Use of a surgical safety checklist after implementation in an academic veterinary hospital.

OBJECTIVE: To determine the use and barriers to uptake of a surgical safety checklist (SSC) after implementation in a veterinary teaching hospital. STUDY DESIGN: Voluntary online survey and retrospective study. SAMPLE POPULATION: All personnel actively involved in the Ontario Veterinary College Health Sciences Centre small animal surgery service between October 2, 2018 and June 28, 2019. METHODS: Surgical case logs and electronically initiated SSC were reviewed to calculate checklist use. The sample population was surveyed to identify factors and barriers associated with use of the SSC. Participants were allowed 1 month to respond, and five reminder emails were sent. RESULTS: Forth-one of 50 (82%) participants completed the survey. The SSC was used in 374 of 784 (47.7%) surgeries. Use rates declined over sequential three-month intervals (P < .0001). Twenty-six of 41 (63%) respondents overestimated checklist use. Staff attitudes were largely supportive of the SSC, with 29 of 41 respondents suggesting mandatory application. Forgetfulness, hierarchal concerns, timing issues, perceived delays in care, lack of clarity regarding roles, and inadequate training were identified as obstacles to use of the SSC. CONCLUSION: The SCC tested in this study was used in approximately half of the surgical procedures performed after its implementation. Hospital personnel were supportive of the SSC; forgetting to use the SSC was the most common barrier identified by respondents (24/41 [59%]). CLINICAL SIGNIFICANCE: The SSC implementation experience and user feedback described here should be taken into consideration to improve design and implementation of future SSC.

Contributing factors to surgical site infection after tibial plateau leveling osteotomy: A follow-up retrospective study.

OBJECTIVE: To identify factors associated with surgical site infection (SSI) after tibial plateau leveling osteotomy (TPLO). STUDY DESIGN: Retrospective case series. ANIMALS: Dogs (n = 541) that underwent TPLO (n = 659). METHODS: Medical records of dogs that underwent TPLO from 2011-2018 were reviewed. Data collected included perioperative and postoperative antimicrobial administration, stifle inspection, duration of surgery and anesthesia, comorbidities, and development of SSI including timing, microbiological investigation, and implant removal. Referring veterinarians were contacted for all dogs without a recorded return visit. Risk factors for SSI were assessed by using a multivariable logistic regression model built by using a stepwise approach. RESULTS: Surgical site infection was documented in 71 of 659 (11%) TPLO, with methicillin-resistant Staphylococcus pseudintermedius accounting for 20 of 71 (28%) infections. Protective factors against SSI included administration of postoperative antimicrobials (odds ratio [OR] 0.263; 95% CI = 0.157, 0.442) and timing of preoperative antimicrobial administration. Preoperative antimicrobial timing was protective against SSI when it was administered more than 60 minutes before the first incision compared with administration within 30 minutes (OR 0.275; 95% CI = 0.112, 0.676) or within 60 minutes (OR 0.419; 95% CI = 0.189, 0.929) of the first incision. CONCLUSION: Early administration of perioperative antimicrobials and postoperative antimicrobial administration were protective against SSI after TPLO. CLINICAL SIGNIFICANCE: Antimicrobials can influence the risk of SSI after TPLO. Perioperative and postoperative antimicrobial administration timing should be considered to reduce SSI.

Body composition of medium to giant breed dogs with or without cranial cruciate ligament disease.

OBJECTIVE: To describe the body composition of dogs with or without cranial cruciate ligament (CCL) disease. STUDY DESIGN: Cross-sectional. ANIMALS: Adult dogs in which CCL disease was diagnosed (n = 30) and adult dogs without clinical signs of orthopedic disease (n = 30). METHODS: Body weight, body condition score, and muscle condition score (MCS) were recorded. Body composition of the whole body and pelvic limbs were assessed by dual-energy x-ray absorptiometry. Body condition score, whole body, and pelvic limb body composition measurements were compared by using general linear mixed-model analysis of variance. Muscle condition score between groups was assessed by using a Mann-Whitney U test, while paired data were analyzed by using a Wilcoxon signed-rank test. RESULTS: Body fat percentage (P < .0001) was higher in affected dogs (38.78% ± 1.40) than in control dogs (27.49% ± 1.24). Affected dogs had lower MCS (1.90 ± 0.13, P < .0001) compared with control dogs (2.77 ± 0.08). The affected pelvic limb of affected dogs contained less lean soft tissues (P < .0001) but more fat (P = .0451) compared with the contralateral pelvic limb. CONCLUSION: Dogs with CCL disease were overweight compared with the control group. CLINICAL SIGNIFICANCE: Dogs that are overweight may be predisposed to developing CCL disease. Body composition changes in the pelvic limbs should be considered when managing the care of these dogs.

In vitro elution of amikacin and Dispersin B from a polymer hydrogel.

OBJECTIVE: To characterize the in vitro elution of amikacin and Dispersin B (ß-N-acetylglucosaminidase) in a degradable hydrogel. STUDY DESIGN: In vitro, prospective study. METHODS: Amikacin (group A; 40 mg/mL), Dispersin B (group D; 70 µg/mL), or combined amikacin and Dispersin B (group AD; 40 mg/mL and 70 µg/mL, respectively) were added to a hydrogel. Ten aliquots per group were incubated in phosphate-buffered saline that was exchanged at 1, 4, 8, 12, and 24 hours and then once daily for 10 days. Eluted amikacin and Dispersin B were quantitated by using an amikacin reagent kit and a Dispersin B enzyme-linked immunosorbent assay kit, respectively. Time point drug concentrations were compared between groups by using repeated-measures analysis of variance, and total drug elution was compared by using an area under the curve calculation. RESULTS: Amikacin alone, Dispersin B alone, and amikacin and Dispersin B combined together underwent rapid elution in the first 24 hours, followed by a gradual decrease over 10 days. The concentration of Dispersin B eluted in group D was higher at 1 day and lower from day 5 to day 10 compared with that in group AD. The concentration of amikacin eluted in group A was higher at 1, 4, and 8 hours and on day 10 and lower on day 1 compared with that in group AD. The total elution of amikacin was greater from group AD compared with that from group A (P = .02). CONCLUSION: Combining amikacin and Dispersin B had an affect on the total elution of amikacin but not Dispersin B. CLINICAL SIGNIFICANCE: The combination of amikacin and Dispersin B in a degradable hydrogel could allow local treatment of complex infections without the requirement for multiple invasive procedures.

Quantitative analysis of enamel on debonded orthodontic brackets.

INTRODUCTION: Iatrogenic damage to the tooth surface in the form of enamel tearouts can occur during removal of fixed orthodontic appliances. The aim of this study was to assess debonded metal and ceramic brackets attached with a variety of bonding materials to determine how frequently this type of damage occurs. METHODS: Eighty-one patients close to finishing fixed orthodontic treatment were recruited. They had metal brackets bonded with composite resin and a 2-step etch-and-bond technique or ceramic brackets bonded with composite resin and a 2-step etch-and- bond technique, and composite resin with a self-etching primer or resin-modified glass ionomer cement. Debonded brackets were examined by backscattered scanning electron microscopy with energy dispersive x-ray spectroscopy to determine the presence and area of enamel on the base pad. RESULTS: Of the 486 brackets collected, 26.1% exhibited enamel on the bonding material on the bracket base pad. The incidences of enamel tearouts for each group were metal brackets, 13.3%; ceramic brackets, 30.2%; composite resin with self-etching primer, 38.2%; and resin-modified glass ionomer cement, 21.2%. The percentage of the bracket base pad covered in enamel was highly variable, ranging from 0% to 46.1%. CONCLUSIONS: Enamel damage regularly occurred during the debonding process with the degree of damage being highly variable. Damage occurred more frequently when ceramic brackets were used (31.9%) compared with metal brackets (13.3%). Removal of ceramic brackets bonded with resin-modified glass ionomer cement resulted in less damage compared with the resin bonding systems.

Comparison of methicillin-resistant Staphylococcus pseudintermedius adherence to 2 canine limb salvage endoprosthesis implants.

The objective of our study was to compare adhesion of methicillin-resistant Staphylococcus pseudintermedius (MRSP) to stainless steel (SS) and to tantalum (TA) canine limb salvage endoprosthesis implants in an in vitro experimental study. The median of the mean log10 colony forming units/mL for adherent MRSP was 4.96 (range: 4.63 to 6.33) for the TA endoprosthesis and 4.31 (range: 3.86 to 5.05) for the SS endoprosthesis (P = 0.009). Although the trabecular and porous design of the TA endoprosthesis provides mechanical benefits over the SS endoprosthesis, it may increase the risk of developing infection due to higher levels of bacterial adherence.

Comparaison de l'adhérence deStaphylococcus pseudintermediusrésistant à la méthicilline à deux implants d'endoprothèse pour sauver des membres canins. L'objectif de notre étude consistait à comparer l'adhésion de Staphylococcus pseudintermedius résistant à la méthicilline (MRSP) à des implants d'endoprothèse en acier inoxydable (AI) et en tantale (TA) pour sauver des membres canins lors d'une étude expérimentale in vitro. La médiane des moyennes en log10 des unités formatrices de colonies/mL pour le MRSP adhérent était de 4,96 (écart : de 4,63 à 6,33) pour l'endoprothèse TA et 4,31 (écart : de 3,86 à 5,05) pour l'endoprothèse d'AI (P = 0,009). Même si la conception trabéculaire et poreuse de l'endoprothèse de TA offre des avantages mécaniques par rapport à l'endoprothèse d'AI, elle peut accroître le risque de développer une infection en raison des taux supérieurs d'adhérence bactérienne.(Traduit par Isabelle Vallières).

An in vitro method to test the safety and efficacy of low-level laser therapy (LLLT) in the healing of a canine skin model.

BACKGROUND: Low-level laser therapy (LLLT) has been used clinically as a treatment modality for a variety of medical conditions including wound-healing processes. It is an attractive and emerging method to enhance wound healing and improve clinical outcomes both in human and veterinary medicine. Despite the fact that the use of LLLT continues to gain in popularity, there is no universally accepted theory that defends all its cellular effects and beneficial biological processes in tissue repair. The present study was designed to evaluate the effect of LLLT on cellular migration and proliferation of cultured canine epidermal keratinocytes (CPEK) in an in vitro wound healing model. RESULTS: Keratinocyte migration and proliferation were assessed using a scratch migration assay and a proliferation assay, respectively. Fifteen independent replicates were performed for each assay. Canine epidermal keratinocyte cells exposed to LLLT with 0.1, 0.2, and 1.2 J/cm(2) migrated significantly more rapidly (p < 0.03) and showed significantly higher rates of proliferation (p < 0.0001) compared to non-irradiated cells cultured in the same medium and cells exposed to the higher energy dose of 10 J/cm(2). Irradiation with 10 J/cm(2) was characterized by decreased cellular migration and proliferation. These results revealed that LLLT has a measurable, dose-dependent effect on two different aspects of keratinocyte biology in vitro. CONCLUSION: In this in vitro wound-healing model, LLLT increased cellular migration and proliferation at doses of 0.1, 0.2, and 1.2 J/cm(2) while exposure to 10 J/cm(2) decreased cellular migration and proliferation. These data suggest that the beneficial effects of LLLT in vivo may be due, in part, to effects on keratinocyte behavior.

Biofilm-Associated Gene Expression in Staphylococcus pseudintermedius on a Variety of Implant Materials.

OBJECTIVE: To evaluate the expression of biofilm-associated genes in Staphylococcus pseudintermedius on multiple clinically relevant surfaces. STUDY DESIGN: In vitro experimental study. SAMPLE POPULATION: Two strains of methicillin-resistant S. pseudintermedius isolated from clinical infections representing the most common international isolates. METHODS: A quantitative polymerase chain reaction (qPCR) assay for expression of genes related to biofilm initial adhesion, formation/maturation, antimicrobial resistance, and intracellular communication was developed and validated. S. pseudintermedius biofilms were grown on 8 clinically relevant surfaces (polymethylmethacrylate, stainless steel, titanium, latex, silicone, polydioxanone, polystyrene, and glass) and samples of logarithmic and stationary growth phases were collected. Gene expression in samples was measured by qPCR. RESULTS: Significant differences in gene expression were identified between surfaces and between bacterial strains for most gene/strain/surface combinations studied. Expression of genes responsible for production of extracellular matrix were increased in biofilms. Expression of genes responsible for initial adhesion and intracellular communication was markedly variable. Antimicrobial resistance gene expression was increased on multiple surfaces, including stainless steel and titanium. CONCLUSION: A method for evaluation of expression of multiple biofilm-associated genes in S. pseudintermedius was successfully developed and applied to the study of biofilms on multiple surfaces. Variations in expression of these genes have a bearing on understanding the development and treatment of implant-associated biofilm infections and will inform future clinical research.

Evaluation of the Impact of Methicillin-Resistant Staphylococcus pseudintermedius Biofilm Formation on Antimicrobial Susceptibility.

OBJECTIVE: To compare the minimum inhibitory concentration (MIC) of four antimicrobials in planktonic vs. biofilm-associated Staphylococcus pseudintermedius. STUDY DESIGN: In vitro study. SAMPLE POPULATION: 78 isolates from dogs colonized or infected with methicillin-resistant S. pseudintermedius (MRSP, n=39) or methicillin-susceptible S. pseudintermedius (MSSP, n=39). METHODS: Agar dilution was used to determine the MIC of amikacin, cefazolin, enrofloxacin, and gentamicin for planktonic bacteria. A modified broth microdilution assay was used to assess the MIC of biofilm-associated bacteria. RESULTS: MIC were significantly higher in biofilm-associated vs. planktonic bacteria for all antimicrobials; amikacin (median MIC: biofilm >2,000 µg/mL vs. planktonic 3 µg/mL, P<.0001), cefazolin (>1,000 vs. 0.5 µg/mL, P<.0001), enrofloxacin (>1,000 vs. 0.25 µg/mL, P<.0001), and gentamicin (>1,000 vs. 0.3 µg/mL, P<.001). For all antimicrobials, there were significant differences in planktonic MIC for MRSP and MSSP (all P<.0001) but no differences between biofilm MIC for MRSP and MSSP (P=.08-1.0). CONCLUSION: The MIC for biofilm-associated S. pseudintermedius are significantly higher than for planktonic bacteria. Standard methods for determining MIC are not appropriate for biofilm-associated infections. This must be considered when determining treatment regimens for infections that potentially involve biofilms, and further study of methods to control biofilm-associated infections is needed.

Internal hemipelvectomy for treatment of obstipation secondary to pelvic malunion in 3 cats.

Pelvic fractures are a common injury in cats, and both surgical and conservative management approaches have been described. One of the major complications of pelvic fractures managed conservatively is narrowing of the pelvic canal. Severe pelvic canal narrowing can result in constipation and subsequent megacolon. The purpose of this case series is to describe the long-term outcome for 3 cats with obstipation treated with internal hemipelvectomy because of megacolon secondary to pelvic canal narrowing after conservative management. All cats had a good functional outcome of the affected limb. Two cats required ongoing medical management for recurrent constipation. Overall, internal hemipelvectomy offers good long-term limb function; however, its success in relieving clinical signs of constipation requires additional research.

Hémipelvectomie interne pour le traitement de la constipation opiniâtre secondaire à un cal vicieux pelvien chez 3 chats. Les fractures pelviennes sont une blessure commune chez les chats et les approches chirurgicales et prudentes ont toutes deux été décrites. L'une des complications majeures des fractures pelviennes gérées de manière prudente consiste à raccourcir le canal pelvien. Un rétrécissement sévère du canal pelvien peut se traduire par la constipation et un mégacôlon subséquent. Le but de cette série de cas consiste à décrire le résultat à long terme pour 3 chats souffrant de constipation opiniâtre traitée par une hémipelvectomie interne en raison d'un mégacôlon secondaire au rétrécissement du canal pelvien après une gestion prudente. Tous les chats ont obtenu de bons résultats fonctionnels du membre affecté. Deux chats ont nécessité une gestion médicale permanente pour une constipation opiniâtre. En général, l'hémipelvectomie interne offre une bonne fonction du membre, mais son succès dans le soulagement des signes cliniques de la constipation exige de la recherche additionnelle.(Traduit par Isabelle Vallières).

Intra-articular pharmacokinetics of a gentamicin impregnated collagen sponge in the canine stifle: an experimental study.

OBJECTIVE: To investigate local and systemic pharmacokinetics of gentamicin after intra-articular implantation of a gentamicin impregnated collagen sponge (GICS) in the inflamed canine joint. STUDY DESIGN: Descriptive repeated measures experimental study. ANIMALS: Dogs (n = 9). METHODS: Stifle joint inflammation was caused by urate injection. Twenty-four hours later a GICS (gentamicin dose, 6 mg/kg) was arthroscopically implanted. Synovial fluid and plasma gentamicin concentrations were measured for 14 days after implantation, and pharmacokinetic parameters modeled using statistical moment analyses. RESULTS: Intra-articular gentamicin concentrations fell to sub-MIC for Staphylococcus sp. (4 µg/mL) by 22.4 hours (95% CI: 18.6-26.2) after sponge implantation. Cmax synovial was 2397 µg/mL (95%CI: 1161-3634 µg/mL) at 1.2 hours (95%CI: 0.5-1.8 hours). Plasma gentamicin concentrations achieved levels of Cmax plasma = 8.0 µg/mL (95%CI: 6.1-10.0 µg/mL) at 1.5 hours (95%CI: 0.8-2.1) after GICS placement and fell below target trough of 2.0 µg/mL by 5.6 hours (95%CI: 4.7-6.5 hours) after GICS placement. CONCLUSIONS: Intra-articular gentamicin concentration after GICS placement at an IV-equivalent dose reached high levels and declined rapidly. The maximum plasma levels attained were ∼1/3 of the recommended sub-toxic target for people after parenteral gentamicin administration.

Investigation of incidence and risk factors for surgical glove perforation in small animal surgery.

OBJECTIVE: To identify incidence and risk factors for surgical glove perforation in small animal surgery. STUDY DESIGN: Observational cohort study. SAMPLE POPULATION: Surgical gloves (n = 2132) worn in 363 surgical procedures. METHODS: All gloves worn by operative personnel were assessed for perforation at end-procedure using a water leak test. Putative risk factors were recorded by a surgical team member. Associations between risk factors and perforation were assessed using multivariable multi-level random-effects logistic regression models to control for hierarchical data structure. RESULTS: At least 1 glove perforation occurred in 26.2% of procedures. Identified risk factors for glove perforation included increased surgical duration (surgery >1 hour OR = 1.79, 95% CI = 1.12-2.86), performing orthopedic procedures (OR = 1.88; 95% CI = 1.23-2.88), any procedure using powered instruments (OR = 1.93; 95% CI = 1.21-3.09) or surgical wire (OR = 3.02; 95% CI = 1.50-6.05), use of polyisoprene as a glove material (OR = 1.59, 95% CI = 1.05-2.39), and operative role as primary surgeon (OR = 2.01; 95% CI = 1.35-2.98). The ability of the wearer to detect perforations intraoperatively was poor, with a sensitivity of 30.8%. CONCLUSIONS: There is a high incidence of unrecognized glove perforations in small animal surgery.

Corrigendum: Extracorporeal Shock Wave Therapy Enhances the In Vitro Metabolic Activity and Differentiation of Equine Umbilical Cord Blood Mesenchymal Stromal Cells.

[This corrects the article DOI: 10.3389/fvets.2020.554306.].

Regulation of angiotensinogen by angiotensin II in mouse primary astrocyte cultures.

Astrocytes are the major source of angiotensinogen in the brain and play an important role in the brain renin-angiotensin system. Regulating brain angiotensinogen production alters blood pressure and fluid and electrolyte homeostasis. In turn, several physiological and pathological manipulations alter expression of angiotensinogen in brain. Surprisingly, little is known about the factors that regulate astrocytic expression of angiotensinogen. There is evidence that angiotensinogen production in both hepatocytes and cardiac myocytes can be positively regulated via the angiotensin type 1 receptor, but this effect has not yet been studied in astrocytes. Therefore, the aim of this project was to establish whether angiotensin II modulates angiotensinogen production in brain astrocytes. Primary astrocyte cultures, prepared from neonatal C57Bl6 mice, expressed angiotensinogen measured by immunocytochemistry and real-time PCR. Using a variety of approaches we were unable to identify angiotensin receptors on cultured astrocytes. Exposure of cultured astrocytes to angiotensin II also did not affect angiotensinogen expression. When astrocyte cultures were transduced with the angiotensin type 1A receptor, using adenoviral vectors, angiotensin II induced a robust down-regulation (91.4% ± 1.8%, p < 0.01, n = 4) of angiotensinogen gene expression. We conclude that receptors for angiotensin II are present in extremely low levels in astrocytes, and that this concurs with available data in vivo. The signaling pathways activated by the angiotensin type 1A receptor are negatively coupled to angiotensinogen expression and represent a powerful pathway for decreasing expression of this protein, potentially via signaling pathways coupled to Gα(q/11) .

Indirect colonic injury after military wounding: a case series.

BACKGROUND: Colonic trauma in wartime most commonly results from direct injury along the path of a penetrating missile. Rarely, the colon may be injured by primary blast effect or by propagation of energy by the missile, remote from the track of the projectile. METHODS/RESULTS: This article describes the clinical presentation and operative findings in five patients who sustained high energy-transfer gunshot wounds (GSWs) or fragmentation injuries from blast who were found to have sustained colonic injuries anatomically remote from the missile track/s. CONCLUSIONS: Military surgeons should be aware of the phenomenon of indirect injury to the colon after high-energy transfer GSW and blast injury. A high index of suspicion should be maintained and cross-sectional imaging used where feasible. Primary colonic reconstruction was used safely in these patients with indirect colonic injuries.

Extracorporeal Shock Wave Therapy Enhances the In Vitro Metabolic Activity and Differentiation of Equine Umbilical Cord Blood Mesenchymal Stromal Cells.

Extracorporeal shock wave therapy (ESWT) has been shown to induce different biological effects on a variety of cells, including regulation and stimulation of their function and metabolism. ESWT can promote different biological responses such as proliferation, migration, and regenerations of cells. Recent studies have shown that mesenchymal stromal cells (MSCs) secrete factors that enhance the regeneration of tissues, stimulate proliferation and differentiation of cells, and decrease inflammatory and immune reactions. Clinically, the combination of these two therapies has been used as a treatment for tendon and ligament lesions in horses; however, there is no scientific evidence supporting this combination of therapies in vivo. Therefore, the objectives of the study were to evaluate the effects of ESWT on equine umbilical cord blood mesenchymal stromal cells (CB-MSCs) proliferative, metabolic, migrative, differentiation, and immunomodulatory properties in vitro. Three equine CB-MSC cultures from independent donors were treated using an electrohydraulic shock wave generator attached to a water bath. All experiments were performed as triplicates. Proliferation, viability, migration and immunomodulatory properties of the cells were evaluated. Equine CB-MSCs were induced to evaluate their trilineage differentiation potential. ESWT treated cells had increased metabolic activity, showed positive adipogenic, osteogenic, and chondrogenic differentiation, and showed higher potential for differentiation toward the adipogenic and osteogenic cell fates. ESWT treated cells showed similar immunomodulatory properties to none-ESWT treated cells. Equine CB-MSCs are responsive to ESWT treatment and showed increased metabolic, adipogenic and osteogenic activity, but unaltered immunosuppressive properties. In vivo studies are warranted to determine if synergistic effects occur in the treatment of musculoskeletal injuries if ESWT and equine CB-MSC therapies are combined.

Adenovirus-mediated delivery of relaxin reverses cardiac fibrosis.

We have evaluated the effectiveness of systemic adenovirally delivered mouse relaxin on reversing fibrosis in a transgenic murine model of fibrotic cardiomyopathy due to beta(2)-adrenergic receptor (beta(2)AR) overexpression. Recombinant adenoviruses expressing green fluorescent protein (Ad-GFP), rat relaxin (Ad-rRLN) and mouse relaxin (Ad-mRLN) were generated and Ad-rRLN and Ad-mRLN were demonstrated to direct the expression of bioactive relaxin peptides in vitro. A single systemic injection of Ad-mRLN resulted in transgene expression in the liver and bioactive relaxin peptide in the plasma. Ad-mRLN, but not Ad-GFP, treatment reversed the increased left ventricular collagen content in beta(2)AR mice to control levels without affecting collagen levels in other heart chambers or in the lung and kidney. Hence a single systemic injection of adenovirus producing mouse relaxin reverses cardiac fibrosis without adversely affecting normal collagen levels in other organs and establishes the potential for the use of relaxin gene therapy for the treatment of cardiac fibrosis.

Characterization and Immunomodulatory Effects of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells.

BACKGROUND: Mesenchymal stromal cells (MSC) hold promise for both cell replacement and immune modulation strategies owing to their progenitor and non-progenitor functions, respectively. Characterization of MSC from different sources is an important and necessary step before clinical use of these cells is widely adopted. Little is known about the biology and function of canine MSC compared to their mouse or human counterparts. This knowledge-gap impedes development of canine evidence-based MSC technologies. HYPOTHESIS AND OBJECTIVES: We hypothesized that canine adipose tissue (AT) and bone marrow (BM) MSC (derived from the same dogs) will have similar differentiation and immune modulatory profiles. Our objectives were to evaluate progenitor and non-progenitor functions as well as other characteristics of AT- and BM-MSC including 1) proliferation rate, 2) cell surface marker expression, 3) DNA methylation levels, 4) potential for trilineage differentiation towards osteogenic, adipogenic, and chondrogenic cell fates, and 5) immunomodulatory potency in vitro. RESULTS: 1) AT-MSC proliferated at more than double the rate of BM-MSC (population doubling times in days) for passage (P) 2, AT: 1.69, BM: 3.81; P3, AT: 1.80, BM: 4.06; P4, AT: 2.37, BM: 5.34; P5, AT: 3.20, BM: 7.21). 2) Canine MSC, regardless of source, strongly expressed cell surface markers MHC I, CD29, CD44, and CD90, and were negative for MHC II and CD45. They also showed moderate expression of CD8 and CD73 and mild expression of CD14. Minor differences were found in expression of CD4 and CD34. 3) Global DNA methylation levels were significantly lower in BM-MSC compared to AT-MSC. 4) Little difference was found between AT- and BM-MSC in their potential for adipogenesis and osteogenesis. Chondrogenesis was poor to absent for both sources in spite of adding varying levels of bone-morphogenic protein to our standard transforming growth factor (TGF-ß3)-based induction medium. 5) Immunomodulatory capacity was equal regardless of cell source when tested in mitogen-stimulated lymphocyte reactions. Priming of MSC with pro-inflammatory factors interferon-gamma and/or tumour necrosis factor did not increase the lymphocyte suppressive properties of the MSC compared to untreated MSC. CONCLUSIONS/SIGNIFICANCE: No significant differences were found between AT- and BM-MSC with regard to their immunophenotype, progenitor, and non-progenitor functions. Both MSC populations showed strong adipogenic and osteogenic potential and poor chondrogenic potential. Both significantly suppressed stimulated peripheral blood mononuclear cells. The most significant differences found were the higher isolation success and proliferation rate of AT-MSC, which could be realized as notable benefits of their use over BM-MSC.

Association of beta-Arrestin 1 with the type 1A angiotensin II receptor involves phosphorylation of the receptor carboxyl terminus and correlates with receptor internalization.

Arrestins bind to phosphorylated G protein-coupled receptors and participate in receptor desensitization and endocytosis. Although arrestins traffic with activated type 1 (AT(1A)) angiotensin II (AngII) receptors, the contribution of arrestins to AT(1A) receptor internalization is controversial, and the physical association of arrestins with the AT(1A) receptor has not been established. In this study, by coimmunoprecipitating AT(1A) receptors and beta-arrestin 1, we provide direct evidence for an association between arrestins and the AT(1A) receptor that was agonist- and time-dependent and contingent upon the level of beta-arrestin 1 expression. Serial truncation of the receptor carboxyl terminus resulted in a graded loss of beta-arrestin 1 association, which correlated with decreases in receptor phosphorylation. Truncation of the AT(1A) receptor to lysine(325) prevented AngII-induced phosphorylation and beta-arrestin 1 association as well as markedly inhibiting receptor internalization, indicating a close correlation between these receptor parameters. AngII-induced association was also dramatically reduced in a phosphorylation- and internalization-impaired receptor mutant in which four serine and threonine residues in the central portion of the AT(1A) receptor carboxyl terminus (Thr(332), Ser(335), Thr(336), Ser(338)) were substituted with alanine. In contrast, substitutions in another serine/threonine-rich region (Ser(346), Ser(347), Ser(348)) and at three PKC phosphorylation sites (Ser(331), Ser(338), Ser(348)) had no effect on AngII-induced beta-arrestin 1 association or receptor internalization. While AT(1A) receptor internalization could be inhibited by a dominant-negative beta-arrestin 1 mutant (beta arr1(319-418)), treatment with hyperosmotic sucrose to inhibit internalization did not abrogate the differences in arrestin association observed between the wild-type and mutant receptors, indicating that arrestin binding precedes, and is not dependent upon, receptor internalization. Interestingly, a substituted analog of AngII, [Sar(1)Ile(4)Ile(8)]-AngII, which promotes robust phosphorylation of the receptor but does not activate receptor signaling, stimulated strong beta-arrestin 1 association with the full-length AT(1A) receptor. These results identify the central portion of the AT(1A) receptor carboxyl terminus as the important determinant for beta-arrestin 1 binding and internalization and indicate that AT(1A) receptor phosphorylation is crucial for beta-arrestin docking.

Phosphorylation of the angiotensin II (AT1A) receptor carboxyl terminus: a role in receptor endocytosis.

The molecular mechanism of angiotensin II type I receptor (AT1) endocytosis is obscure, although the identification of an important serine/threonine rich region (Thr332Lys333Met334Ser335Thr336Leu337 Ser338) within the carboxyl terminus of the AT1A receptor subtype suggests that phosphorylation may be involved. In this study, we examined the phosphorylation and internalization of full-length AT1A receptors and compared this to receptors with truncations and mutations of the carboxyl terminus. Epitope-tagged full-length AT1A receptors, when transiently transfected in Chinese hamster ovary (CHO)-K1 cells, displayed a basal level of phosphorylation that was significantly enhanced by angiotensin II (Ang II) stimulation. Phosphorylation of AT1A receptors was progressively reduced by serial truncation of the carboxyl terminus, and truncation to Lys325, which removed the last 34 amino acids, almost completely inhibited Ang II-stimulated 32P incorporation into the AT1A receptor. To investigate the correlation between receptor phosphorylation and endocytosis, an epitope-tagged mutant receptor was produced, in which the carboxyl-terminal residues, Thr332, Ser335, Thr336, and Ser338, previously identified as important for receptor internalization, were substituted with alanine. Compared with the wild-type receptor, this mutant displayed a clear reduction in Ang II-stimulated phosphorylation. Such a correlation was further strengthened by the novel observation that the Ang II peptide antagonist, Sar(1)Ile8-Ang II, which paradoxically causes internalization of wild-type AT1A receptors, also promoted their phosphorylation. In an attempt to directly relate phosphorylation of the carboxyl terminus to endocytosis, the internalization kinetics of wild-type AT1A receptors and receptors mutated within the Thr332-Ser338 region were compared. The four putative phosphorylation sites (Thr332, Ser335, Thr336, and Ser338) were substituted with either neutral [alanine (A)] or acidic amino acids [glutamic acid (E) and aspartic acid (D)], the former to prevent phosphorylation and the latter to reproduce the acidic charge created by phosphorylation. Wild-type AT1A receptors, expressed in Chinese hamster ovary cells, rapidly internalized after Ang II stimulation [t1/2 2.3 min; maximal level of internalization (Ymax) 78.2%], as did mutant receptors carrying single acidic substitutions (T332E, t1/2 2.7 min, Ymax 76.3%; S335D, t1/2 2.4 min, Ymax 76.7%; T336E, t1/2 2.5 min, Ymax 78.2%; S338D, t1/2 2.6 min, Ymax 78.4%). While acidic amino acid substitutions may simply be not as structurally disruptive as alanine mutations, we interpret the tolerance of a negative charge in this region as suggestive that phosphorylation may permit maximal internalization. Substitution of all four residues to alanine produced a receptor with markedly reduced internalization kinetics (T332A/S335A/T336A/S338A, t1/2 10.1 min, Ymax 47.9%), while endocytosis was significantly rescued in the corresponding quadruple acidic mutant (T332E/S335D/T336E/S338D, t1/2 6.4 min, Ymax 53.4%). Double mutation of S335 and T336 to alanine also diminished the rate and extent of endocytosis (S335A/T336A, 3.9 min, Ymax 69.3%), while the analogous double acidic mutant displayed wild type-like endocytotic parameters (S335D/T336E, t1/2 2.6 min, Ymax 77.5%). Based on the apparent rescue of internalization by acidic amino acid substitutions in a region that we have identified as a site of Ang II-induced phosphorylation, we conclude that maximal endocytosis of the AT1A receptor requires phosphorylation within this serine/threonine-rich segment of the carboxyl terminus.