast differentiation, cells were incubated with L-ascorbic acid-2-phosphate and glycerol-2-phosphate in the presence or absence of human bone morphogenetic protein2 （BMP2）, dexamethasone （DEX）, or combination of the two. Alkaline phosphatase （ALP） activity, a marker of osteoblast differentiation, was determined by ELISA. Total RNA was isolated from differentiating BMSC between d 0 to 18 to determine expression of runt-reloted tronscrJption foctor2 （Runx2）, osterix （Osx）, and T-box3 （Tbx3）. Data were analyzed by ANOVA. Results： Relative to control, FBS and HS increased cell number （133 ± 5 and 116 ± 5%, respectively; P 〈 0.001） and 5-bromo- 2＇-deoxyuridine （BrdU） incorporation （167 ± 6 and 120 ± 6%, respectively; P 〈 0.001）. Treatment with DEX increased ALP activity compared with control （1,638 ± 38%; P 〈 0.001）. In the absence and presence of Dex, BMP-2 did not alter ALP activity （P 〉 0.8）. Runt-reloted transcription foctor2 expression increased 3-fold （P 〈 0.001） by d 6 of culture. Osterix expression increased 94old （P 〈 0.05） by d 18 of culture. Expression of Tbx3 increased 1.8-fold at d 3 （P 〈 0.01）; however expression was reduced 4-fold at d 18 （P 〈 0.01）. Conclusions： Dexamethasone, but not BMP-2, is required for differentiation of Equine BMSC into osteo

关键词： Bone marrow mesenchymal stem cells Cell culture Equine Osteoblasts Transcription factors

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Changes of Biomarkers in Synovial Fluid in Equine OA Model

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Journal of Northeast Agricultural University(English Edition) 2017年第2期24卷 59-65页

作者： Li Xin-ran Wang Guan-ying Jiang Ren-li Wang Hao Gao Li College of Veterinary Medicine Northeast Agricultural University Harbin 150030 China

The pathogenesis of Equine Osteoarthritis（OA） is more complex, and the disease in the early stage is not easy to be found, therefore, the early diagnosis and treatment are very important. Based on this, this experiment established OA model induced by Equine, aimed to study the changes of contents of Matrix Metalloproteinases-3（MMP-3）, Matrix Metalloproteinases-13（MMP-13）, Aggrecanase（ADAMTS-5）, Hyaluronic Acid（HA） and Osteocalcin（OCN） in synovial fluid, and establish rapid diagnostic technique for the Equine OA. Thirteen Mongolian Equines were used in these induction studies. Equines were randomly divided into two groups： the experimental group contained eight Equines and the control group contained five Equines. The experimental group was to build the Equine osteoarthritis model. The induction was done through Intra-articular（IA） injection of 2 m L Amphotericin-B in Equines’ left carpal joints. The Equine of the control group was injected into 2 m L physiological saline in Equines’ left carpal joints. Synovial fluid was collected every week until the 9th week. The contents of MMP-3, MMP-13, ADAMTS-5, HA and OCN in synovial fluid were evaluated by using ELISA kits. Equine OA model, compared with the control group, starting from the 1st to the 2nd week after induction model, the content of MMP-3, MMP-13, ADAMTS-5, HA and OCN tended to increase, but there was no significant increase, from the 2nd to the 3rd week they significantly increased（p〈0.05） and kept increasing trend until the 9th week. In OA model, MMP-3, MMP-13, ADAMTS-5, HA and OCN showed a rising trend in joint fluid, which would accelerate the cartilage, subchondral bone degradation and metabolism of these proteases increased, and ADAMTS-5 and HA in the early stage increased significantly.

关键词： Osteoarthritis （OA） Equine Matrix Metalloproteinases （MMPs）

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A Review: Interactions of Equine Herpesvirus-1 with Immune System and Equine Lymphocyte

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Open Journal of Veterinary Medicine 2014年第12期4卷 294-307页

作者： Nor Dini Rusli Khairiyah Binti Mat Hasnita Che Harun Faculty of Agro Based Industry Universiti Malaysia Kelantan Jeli Campus Kelantan Malaysia

Equine herpesvirus-1 (EHV-1) remains one of the most common viral pathogens affecting horses worldwide presenting as a persistent infection which can establish latency in nerve ganglia (trigeminal ganglion), lymphoid tissues of the respiratory tract and peripheral blood lymphocytes. EHV-1 infection induces both humoral and cellular immune responses in horses. Virus neutralising antibody, particularly in the nasopharynx, is to kill free virus shed from infected epithelial cells. Hence this antibody has important functions in reducing virus shedding and spreading infection to cohorts. Cellular immune responses, particularly those carried out by cytotoxic T lymphocyte (CTL), have been shown to be effective in killing virus-infected cells in vitro. This review underlines the state of knowledge regarding immunity to EHV-1 and also its interaction with Equine lymphocyte. Finally, the review also includes the importance of the viral immediate early (IE) protein in the pathogenesis of EHV-1. This information can be used as the basis for future research.

关键词： Equine Herpesvirus-1 (EHV-1) Lymphocyte Cytotoxic T Lymphocyte (CTL) Immune Response Peripheral Blood Mononuclear cell (PBMC)

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Doping Control Analysis of 16 Non-Steroidal Anti-Inflammatory Drugs in Equine Plasma Using Liquid Chromatography-Tandem Mass Spectrometry

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American Journal of Analytical Chemistry 2014年第17期5卷 1184-1199页

作者： Youwen You Cornelius E. Uboh Fuyu Guan Lawrence R. Soma Department of Clinical Studies School of Veterinary Medicine University of Pennsylvania Kennett Square PA USA PA Equine Toxicology & Research Center West Chester University West Chester PA USA

Non-steroidal anti-inflammatory drugs (NSAIDs) are classified as Class 4 agents by the Association of Racing Commissioners International and are banned in racehorses during competition in Pennsylvania (PA). To control the abuse of these agents in racehorses competing in PA, a forensic method for screening and confirmation of the presence of these agents is needed. Equine plasma (0.5 mL) was acidified with 75 μL 1M H3PO4 to increase recovery of the analytes by liquid-liquid extraction using methyl tert-butyl ether (MTBE). Extracted analytes were separated by reversed-phase liquid chromatography using a C8 column under gradient condition. All 16 analytes were detected, quantified and confirmed using a triple quadrupole tandem mass spectrometry with selected reaction monitoring (SRM) in both negative and positive electrospray ionization modes. The limit of detection, quantification and confirmation of the analytes were 1.0 - 5.0 ng/mL, 1.0 - 5.0 ng/mL and 1.0 - 20 ng/mL, respectively. The linear dynamic range of quantification was 5.0 - 200 ng/mL. The method is routinely used in anti-doping analysis to control the abuse of NSAIDs in racehorses competing in PA.

关键词： Equine Plasma Doping Control Non-Steroidal Anti-Inflammatory Drugs Liquid

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Immune Control of Equine Infectious Anemia Virus Infection by Cell-Mediated and Humoral Responses

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Applied Mathematics 2013年第8期4卷 171-177页

作者： Elissa J. Schwartz Kasia A. Pawelek Karin Harrington Richard Cangelosi Silvia Madrid Department of Mathematics and Computational Science University of South Carolina Beaufort Bluffton SC USA Department of Mathematics Washington State University Pullman WA USA Department of Mathematics Washington State University Pullman WA USASchool of Biological Sciences Washington State University Pullman WA USA School of Biological Sciences Washington State University Pullman WA USA

Equine Infectious Anemia Virus (EIAV) is a retrovirus that establishes a persistent infection in horses and ponies. The virus is in the same lentivirus subgroup that includes human immunodeficiency virus (HIV). The similarities between these two viruses make the study of the immune response to EIAV relevant to research on HIV. We developed a mathematical model of within-host EIAV infection dynamics that contains both humoral and cell-mediated immune responses. Analysis of the model yields results on thresholds that would be necessary for a combined immune response to successfully control infection. Numerical simulations are presented to illustrate the results. These findings have the potential to lead to immunological control measures for lentiviral infection.

关键词： Deterministic Model Virus Infection Equine Infectious Anemia Virus Immune Response Antibodies Cy-totoxic T Lymphocytes

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Phylogenetic analysis of the five internal genes and evolutionary pathways of the Greek H3N8 Equine influenza virus

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Natural Science 2012年第11期4卷 839-847页

作者： Maria Bountouri Vasileios Ntafis Eirini Fragkiadaki Theo Kanellos Eftychia Xylouri Department of Anatomy and Physiology of Farm Animals Faculty of Animal Science and Aquaculture Agricultural University of Athens Athens Greece Department of Anatomy and Physiology of Farm Animals Faculty of Animal Science and Aquaculture Agricultural University of Athens Athens Greece Pfizer Animal Health Biologicals Development Sandwich UK

To amplify the NS, NP, PB1, PB2 and PA internal genes of two Equine H3N8 influenza A viruses isolated in Greece in 2003 and 2007 five primer pairs were designed. The derived sequences were analysed from a phylogenetic point of view and compared with the evolutionary patters of the HA and NA proteins. Comparison of nucleotide sequences of the five internal genes of the Greek strains showed high similarity (99.3% - 99.7%) to strains isolated from outbreaks in Europe and Asia during 2002-2008. A total of 11 amino acid substitutions of the surface protein NA and the RNP complex proteins were identified in the Greek strains compared to those of progenitor viruses circulating up to 2003. These substitutions were repeated in Chinese and Mongolian isolates from outbreaks in 2007-2008. Notably NS1 protein did not acquired amino acid substitutions and moreover, a stop codon introduced at position 220 was stably maintained in the Greek strains. Phylogenetic trees of the five internal genes did not show the same separation in clades. Greek strains classified them into the American sublineage (as for the PA) Florida clade II (as for the NP, NS1 and PB1) and among Chinese strains of 2007-2008 outbreaks (as for the PB2). Additionally, evolutionary profiles of these internal proteins, except PB2, indicated a parallel evolution fashion to the HA protein, suggesting the possible occurrence of genetic reassortment between H3N8 viruses of district evolutionary lineages. In conclusion, phylogenetic analysis of the internal genes reported in this study could establish a candidate framework for future scientific communications on the phylogenetic diversity and evolution of the Equine influenza viruses.

关键词： Equine Influenza Internal Genes Evolution Pathways

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Proviral genomic sequence analysis of Chinese donkey leukocyte attenuated Equine infectious anemia virus vaccine and its parental virus strain Liaoning

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Science China(Life Sciences) 2002年第1期45卷 57-67页

作者：王柳杨志彪王玫童光志仇华吉刘红全孔宪刚 National Key Laboratory of Veterinary Biotechnology Harbin Veterinary Research Institute Chinese Academy of Agricultural Sciences Harbin 150001 China

Proviral DNA was extracted from donkey leukocyte infected with Chinese donkey leukocyte attenuated Equine infectious anemia virus(DLA-EIAV), and peripheral blood lymphocytes(PBL) from a horse infected with the virulent EIAV strain Liaoning(EIAV L). The entire proviral DNA from both viruses was cloned and sequenced. The lengths of complete genomic sequences of DLA-EIAV and EIAV L provirus were 8266 bp and 8235 bp, respectively. Sequence comparison indicated that DLA-EIAV shares 97.0% and 97.5% in sequence homology with EIAV L and donkey-adapted EIAV(DA-EIAV), respectively. Lots of variations occurred in long terminal repeat(LTR, consisting of U3, R, U5), ORF S2, and env regions between DLA-EIAV and EIAV L. The nucleotide sequence differences of the two viruses in U3, R, U5, ORF S2, and env are 13.2%, 7.5%, 5.1%, 3.9%, and 2.7%, respectively, and predicted amino acid sequence differences in env and S2 coding regions are 4.4% and 8.8%, respectively. Six conserved regions are characterized in Gp90. There is a cis-activating GATA motif in ENH of DLA-EIAV and EIAV L. Two N-linked glycosylation sites disappeared in DLA-EIAV Gp90 in comparison with that of EIAV L. A bHLH transcription factor binding consensus sequence was found in LTR of DLA-EIAV but not in EIAV L. Furthermore, there is a mutation in the stem of DLA-EIAV TAR resulting in formation of a uridine tuber. Further study is needed to uncover the relationship between sequence changes and their biological functions of DLA-EIAV and L.

关键词： Equine infectious anemia virus(EIAV), donkey leukocyte attenuated(DLA) strain, L strain, sequence analysis.

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Selection of Reference Genes in Equine White Blood Cells for Real Time PCR Normalization Following Extracorporeal Shock Wave Therapy

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American Journal of Molecular Biology 2014年第2期4卷 72-80页

作者： Zibin Jiang Jinwen Chen Cornelius E. Uboh Mary A. Robinson Lawrence R. Soma Department of Clinical Studies New Bolton Center Campus University of Pennsylvania School of Veterinary Medicine Philadelphia USA Department of Clinical Studies New Bolton Center Campus University of Pennsylvania School of Veterinary Medicine Philadelphia USA Pennsylvania Equine Toxicology & Research Center Department of Chemistry West Chester University West Chester USA

Selection of proper reference genes (RGs) is an essential step needed for accurate normalization of results from genomic studies. Expression of RGs is regulated by many factors such as species, age, gender, type of tissue, the presence of disease, and the administration of therapeutic treatment. The aim of the present study was to identify optimal RGs in a set of blood samples collected at different time points (0, 24, 48, 72 h) from horses following administration of extracorporeal shock wave therapy (ESWT). The mRNA expression of twelve RGs: HPRT1, ACTB, HSP90A, SDHA, GUSB, B2M, UBC, NONO, TBP, H6PD, RPL32, GAPDH was determined using real time quantitative polymerase chain reaction (qPCR). An SAS program developed on the algorithm of geNorm, SASqPCR, was used to determine stability of the expression and the number of optimal RGs. The results showed that the range of quantification cycle (Cq) values of the evaluated genes varied between 17 and 26 cycles, and that one optimal RG, ACTB, was sufficient for normalization of gene expression. Results of stability of expression demonstrated that ACTB was the optimal choice for all the samples studied. Notably, in samples collected at 72 h post ESWT, TBP showed a significant change in the expression level, and was not suitable for use as a RG. These results substantiate the importance of validating and selecting an appropriate RG.

关键词： Reference Genes Real Time PCR Normalization Equine White Blood Cell Extracorporeal Shock Wave Therapy

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Fast and Sensitive Chiral Analysis of Amphetamines and Cathinones in Equine Urine and Plasma Using Liquid Chromatography Tandem Mass Spectrometry

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American Journal of Analytical Chemistry 2015年第13期6卷 995-1003页

作者： Caroline C. Wang Petra Hartmann-Fischbach Tim R. Krueger Alisha Lester Aaron Simonson Terry L. Wells Max O. Wolk Nick J. Hidlay Industrial Laboratories Wheat Ridge USA

A simple, rapid, sensitive and reproducible method for enantiomer analysis of methamphetamine, amphetamine, cathinone and methcathinone was developed and validated. The compounds were extracted from Equine plasma and urine using a fast liquid-liquid extraction procedure. Only one milliliter plasma and one hundred microliter urine sample is needed for analysis. The extraction procedure had good recovery (>70%) and the matrix effect was negligible. Enantiomer differentiation and confirmation were achieved using liquid chromatography with chiral stationary phase and mass spectrometry detection. The method demonstrated excellent reproducibility with intra-day and inter-day precision of lower than 5%. The lower limits of detection for all of the compounds studied here were at low pg/mL level for both plasma and urine. This is the first report of the analysis of four chiral compounds in Equine plasma and urine. Routine application was demonstrated for (S)- and (R)-enantiomer differentiation.

关键词： Amphetamines and Cathinones Equine Plasma and Urine Chiral Analysis Liquid Chromatography Tandem Mass Spectrometry

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Pathophysiology, diagnosis and treatment of intermittent claudication in patients with lumbar canal stenosis

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World Journal of Orthopedics 2014年第2期5卷 134-145页

作者： Shigeru Kobayashi Department of Orthopaedics and Rehabilitation Medicine Faculty of Medical Sciences and the Research and Education Program for Life Science the University of Fukui Fukui 910-1193 Japan

Spinal nerve roots have a peculiar structure, different from the arrangements in the peripheral nerve. The nerve roots are devoid of lymphatic vessels but are immersed in the cerebrospinal fluid(CSF) within the subarachnoid space. The blood supply of nerve roots depends on the blood flow from both and peripheral direction(ascending) and the spinal cord direction(descending). There is no hypovascular region in the nerve root, although there exists a so-called water-shed of the bloodstream in the radicular artery itself. Increased mechanical compression promotes the disturbance of CSF flow, circulatory disturbance starting from the venous congestion and intraradicular edema formation resulting from the breakdown of the blood-nerve barrier. Although this edema may diffuse into CSF when the subarachnoid space is preserved, the endoneurial fluid pressure may increase when the area is closed by increased compression. On the other hand, the nerve root tissue has already degenerated under the compression and the numerous macrophages releasing various chemical mediators, aggravating radicular symptomsthat appear in the area of Wallerian degeneration. Prostaglandin E1(PGE1) is a potent vasodilator as well as an inhibitor of platelet aggregation and has therefore attracted interest as a therapeutic drug for lumbar canal stenosis. However, investigations in the clinical setting have shown that PGE1 is effective in some patients but not in others, although the reason for this is unclear.

关键词： Lumbar canal stenosis Cauda Equine Nerve root Prostaglandin E1 Blood flow

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