Gareth Trope

Efficient transduction of equine adipose-derived mesenchymal stem cells by VSV-G pseudotyped lentiviral vectors

Equine adipose-derived mesenchymal stem cells (EADMSC) provide a unique cell-based approach for treatment of a variety of equine musculoskeletal injuries, via regeneration of diseased or damaged tissue, or the secretion of immunomodulatory molecules. These capabilities can be further enhanced by genetic modification using lentiviral vectors, which provide a safe and efficient method of gene delivery. We investigated the suitability of lentiviral vector technology for gene delivery into EADMSC, using GFP expressing lentiviral vectors pseudotyped with the G glycoprotein from the vesicular stomatitis virus (V-GFP) or, for the first time, the baculovirus gp64 envelope protein (G-GFP). In this study, we produced similarly high titre V-GFP and G-GFP lentiviral vectors. Flow cytometric analysis showed efficient transduction using V-GFP; however G-GFP exhibited a poor ability to transduce EADMSC. Transduction resulted in sustained GFP expression over four passages, with minimal effects on cell viability and doubling time, and an unaltered chondrogenic differentiation potential.

Direct Conversion of Equine Adipose-Derived Stem Cells into Induced Neuronal Cells Is Enhanced in Three-Dimensional Culture

The ability to culture neurons from horses may allow further investigation into equine neurological disorders. In this study, we demonstrate the generation of induced neuronal cells from equine adipose-derived stem cells (EADSCs) using a combination of lentiviral vector expression of the neuronal transcription factors Brn2, Ascl1, Myt1l (BAM) and NeuroD1 and a defined chemical induction medium, with βIII-tubulin-positive induced neuronal cells displaying a distinct neuronal morphology of rounded and compact cell bodies, extensive neurite outgrowth, and branching of processes. Furthermore, we investigated the effects of dimensionality on neuronal transdifferentiation, comparing conventional two-dimensional (2D) monolayer culture against three-dimensional (3D) culture on a porous polystyrene scaffold. Neuronal transdifferentiation was enhanced in 3D culture, with evenly distributed cells located on the surface and throughout the scaffold. Transdifferentiation efficiency was increased in 3D culture, with an increase in mean percent conversion of more than 100% compared to 2D culture. Additionally, induced neuronal cells were shown to transit through a Nestin-positive precursor state, with MAP2 and Synapsin 2 expression significantly increased in 3D culture. These findings will help to increase our understanding of equine neuropathogenesis, with prospective roles in disease modeling, drug screening, and cellular replacement for treatment of equine neurological disorders.

Survey of equine castration techniques, preferences and outcomes among Australian veterinarians

OBJECTIVES (1) To collect the perceptions of veterinarians performing equine castrations in Australia on techniques, preferences and outcomes, (2) to investigate veterinarian use and experience with the Henderson castrating instrument and (3) to investigate potential associations between demographics, castration methods and techniques, and complications. DESIGN Online survey of members of the Australian Veterinary Associations Special Interest Group, Equine Veterinarians Australia (EVA). METHODS A link to the survey was included in the EVA e-newsletter and practices on the EVA website were contacted by telephone and follow-up email. Fishers exact test was used to determine associations between ligation and complications. A generalised linear model with a negative binomial family was used to determine associations between count response variables and categorical independent variables. RESULTS Responses were obtained from 138 veterinarians (response rate, 13.1%) who performed 5330 castrations over 12 months. Castrations were most commonly performed in the field, on anaesthetised horses, using emasculators, via an open approach and without ligation of the spermatic cord. Estimated complications after use of emasculators were swelling (25%), haemorrhage (5%) and infection (5%). The Henderson instrument was used by approximately 10% of respondents and its use for castration was associated with fewer reports of postoperative swelling compared with emasculators (P = 0.002). Rates of evisceration with the Henderson and emasculator methods were comparable (0.43% and 0.9%, respectively). CONCLUSION Castration preferences varied widely among survey participants. Reported complication types and rates were comparable to those reported previously in other countries. Perceptions that the Henderson instrument was associated with less swelling should be investigated further via a prospective controlled investigation.

Generation of Immortalised Equine Chondrocytes With Inducible Sox9 Expression Allows Control of Hypertrophic Differentiation.

Immortalization of chondrocytes enables long term in vitro culture; however, the chondrogenic capacity of transformed cells varies, thus highlighting the need to develop a proliferative and tuneable chondrocyte cell line where hypertrophic differentiation can be controlled. In this study the SV40 large T antigen and human telomerase reverse transcriptase were employed to immortalize pooled equine chondrocytes through lentiviral vector mediated transduction either singly or on combination. Transformed chondrocytes proliferated stably over multiple passages, but resulted in significantly lower expression of chondrocyte specific collagen II mRNA (P < 0.0001) and up regulation of the hypertrophic marker collagen X (P < 0.0001) in three dimensional cultures. A Col2a1 promoter driven GFP reporter was constructed for real time monitoring of chondrogenic differentiation and a significant increase in promoter activation was observed in cultures treated with the growth factor TGFβ‐3 (P < 0.05). To recapitulate the native articular chondrocyte phenotype we further transduced large T antigen immortalized chondrocytes with lentiviral vectors allowing either constitutive or doxycycline inducible expression of Sox9. In 3D cultures, the Sox9 over‐expressing chondrocytes secreted significantly higher levels of extracellular matrix polysaccharide glycosaminoglycan (P < 0.05), while up‐regulating collagen II and Aggrecan mRNA (P < 0.05) in both expression systems with a similar patterns observed with imunohistochemical staining. High levels of collagen X mRNA and protein were maintained with constitutive sox9 reflecting hypetrophic differentiation but significantly lower expression could be achieved with inducible Sox9. In conclusion, immortalization of equine chondrocytes results in stable proliferation but a reduction of chondrogenic potential whilst modulation of sox9 expression enabled control of hypertrophic characteristics. J. Cell. Biochem. 118: 1201–1215, 2017.

Palatal dysfunction in horses: where next?

PALATAL dysfunction, the term which describes both intermittent dorsal displacement of the soft palate and palatal instability, is one of the most commonly identified causes of upper airway obstruction in horses (Barakzai and Hawkes 2010). The reported prevalence of palatal dysfunction ranges from 10 to 20 per cent in young racehorses (Pollock and others 2009, Ducharme 2012). Dynamic endoscopic examination (high speed treadmill endoscopy or overground endoscopy) is required for definitive diagnosis of this condition (Barakzai and Dixon 2011). The reported incidence of palatal dysfunction in horses presented for poor performance or abnormal noise during exercise following dynamic endoscopy ranges from 20 to 50 per cent (Kannegieter and Dore 1995, Tan and others 2005, Lane and others 2006, Pollock and others 2009, Allen and Franklin 2010, Kelly and others 2013). The laryngeal tie-forward (LTF) procedure using metallic implants. The distance of laryngeal advancement is being measured Numerous options exist for the treatment of palatal dysfunction in horses including, but not limited to, conservative management, the use of …

Dynamic endoscopy of the equine upper airway – what is significant?

DYNAMIC upper respiratory collapse is a common cause of poor performance in horses (Dart and others 2001, Parente and others 2002, Tan and others 2005, Lane and others 2006). Dynamic endoscopy of the upper respiratory tract allows the identification of various obstructive conditions that endoscopy at rest is unable to identify. Until recently, this has only been achievable using high speed treadmill endoscopy (HSTE). This technique has allowed the identification of numerous obstructive conditions, including recurrent laryngeal neuropathy, intermittent dorsal displacement of the soft palate, axial deviation of the aryepiglottic folds, vocal fold collapse and, more recently, ventroaxial luxation of the apex of the corniculate process of the arytenoid cartilage (Kannegieter and Dore 1995, Dart and others 2001, 2005, Parente and others 2002, Tan and others 2005, Lane and others 2006, Davidson and others 2011). Some of the main criticisms of HSTE include an inability to recreate true racing/training conditions, the cost of the equipment, limited availability of high speed treadmills and the safety risk to both horse and handlers. The development …

Lentiviral vector expression of Klf4 enhances chondrogenesis and reduces hypertrophy in equine chondrocytes

Monolayer expansion of chondrocytes in culture results in the dedifferentiation of chondrocytes with inferior cartilage specific extracellular matrix synthesis and proliferation when compared with its native counterpart. We aimed to enhance chondrocyte proliferation and articular cartilage specific gene expression through ectopic expression of the major pluripotency transcription factors (Oct4, Sox2, Klf4 and c-Myc). We also aimed to provide insights to the modulation of TGFβ receptor mRNA with Klf4 overexpression. Equine chondrocytes pooled from three donors were transduced with lentiviral vectors expressing the induced pluripotency factors, Oct4, Sox2. Klf4 and c-Myc (OSKM), singly, or in combination or together with green fluorescent protein (GFP) as a control. Klf4 and c-Myc overexpressing chondrocytes showed a significant increase in mitosis when compared to the control (P < 0.01 and P < 0.0001 respectively). Furthermore, overexpression of Klf4 or OSKM in three dimensional (3D) culture of equine chondrocytes resulted in a significant increase in Col2a1 mRNA levels relative to the controls (P < 0.05 and P < 0.01 respectively) while all transcription factors significantly lowered the mRNA of the fibrocartilage marker Col1a1. We also employed a Col2a1 promoter driven GFP reporter for real time monitoring of Col2a1 gene activation in 3D micromass culture, which showed significantly higher promoter activity when cultures were treated with the growth factor TGFβ3 (P < 0.05). The chondrogenic properties of Klf4 transduced chondrocytes at a lower passage (P4) showed significant increases in Sox9 (P < 0.001), Col2a1 (P < 0.05) and TGFβ receptor I (P < 0.05) and II (P < 0.001) expression relative to the DS-Red expressing control. The chondrocyte dedifferentiation marker Col1a1 and hypertrophic marker Col10a1 were significantly downregulated with the inclusion of Klf4 (P < 0.01 and P < 0.05 respectively). In Conclusion, chondrogenic re-differentiation and proliferation of equine chondrocytes is promoted through ectopic expression of Klf4 while suppressing chondrocyte dedifferentiation.

Pharmacokinetic and pharmacodynamic effects of two omeprazole formulations on stomach pH and gastric ulcer scores

REASONS FOR PERFORMING THE STUDY Limited data are available on the relative pharmacokinetics and pharmacodynamics of different omeprazole formulations. OBJECTIVES To compare pharmacokinetic and pharmacodynamic effects of a novel omeprazole formulation against a currently registered product. STUDY DESIGN Masked 2 period, 2 treatment crossover. METHODS Twelve clinically healthy horses were studied over two 6-day treatment periods. Horses were randomly assigned to receive a novel omeprazole paste (Ulcershield: ULS) or a currently registered reference omeprazole product (OMO). Gastric pH was measured continuously for 10 h on the day prior to commencing treatment (Day -1) and after 6 days of oral treatment (Day 5) using in situ antimony pH probes within an indwelling nasogastric tube. Plasma pharmacokinetics were determined on Days 0 and 6. RESULTS Treatment significantly (P<0.005) increased gastric pH on Day 5, compared to results obtained prior to treatment (Day -1) and there was no significant difference between products (P = 0.773). Similarly, comparison of median hourly gastric pH (P = 0.593), mean gastric pH (P = 0.154), percentage time pH<4 (P = 0.259) and area under the time-gastric pH response curve (P = 0.734) did not discriminate between products. Both treatments resulted in significantly lower gastric ulcer severity scores (both P = 0.004), with no difference between treatments (P = 0.688). Comparison of mean log area under time-plasma concentration curves demonstrated that, although the lower limit of the 90% confidence interval was within the -20% limit for bioequivalence, the upper limit was exceeded, suggesting that the test product could have greater bioavailability than the reference product. MAIN LIMITATIONS The small sample size, large interhorse plasma omeprazole concentrations, and low bioavailability of omeprazole impacted the sensitivity of the bioequivalence analysis. CONCLUSIONS ULS matched or slightly exceeded OMO plasma concentrations. Both products resulted in equivalent increases in gastric pH, gastric pH profiles and decrease in gastric ulcer scores. Thus, ULS was pharmacodynamically equivalent to OMO and was associated with an equivalent beneficial effect on gastric squamous mucosal ulceration.

Oesophageal lumen pH in yearling horses and effects of management and administration of omeprazole.

BACKGROUND In human subjects, arytenoid chondritis can be caused by chemical trauma of mucosa attributable to gastro-oesophageal reflux. Although a similar process may be involved in the aetiopathogenesis of arytenoid chondritis in horses, the oesophageal lumen pH in this species is poorly understood. OBJECTIVES To determine if gastro-oesophageal reflux occurs in horses by characterising oesophageal lumen pH. STUDY DESIGN Blinded, randomised, placebo-controlled, crossover, experimental study. METHODS Luminal oesophageal pH in six yearling horses was recorded over four 24 h periods using an ambulatory pH recorder attached to a catheter with two electrodes (proximal and distal) inserted into the oesophagus. Recordings of pH were made during three management protocols. Initially, horses grazed in a paddock (Protocol A). Horses were then moved to stables to simulate sale preparation of Thoroughbred yearlings, and were given either omeprazole (Protocol B) or placebo paste (Protocol C) orally once per day. Protocol A was repeated for each horse (after a 13 day washout period) between Protocols B and C. Summary statistics described pH range and frequency of pH changes. Associations with predictor variables were investigated using linear mixed-effects models. Data are presented as the mean ± s.d. RESULTS Oesophageal lumen pH ranged from 4.90 to 9.70 (7.36 ± 0.27 and 7.18 ± 0.24 for the proximal and distal electrodes, respectively) and varied frequently (1.2 ± 0.9 changes/min and 0.8 ± 0.8 changes/min for the proximal and distal electrodes, respectively). Oesophageal lumen pH was associated with time since concentrate feeding, activity and time of day, but not with treatment of omeprazole. MAIN LIMITATIONS A small number of horses were used and measurement periods were limited. CONCLUSIONS Gastro-oesophageal reflux occurs in clinically normal yearling horses. Although omeprazole had no detectable effect, oesophageal lumen pH recorded during this study did not fall within the therapeutic range of omeprazole.