Characterization and clinical application of mesenchymal stem cells from equine umbilical cord blood

Tendinitis of the superficial digital flexor tendon (SDFT) is a significant cause of lameness in horses; however, recent studies have shown that stem cells could be useful in veterinary regenerative medicine. Therefore, we isolated and characterized equine umbilical cord blood mesenchymal stem cells (eUCB-MSCs) from equine umbilical cord blood obtained from thoroughbred mares during the foaling period. Horses that had tendinitis of the SDFT were treated with eUCB-MSCs to confirm the therapeutic effect. After eUCB-MSCs transplantation, the core lesion in the SDFT was found to decrease. These results suggest that transplantation using eUCB-MSCs could be another source of cell treatment.     

Three types of anomalous vasculature in the equine umbilical cord

Anomalies in the number of major blood vessels in the umbilical cord are well documented in man and have been associated with both placental abnormalities and congenital problems in the neonate. This paper describes 5 cases of anomalies in the number of umbilical arteries or veins within the equine umbilical cord at term. In 4 cases, the anomalies affected vessels in the amniotic part of the cord, and in one case, vessels in the allantoic part. In all the cases placental morphology was essentially normal and fetal health and post natal development were not compromised as a result of the unusual arrangement of blood vessels within the cord.     

In vitro neuronal and osteogenic differentiation of mesenchymal stem cells from human umbilical cord blood

Mesenchymal stem cells (MSCs) have the capabilities for self-renewal and differentiation into cells with the phenotypes of bone, cartilage, neurons and fat cells. These features of MSCs have attracted the attention of investigators for using MSCs for cell-based therapies to treat several human diseases. Because bone marrow-derived cells, which are a main source of MSCs, are not always acceptable due to a significant drop in their cell number and proliferative/differentiation capacity with age, human umbilical cord blood (UCB) cells are good substitutes for BMCs due to the immaturity of newborn cells. Although the isolation of hematopoietic stem cells from UCB has been well established, the isolation and characterization of MSCs from UCB still need to be established and evaluated. In this study, we isolated and characterized MSCs. UCB-derived mononuclear cells, which gave rise to adherent cells, exhibited either an osteoclast or a mesenchymal-like phenotype. The attached cells with mesenchymal phenotypes displayed fibroblast-like morphologies, and they expressed mesenchym-related antigens (SH2 and vimentin) and periodic acid Schiff activity. Also, UCB-derived MSCs were able to transdifferentiate into bone and 2 types of neuronal cells, in vitro. Therefore, it is suggested that the MSCs from UCB might be a good alternative to bone marrow cells for transplantation or cell therapy.     

Characterization and quantification of blood cells from the umbilical cord of dogs

BACKGROUND: Models for the study of hematopoietic stem cells in dogs provide important information for bone marrow transplantation in humans. Recent studies have reported the importance of human umbilical cord blood (UCB) as an alternative to allogenic bone marrow for hematopoietic reconstitution. However, there are no studies on the UCB cells of dogs. OBJECTIVE: The aim of this experiment was to characterize and quantify the blood cells of the umbilical cord of dogs. METHODS: The blood of the umbilical cord of 20 neonatal dogs, delivered at term, with a median gestation time of 58 days, was collected with a 5-mL syringe containing EDTA. Total RBC, WBC, and platelet counts, HCT, hemoglobin (Hgb) concentration, and RBC indices were determined using an automatic cell counter. The differential leukocyte count was determined manually in blood smears stained with May-Grünwald-Giemsa. Reticulocyte percentages were determined on blood smears stained with brilliant cresyl blue and counterstained with May-Grünwald Giemsa. RESULTS: The MCHC and numbers of RBCs, WBCs, neutrophils, and eosinophils in UCB were lower as compared with reference values for the peripheral blood of healthy neonatal and adult dogs; whereas, the MCV and reticulocyte percentages were higher. CONCLUSION: Erythrocyte macrocytosis and hypochromasia in UCB were consistent with marked reticulocytosis and indicative of high erythropoietic activity. The results of this study are an important first step in the characterization of UCB from neonatal dogs.     

Use of ultrasonography to detect pulmonary lesions in Thoroughbred foals in Argentina

Rhodococcus equi is an important cause of pulmonary disease in foals often related to economic losses and death. A study was performed on a Thoroughbred farm (Buenos Aires Province, Argentina) where this disease was enzootic causing 1–2% of foal deaths every year. One hundred foals were monitored by thoracic ultrasonography at ages 1–5 months to detect pulmonary lesions in order to initiate an early treatment. This strategy allowed reducing the number of foal deaths almost totally. In our experience, thoracic ultrasonography was proved to be very efficient in detection of early cases of pneumonia on a farm with high density of horses and intensive management.     

Isolation and characterization of canine umbilical cord blood-derived mesenchymal stem cells

Human umbilical cord blood-derived mesenchymal stem cells (MSCs) are known to possess the potential for multiple differentiations abilities in vitro and in vivo. In canine system, studying stem cell therapy is important, but so far, stem cells from canine were not identified and characterized. In this study, we successfully isolated and characterized MSCs from the canine umbilical cord and its fetal blood. Canine MSCs (cMSCs) were grown in medium containing low glucose DMEM with 20% FBS. The cMSCs have stem cells expression patterns which are concerned with MSCs surface markers by fluorescence-activated cell sorter analysis. The cMSCs had multipotent abilities. In the neuronal differentiation study, the cMSCs expressed the neuronal markers glial fibrillary acidic protein (GFAP), neuronal class III β tubulin (Tuj-1), neurofilament M (NF160) in the basal culture media. After neuronal differentiation, the cMSCs expressed the neuronal markers Nestin, GFAP, Tuj-1, microtubule-associated protein 2, NF160. In the osteogenic & chondrogenic differentiation studies, cMSCs were stained with alizarin red and toluidine blue staining, respectively. With osteogenic differentiation, the cMSCs presented osteoblastic differentiation genes by RT-PCR. This finding also suggests that cMSCs might have the ability to differentiate multipotentially. It was concluded that isolated MSCs from canine cord blood have multipotential differentiation abilities. Therefore, it is suggested that cMSCs may represent a be a good model system for stem cell biology and could be useful as a therapeutic modality for canine incurable or intractable diseases, including spinal cord injuries in future regenerative medicine studies.     

Effect of altrenogest‐treatment of mares in late gestation on adrenocortical function,blood count and plasma electrolytes in their foals

Reasons for performing study: Mares with compromised pregnancies are often treated with altrenogest to prevent abortion. However, there is only limited information about effects on the foal when altrenogest treatment is continued during final maturation of the fetus. Objectives: To determine effects of altrenogest treatment during late gestation in mares on maturity, haematology changes, adrenocortical function and serum electrolytes in their newborn foals. Methods: Six mares were treated with altrenogest (0.088 mg/kg bwt) once daily from Day 280 of pregnancy until foaling and 7 mares served as controls. Results: Foals born to altrenogest‐treated mares had a significantly lower neutrophil/lymphocyte ratio on the first day after birth than control foals (P<0.05). Basal plasma cortisol concentrations immediately after birth were higher in foals of altrenogest‐treated mares than in control foals (P<0.05). Cortisol release in response to exogenous adrenocorticotropic hormone (ACTH) ‐ except for higher values 15 min after ACTH injection in foals of altrenogest‐treated mares on Day 1 ‐ revealed no differences in adrenocortical function between the groups of foals. Plasma potassium concentration in foals from altrenogest‐treated mares compared to control foals was significantly lower immediately after birth (P<0.05) and plasma ionised calcium concentration was significantly lower 3 h after birth (P = 0.01). Conclusions and potential relevance: Altrenogest treatment of pregnant mares prolonged labour had no major effects on adrenocortical function in foals. A reduced neutrophil/ lymphocyte ratio in these foals may suggest either immunomodulatory effects of altrenogest or dysmaturity of the foals.     

Assessment of the equine neonate in ambulatory practice

The ambulatory practitioner is usually the first to evaluate the equine neonate and must assess the foal and determine its health status. If there is suspicion of one or more abnormalities, then the ambulatory veterinarian must initiate appropriate treatment and determine if the foal can be successfully treated in the field or should be referred to a specialty hospital. This article will describe the examination procedure including an accurate history, a comprehensive physical examination, laboratory evaluation and application of point‐of‐care diagnostics. The initial examination is the basis for the correct determination of health or illness and informing the owner of the correct course of action for the neonatal foal.     

Stability of common biochemistry analytes in equine blood stored at room temperature

Reasons for performing study: Time delays between collection of blood samples and biochemical analysis of equine blood are unavoidably common in equine practice. The effect that delays may have on the accuracy of results of blood biochemical analyses is not well established. Hypothesis: Delays in processing of blood of up to 72h results in alterations in measured levels of common biochemical analytes that are of potential clinical relevance. Separation of serum prior to storage is protective against the effects of time delays. Methods: Samples of clotted blood, separated serum and oxalate fluoride plasma from 20 horses were stored and analysed at 0, 24, 48 and 72 h. Graphical exploration of each analyte was undertaken. General linear models with fixed effects were fitted for the whole blood data. The mean bias and 95% limits of agreement were calculated, using bootstrapped data, to assess agreement between pairs of samples analysed at 0 h and other time points. Bland‐Altman plots were used to explore general trends in the data. Paired t tests were used to compare the results from whole blood and separated serum. Results: Delays in processing equine blood resulted in significant increases in measured concentrations of aspartate aminotransferase, creatine kinase, lactate dehydrogenase, total bile acids and magnesium. A significant decrease in concentration was identified for glucose (serum and oxalate fluoride preserved plasma). Separation of serum immediately following clot formation resulted in nonsignificant increases in accuracy for some analytes. Conclusions and practical significance: Delays in processing of blood samples may result in biochemical changes of clinical relevance in individual cases; however, in the majority of cases, where delays are only a few days and a number of analytes are assessed concurrently, delays are unlikely to have an effect on the interpretation of results. Separation of serum following clot formation is of limited benefit. Clinical samples in which a delay in processing has occurred may be interpreted with reference to the data presented.     

Management and treatment of the sick equine neonate in ambulatory practice

The ambulatory practitioner is usually the first to evaluate the equine neonate and must assess the foal and determine its health status. If one or more abnormalities are identified, the practitioner must initiate appropriate treatment and determine whether the foal can be managed successfully on the farm or referred to a specialty centre. This article describes the basic requirements that should be met and the limitations involved in treating compromised neonates on the farm. Treatments for common foal disorders are discussed.     

Osteogenic potential of mesenchymal cells derived from canine umbilical cord matrix co-cultured with platelet-rich plasma and demineralized bone matrix

Canine mesenchymal cells (MSCs) derived from Wharton''s jelly were co-cultured, then supplemented or not supplemented with platelet rich plasma (PRP) and demineralized bone matrix (DBM) to verify osteogenic differentiation. Osteoblastic differentiation followed by mineralized bone matrix production was found to be significantly higher (p < 0.05) when MSCs were associated with PRP/DBM in culture after 14-21-days of induction. Osteopontin and osteocalcin gene expression were significantly superior (p < 0.05) under the same culture conditions after 21 days of observation. In conclusion, addition of PRP to DBM co-cultured with MSCs successfully induced osteogenesis in vitro.     

Agreement between point-of-care glucometry, blood gas and laboratory-based measurement of glucose in an equine neonatal intensive care unit

Objective: To test the agreement between 3 common methods of glucose measurement in a population of critically ill foals presenting to a neonatal intensive care unit. Design: Prospective clinical study. Setting: University large animal hospital neonatal intensive care unit. Animals: Sequentially admitted critically ill neonatal foals <30 days of age. Interventions: Venous blood obtained from a jugular vein was used for determination of blood glucose concentration using point‐of‐care (POC) glucometry (GLU), a laboratory chemistry technique (CHEM) and a multi‐electrode blood gas analyzer (BG). Paired data were compared using Lin's concordance correlation, Pearson's correlation and robust regression. Bias and limits of agreement were investigated using the technique of Bland and Altman. Measurements and main results: Concordance was significant for all comparisons and was strongest for CHEM‐BG while weakest for GLU‐BG. Pearson's correlation was excellent for all comparisons: CHEM‐BG, 0.98; GLU‐BG, 0.94; GLU‐CHEM, 0.96. All comparisons had significant robust regression coefficients: CHEM‐BG, 0.99; GLU‐BG, 0.80; GLU‐CHEM, 0.79. For Bland–Altman analysis, mean differences (mean±SD, 95% limits of agreement) were: GLU‐BG (?33±18 mg/dL, ?68.6 to 1.5); GLU‐CHEM (?20±16 mg/dL, ?51.0 to 10.9); CHEM‐BG (13±11 mg/dL, ?8.7 to 34.7). Conclusions: This study demonstrates that glucometry has less than ideal agreement with a laboratory standard and another POC test, blood gas analysis. These differences may be clinically important and decisions regarding management of glucose concentrations in critically ill foals should be made with these differences in mind.     

Seroprevalence of equine herpesvirus 1 in Thoroughbred foals before and after weaning

Objective To investigate the seroprevalence of equine herpesvirus 1 in foals around weaning and after weaning on two large Thoroughbred farms using a type-specific enzyme-linked immunosorbent assay to determine exposure to infection.
Design A longitudinal population study in groups of Thoroughbred weanling foals.
Study population Two hundred weanling Thoroughbred foals from a population of about 380 foals were enrolled on two adjacent stud farms in the Hunter Valley of New South Wales. Foals on both farms were weaned from February to May 1995 into randomly selected groups of 10 to 15 foals. Farms were selected because of their willingness to cooperate in the survey and because their detailed records of foals and their movements. They were representative of well-managed large Thoroughbred stud farms in New South Wales. Both studs had upper respiratory tract disease among weanling foals around weaning each year although the sero-prevalence of viral respiratory disease on either farm was not known before the study.
Procedure Serum was collected from foals within each group at fortnightly intervals from 9th February until 1st June 1995, and at a single follow-up period in August 1995. Each sample was tested in triplicate using an antibody-detection ELISA which is type-specific for EHV-1 and EHV-4 antibodies.
Results and conclusions There was serological evidence of EHV-1 infection both before and after weaning. The prevalence of EHV-1 antibody in the sample population increased during the study and individual cases of EHV-1 infection were identified. The increase was caused both by the seroconversion of foals within the groups and by the recruitment into the study of foals with pre-existing EHV-1 antibody. Evidence of EHV-1 infection in Thoroughbred foals after weaning has not been reported previously in Australia and this has implications for vaccination regimens.     

Autologous point‐of‐care cellular therapies variably induce equine mesenchymal stem cell migration,proliferation and cytokine expression

Reasons for performing study: Autologous cellular therapy products including adipose‐derived stromal vascular fraction (SVF), bone marrow mononuclear cells (BMMNs), cord blood mononuclear cells (CBMNs) and platelet rich plasma are options for treatment of acute orthopaedic lesions while mesenchymal stem cells (MSCs) are culture expanded. These products may contribute to healing by secreting matrix proteins or growth factors, but they may also act on endogenous MSCs to facilitate healing. Objectives: To determine the effects of cell therapy products on MSCs function in vitro. The hypothesis was that cell therapy products promote MSCs functions including proliferation, migration and mediator release. Methods: Fat, bone marrow (BM), cord blood and platelets were obtained from 6 Quarter Horses. The BM‐MSCs and their autologous cell therapy products were co‐incubated in transwells. Mesenchymal stem cells proliferation, migration, gene expression and cytokine concentrations were determined. Results: All cell therapy products increased MSCs proliferation, but SVF induced significantly more proliferation than any other product. Also SVF elicited more MSCs chemotaxis and, along with BMMNs, significantly more MSCs chemoinvasion. Cord blood mononuclear cells stimulated MSCs to produce high concentrations of interleukin‐6 (IL‐6), transforming growth factor‐β1 (TGF‐β1), and prostaglandin E₂ (PGE₂). Stromal vascular fraction and platelet lysate did not stimulate MSCs but SVF and platelet lysate themselves contained high concentrations of PGE₂ and IL‐6 (SVF) and TGF‐β1 (platelet lysate). Conclusions: Autologous cell products variably stimulate MSCs functions with 2 primary patterns apparent. Products either contained preformed mediators that may have intrinsic healing function, or products stimulated MSCs to secrete mediators. Potential relevance: The specific clinical indications for these products may differ to include administration as a sole treatment modality prior to MSCs injection for intrinsic cell and cytokine activity (i.e. SVF) or administration concurrently with MSCs to activate MSCs for treatment of chronic lesions (i.e. CBMNs).     

规模化猪场脐带血猪瘟病原检测及遗传变异分析

为了了解某规模化猪场母猪猪瘟病毒(CSFV)带毒及病原遗传变异情况,应用荧光定量PCR对117份分娩母猪的脐带血进行猪瘟病原检测,对阳性样品进行CSFV E2基因RT-PCR扩增,研究CSFV遗传变异特性。结果表明,该猪场猪瘟病原阳性率为7.69%(9/117),5株CSFVE2基因之间的核苷酸同源性为97.8%~100%,与参考株的同源性为80.0%~97.5%,与C株和HCLV株的同源性分别为95.6%~96.7%、96.4%~97.5%;5株CSFVE2基因推导的氨基酸同源性为97.8%~100%,与参考株之间同源性为83.5%~96.7%,与C株和HCLV株同源性均为95.5%~96.7%。遗传进化树分析表明,5株CSFV E2基因属于1.1亚群,与疫苗株HCLV亲缘性最近;氨基酸变异位点分析表明,5株CSFV E2基因在位于抗原决定区内氨基酸发生较小程度的变异。表明该猪场通过胎盘屏障感染仔猪的猪瘟病毒可能来源疫苗株,这种现象应该引起重视。