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Background Necrotizing meningoencephalitis (NME, aka Pug dog encephalitis) is an inflammatory brain condition associated with advanced disease at initial presentation, rapid progression, and poor response to conventional immunomodulatory therapy. Hypothesis/objectives That genetic risk for NME, defined by a common germline DNA haplotype located on chromosome 12, is associated with altered blood cytokine concentrations and leukocyte subsets in asymptomatic Pugs. Animals Forty Pug dogs asymptomatic for NME from a hospital sample. Methods Prospective observational cohort study, including germline genome-wide genotyping, plasma cytokine determination by multiplexed profiling, and leukocyte subset characterization by flow cytometric analysis. Results Seven (18%) dogs were high risk, 10 (25%) medium risk, and 23 (58%) low risk for NME, giving a risk haplotype frequency of 30%. High and medium risk Pugs had significantly lower proportion of CD4+ T cells (median 22% [range, 7.3%-38%] vs 29% [range, 16%-41%], P = .03) and higher plasma IL-10 concentrations than low-risk Pugs (median 14.11 pg/mL [range, 9.66-344.19 pg/mL] vs 12.21 pg/mL [range, 2.59-18.53 pg/mL], P = .001). No other variables were significantly associated with the NME haplotype-based risk. Conclusions and clinical importance These data suggest an immunological underpinning to NME and a biologic rationale for future clinical trials that investigate novel diagnostic, preventative, and therapeutic strategies for this disease.

Made available in DSpace on 2020-12-12T02:46:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-07-22 Background: Canine inflammatory brain disease (IBD) is a severe inflammatory disorder characterized by infiltration of activated immune cell subsets into the brain and spinal cord. Multipotent mesenchymal stromal cells (MSCs) are a promising therapy for IBD, based on their potent pro-angiogenic, neuroprotective, and immunomodulatory properties. The aims of this study were to compare the immunomodulatory attributes of canine adipose-derived MSCs (ASCs) and placenta-derived MSCs (PMSCs) in vitro. These data will serve as potency information to help inform the optimal MSC cell source to treat naturally occurring canine IBD. Methods: Indoleamine 2,3 dioxygenase (IDO) activity and prostaglandin E2 (PGE2) concentration at baseline and after stimulation with interferon gamma (IFNγ) and/or tumor necrosis factor alpha (TNFα) were measured from canine ASC and PMSC cultures. Leukocyte suppression assays (LSAs) were performed to compare the ability of ASCs and PMSCs to inhibit activated peripheral blood mononuclear cell (PBMC) proliferation. IDO activity and PGE2; interleukin (IL)-2, IL-6, and IL-8; TNFα; and vascular endothelial growth factor (VEGF) concentrations were also measured from co-culture supernatants. Cell cycle analysis was performed to determine how ASCs and PMSCs altered lymphocyte proliferation. Results: Activated canine MSCs from both tissue sources secreted high concentrations of IDO and PGE2, after direct stimulation with IFNγand TNFα, or indirect stimulation by activated PBMCs. Both ASCs and PMSCs inhibited activated PBMC proliferation in LSA assays; however, PMSCs inhibited PBMC proliferation significantly more than ASCs. Blocking PGE2 and IDO in LSA assays determined that PGE2 is important only for ASC inhibition of PBMC proliferation. Activated ASCs increased IL-6 and VEGF secretion and decreased TNFα secretion, while activated PMSCs increased IL-6, IL-8, and VEGF secretion. ASCs inhibited lymphocyte proliferation via cell cycle arrest in the G0/G1 and PMSCs inhibited lymphocyte proliferation via induction of lymphocyte apoptosis. Conclusion: Our results demonstrate that ASCs and PMSCs have substantial in vitro potential as a cell-based therapy for IBD; however, PMSCs more potently inhibited lymphocyte proliferation by inducing apoptosis of activated lymphocytes. These data suggest that the mechanism by which ASCs and PMSCs downregulate PBMC proliferation differs. Additional studies may elucidate additional mechanisms by which canine MSCs modulate neuroinflammatory responses. Veterinary Institute for Regenerative Cures Department of Pathology Microbiology and Immunology School of Veterinary Medicine University of California Davis Department of Veterinary Clinics São Paulo State University Julio de Mesquita Filho - UNESP Surgical Bioengineering Laboratory Department of Surgery School of Medicine University of California Davis, 4625 2nd Ave., Research II Institute for Pediatric Regenerative Medicine (IPRM) Shriners Hospitals Pediatric Research Center Northern California Department of Biomedical Engineering University of California Department of Veterinary Clinics São Paulo State University Julio de Mesquita Filho - UNESP

Multipotent stromal cells (MSCs) are widely utilized in therapy for their immunomodulatory properties, but their usage in infectious viral diseases is less explored. This review aimed to collate the current novel use of MSCs in virus-associated conditions, including MSC’s susceptibility to virus infection, antiviral properties of MSCs and their effects on cell-based immune response and implementation of MSC therapy in animal models and human clinical trials of viral diseases. Recent discoveries shed lights on MSC’s capability in suppressing viral replication and augmenting clearance through enhancement of antiviral immunity. MSC therapy may maintain a crucial balance between aiding pathogen clearance and suppressing hyperactive immune response. Graphical Abstract

Osteoarthritis challenges traditional therapies and remains a leading cause of lameness in older dogs. Regenerative medicine offers new strategies, typically involving the injection of autologous adipose-derived mesenchymal stem cells (MSCs). Conversely, allogenic MSCs are appealing candidates to palliate patient morbidity and cell preparation time. Regardless of the source of cells, identifying critical donor characteristics, such as age, is essential to obtain the most competent MSCs. The objectives of this study were to determine the influence of donor's age on proliferation, gene expression, and immunomodulatory properties of MSCs in dogs. Canine adipose tissue-derived MSCs (cAD-MSCs) were isolated from the falciform-ligament adipose tissues of nine pairs of gender-matched young (2 years) or old (7 years) client-owned dogs undergoing abdominal surgery. Growth kinetics, transcriptome before and after stimulation by tumor necrosis factor alpha and interferon gamma, MSC-induced lymphocyte suppression assay, and secretion of prostaglandin E2 (PGE

Objectives The aim of this pilot study was to determine the safety, efficacy and immunomodulatory function of systemically administered adipose-derived mesenchymal stem cells (ASCs) in cats affected by feline chronic gingivostomatitis (FCGS) prior to full-mouth tooth extractions. Methods Five client-owned cats affected with FCGS that did not undergo full-mouth tooth extractions for FCGS treatment received two intravenous injections of 20 million fresh, allogeneic or autologous ASCs. An oral examination with photographs, a complete blood count, blood immune cell phenotyping and a biochemical profile were completed at 0 and 6 months after treatment. Results Four cats completed the study and one cat exited the study 3 months after treatment. While the treatment was determined to be clinically safe, no positive clinical response was observed in three cats and a mild response was noted in two cats. Furthermore, none of the cats exhibited immune modulation, as evidenced by no alteration in circulating CD8+ T cells, normalization of the CD4:CD8 ratio or neutrophil counts. Conclusions and relevance Unlike the reported efficacy of ASCs in treating cats with non-responsive FCGS after full-mouth tooth extraction, the systemic administration of ASCs prior to full-mouth tooth extraction lacks substantial clinical efficacy and is not recommended at this time.

Background Feline adipose-derived mesenchymal stem cells (ASCs) engage with a variety of immune cells and have been used in several clinical trials for the treatment of inflammatory and immune-dysregulated diseases in cats, but the impact they exert on the functional characteristics on T cells, particularly CD8+ T cells, remains to be elucidated. Methods Modified mixed leukocyte reaction was performed between feline ASCs and PBMCs. Changes of cell cycle stages, phenotype and cellular senescence were determined through flow cytometry and gene expression analysis. Cytotoxicity assay was performed to evaluate CD8+ T cell effector function. Results Feline ASCs induce cell cycle arrest on CD8+ T cells in a contact-dependent manner, downregulate CD8 surface expression, and shift their phenotype toward terminally differentiated effector cells (CD57+, CD45R+, CD62L−). CD8 T cells interacted with feline ASCs also upregulated granzyme B, IL-2 and KLRG-1 expression and have enhanced cytotoxic potential, evident by the increased percentage of lysis on target cells. Conclusions Our findings suggest that feline ASCs (1) alter CD8+ T cells toward terminally differentiated, proinflammatory effector phenotype with limited proliferative capacity, and (2) enhance their cytotoxic potential through granzyme B upregulation. These cytotoxic CD8+ T cells could aid in disease cure in cases caused by an underlying, unresolved viral infection.

This study was conducted to investigate the therapeutic effect of allogeneic adipose-derived MSCs on dogs with hip osteoarthritis (OA). Twenty dogs with bilateral osteoarthritis of the coxofemoral (hip) joint, diagnosed by a veterinarian through physical examination and radiographs were randomly allocated into four groups. Group 1 served as a placebo control and were injected with 0.9% sodium chloride (saline) (n = 4). Group 2 were injected with a single dose of 5 million MSCs (n = 5). Group 3 received a single dose of 25 million MSCs (n = 6) and Group 4 received a single dose of 50 million MSCs (n = 5). Intra-articular administration of allogeneic MSCs into multiple joints did not result in any serious adverse events. The average lameness score of the dogs in the placebo control group (−0.31) did not show improvement after 90 days of intra-articular saline administration. However, the average lameness score of the all MSC-treated dogs was improved 2.11 grade at this time point (P < 0.001). Overall, sixty five percent (65%) of the dogs that received various doses of MSCs showed improvement in lameness scores 90 days after intra-articular MSC administration. Our results showed that intra-articular administration of allogeneic adipose derived MSCs was well-tolerated and improved lameness scores and reduced pain in dogs associated with hip OA. All doses of MSCs were effective. Subsequent studies with more animals per group are needed to make a conclusion about the dose response. The improved lameness effect was present up to 90 days post-injection. Serum interleukin 10 was increased in a majority of the dogs that received MSCs and that also had improved lameness.

Bruton's tyrosine kinase (BTK) is important in B-cell signalling. Efficacy has been reported for BTK inhibitors (BTKi) in human autoimmune diseases. Canine pemphigus foliaceus (cPF) is one of the most common canine autoimmune skin diseases.To determine the safety and efficacy of the BTKi PRN1008 in the treatment of cPF.Four privately owned dogs.Four dogs diagnosed with PF were administered BTKi PRN1008. Initial dosages approximated to 15 mg/kg once daily, increased to twice daily if inadequate response was seen. Treatment continued for 20 weeks, attempting to decrease to every other day. Dogs were monitored with complete blood counts, serum biochemistry panels and urinalyses, and evaluated with a modified version of a validated human Pemphigus Disease Activity Index (cPDAI). Serum anti-desmocollin-1 (DSC-1) and desmoglein-1 (DSG-1) immunoglobulin (Ig)G titres were performed before and after the treatment period. Drug bound to target was measured in peripheral blood mononuclear cells (PBMC).All four dogs showed reduction in lesions and cPDAI score during the first two weeks of treatment. Three dogs continued to improve and sustained near complete remission by 20 weeks, at which point three responses were considered "good" and one "fair". Final daily dosages were in the range 17-33 mg/kg. Anti-DSC-1 IgG titre decreased dramatically in one dog, was undetectable in two and was uninterpretable in one dog. No dogs had detectable IgG to DSG1. A possible adverse event occurred in one dog.BTKi PRN1008 monotherapy may have some beneficial effects in some cases of cPF.La BTK (Bruton’s tyrosine kinase) est une importante voie de signal des cellules B. L'efficacité des inhibiteurs de BTK (BTKi) a été rapportée dans les maladies auto-immunes chez l'homme. Le pemphigus foliacé (cPF) est la dermatose auto-immune la plus fréquente chez le chien.Déterminer l'innocuité et l'efficacité de BTKi PRN 1008 dans le traitement de cPF.Quatre chiens de propriétaires. MATÉRIELS ET MÉTHODES: Quatre chiens diagnostiqués PF ont reçus BTKi PRN 1008. Les doses initiales étaient approximativement de 15 mg/kg une fois par jour, augmenté à deux fois par jour si une réponse inadéquate était observée. Le traitement a été poursuivi pendant 20 semaines, avec essai de diminution à un jour sur deux. Les chiens ont été suivis avec une numération formule, biochimie complète et analyses urinaires et évalués avec une version modifiée de cPDAI (human Pemphigus Disease Activity Index). Les titres d'immunoglobulines (Ig) G anti-desmocolline 1 (DSC-1) et desmogléine-1 (DSG-1) ont été réalisés avant et après traitement. Le médicament lié à la cible a été mesuré dans les cellules mononuclées sanguines périphériques (PBMC). RÉSULTATS: Les quatre chiens ont montré une diminution des lésions et du score cPDAI au cours des deux premières semaines de traitement. Trois chiens ont continués à s'améliorer jusqu'à guérison presque complète à la semaine 20, à chaque point, trois réponses ont été considérées "bonne" et une "faible". Les dosages quotidiens finaux étaient dans les valeurs 17-33 mg/kg. Les titres d'IgG anti-DSC-1 ont diminués drastiquement chez un chien, étaient non détectable pour deux et étaient non interprétable pour un chien. Aucun chien n'avait d'IgG détectable à DSG1. Un effet indésirable possible a été observé chez un chien.BTK1 PRN 1008 en monothérapie pourrait avoir des effets bénéfiques dans certains cas de cPF.INTRODUCCIÓN: la tirosina quinasa de Bruton (BTK) es importante en las señales celulares de linfocitos B. Se ha publicado acerca de la eficacia de los inhibidores de BTK (BTKi) en enfermedades autoinmunes humanas. El pénfigo foliáceo canino (cPF) es una de las enfermedades cutáneas autoinmunes caninas más comunes. OBJETIVOS: Determinar la seguridad y eficacia del BTKi PRN1008 en el tratamiento de cPF. ANIMALES: cuatro perros de propietarios particulares. MÉTODOS Y MATERIALES: cuatro perros diagnosticados con PF recibieron BTKi PRN1008. Las dosis iniciales fueron de aproximadamente 15 mg/kg una vez al día, que se aumentaron a dos veces al día si se observaba una respuesta inadecuada. El tratamiento continuó durante 20 semanas, intentando disminuir a cada dos días. Los perros fueron evaluados con recuentos sanguíneos completos, paneles de bioquímica sérica y análisis de orina, y valorados con una versión modificada de un índice de actividad de la enfermedad del pénfigo humano validado (cPDAI). Los títulos séricos de inmunoglobulina (Ig)G anti-desmocolina-1 (DSC-1) y desmogleina-1 (DSG-1) se realizaron antes y después del período de tratamiento. El fármaco unido a la proteína diana se midió en células mononucleares de sangre periférica (PBMC). RESULTADOS: los cuatro perros mostraron una reducción en las lesiones y del valor de cPDAI durante las primeras dos semanas de tratamiento. Tres perros continuaron mejorando y mantuvieron una remisión casi completa a las 20 semanas, momento en el que tres respuestas se consideraron "buenas" y una "regular". Las dosis diarias finales estuvieron en el rango de 17-33 mg/kg. El título de IgG anti-DSC-1 disminuyó drásticamente en un perro, fue indetectable en dos y no fue interpretable en un perro. Ningún perro tenía IgG detectable a DSG1. Un posible evento adverso ocurrió en un perro. CONCLUSIONES E IMPORTANCIA CLÍNICA: la monoterapia con BTKi PRN1008 puede tener algunos efectos beneficiosos en algunos casos de cPF.Die Bruton Tyrosinkinase (BTK) ist wichtig bei der Signalgebung von B-Zellen. Bei Autoimmunerkrankungen des Menschen wurde bereits eine Wirksamkeit für BTK Inhibitoren (BTKi) beschrieben. Der Pemphigus foliaceus (cPF) des Hundes ist eine der häufigsten caninen Autoimmunerkrankungen.Eine Bestimmung der Sicherheit und der Wirksamkeit der BTKi PRN1008 bei der Behandlung des cPF.Vier Hunde in Privatbesitz.Vier Hunden, die mit PF diagnostiziert worden waren, wurde BTKi PRN1008 verabreicht. Anfangsdosen waren annähernd 15 mg/kg einmal täglich, wurden allerdings bei unzureichender Verbesserung auf zweimal täglich erhöht. Die Behandlung wurde 20 Wochen lang fortgesetzt, wobei versucht wurde, auf jeden zweiten Tag zu reduzieren. Die Hunde wurden mittels Blutbild, Serumbiochemie und Urinanalyse kontrolliert, sowie mit einer modifizierten Version eines validierten humanen Pemphigus Disease Activity Index (cPDAI) evaluiert. Es wurden Serum Anti-Desmocollin-1 (DSC-1) und Desmoglein-1 (DSG-1) Immunglobulin (Ig)G Titer vor und nach der Behandlungsperiode bestimmt. Mittels peripherer Blutmononuklearzellen (PBMC) wurde das ans Zielmolekül gebundene Medikament bestimmt.Alle vier Hunde zeigten weniger Veränderungen und einen verringerten cPDAI Wert während der ersten zwei Wochen der Behandlung. Drei Hunde verbesserten sich weiterhin und blieben nach 20 Wochen nahezu in Remission, wodurch zu diesem Zeitpunkt die Verbesserung bei drei als „gut“ und einem als „fair“ eingestuft wurde. Am Ende lagen die Dosierungen im Bereich von 17-33 mg/kg. Anti-DSC-1 IgG Titer verringerten sich bei einem Hund drastisch, waren bei zwei anderen nicht feststellbar und bei einem Hund nicht interpretierbar. Keiner der Hunde hatte messbare IgG auf DSG-1. Eine mögliche Nebenwirkungsreaktion trat bei einem Hund auf.BTKi PRN1008 Monotherapie könnte in einigen Fällen von cPF eine günstige Auswirkung haben.背景: Bruton型チロシンキナーゼ（BTK）は、B細胞シグナル伝達において重要である。ヒト自己免疫疾患におけるBTK阻害剤（BTKi）の有効性が報告されている。犬の天疱瘡（cPF）は、最も一般的な犬の自己免疫性皮膚疾患の1つである。 目的: 本研究の目的は、cPF治療におけるBTKi PRN1008の安全性および有効性を判断することであった。 供試動物: 4頭の飼育犬。 材料と方法: PFと診断した4頭の犬にBTKi PRN1008を投与した。初期投与量は1日1回15 mg/kgと概算され、不十分な反応が見られた場合は1日2回に増量した。治療は20週間継続し、投与量を隔日に減量するよう試みた。供試犬を全血球計算、血清生化学パネルおよび尿検査でモニターし、修正されたヒト天疱瘡疾患活動指数（cPDAI）で評価した。血清抗デスモコリン-1（DSC-1）およびデスモグレイン-1（DSG-1）免疫グロブリン（Ig）G力価を、治療期間の前後で測定した。標的に結合した薬物を末梢血単核細胞（PBMC）で測定した。 結果: 治療開始2週間で、4頭の犬すべてが病変とcPDAIスコアの低下を示した。 3頭の犬は改善を続け、20週間までにほぼ完全寛解を維持した。その時点で、3頭の反応は「良」、1頭は「可」と見なされた。最終的な1日投与量は17〜33 mg/kgの範囲であった。抗DSC-1 IgG力価は1頭の犬で劇的に減少し、2頭では検出できず、1頭の犬では解釈できなかった。 DSG1に対するIgGが検出された犬はいなかった。 1頭の犬で有害事象の可能性が生じた。 結論と臨床的重要性: BTKi PRN1008単剤療法は、cPFの一部の症例でいくつかの有益な効果をもたらす可能性がある。.背景: Bruton酪氨酸激酶(BTK)在B细胞信号转导中很重要。已报告BTK抑制剂(BTKi)在人类自身免疫性疾病中的疗效。犬落叶型天疱疮(cPF)是最常见的犬自身免疫性皮肤病之一。 目的: 确定BTKi PRN1008治疗cPF的安全性和疗效。 动物: 四只私家犬。 方法和材料: 对诊断为PF的4只犬给予BTKi PRN1008。初始剂量约为15mg/kg，每日一次，如果观察到反应不足，则增加至每日两次。治疗持续20周，尝试降至隔日一次。通过全血细胞计数、血清生化全项和尿分析对犬进行监测，并使用经验证的人天疱疮疾病活动指数 (cPDAI) 的改良版进行评价。在治疗期前后进行血清抗桥粒胶糖蛋白-1(DSC-1)和桥粒芯糖蛋白-1(DSG-1)免疫球蛋白(Ig)G滴度检测。在外周血单核细胞(PBMC)中测定与靶标结合的药物。 结果: 在治疗的前两周，所有4只犬均显示病变减轻和cPDAI评分下降。3只犬持续改善并在20周时维持接近完全缓解，此时认为3只效果为“良好”，1只为“一般”。最终日剂量范围为17-33mg/kg。在1只犬中，抗DSC-1 IgG滴度显著降低，在2只犬中检测不到，在1只犬中无法判断。所有犬均未检出DSG1 IgG。一只犬可能发生了不良事件。 结论和临床重要性: BTKi PRN1008单药治疗在某些cPF病例中可能有一定效果。.HISTÓRICO: A tirosina quinase de Bruton (BTK) é importante na sinalização de células B. A eficácia de inibidores da BTK (BTKi) em doenças autoimunes humanas já foi relatada. O pênfigo foliáceo canino (cPF) é uma das doenças cutâneas autoimunes caninas mais comuns.Determinar a segurança e a eficácia do BTKi PRN1008 no tratamento de cPF.Quatro cães de proprietários. MÉTODOS E MATERIAIS: Quatro cães diagnosticados com PF receberam BTKi PRN1008.As dosagens iniciais foram aproximadamente 15 mg / kg uma vez ao dia, aumentadas para duas vezes ao dia se houvesse resposta inadequada. O tratamento continuou por 20 semanas, com a tentativa de diminuir a dose para dias alternados. Os cães foram monitorados por hemograma completo, perfis bioquímicos séricos e urinálise, e avaliados utilizando uma versão modificada de um Índice validado de Atividade de Doença para o Pênfigo humano (cPDAI). Os títulos séricos de anticorpos (Ig) G anti-desmocolina-1 (DSC-1) e anti-desmogleina-1 (DSG-1) foram realizados antes e após o período de tratamento. A ligação do fármaco ao alvo foi mensurada em células mononucleares do sangue periférico (PBMC).Todos os quatro cães apresentaram redução nas lesões e escore de cPDAI durante as duas primeiras semanas de tratamento. Três cães continuaram a melhorar e mantiveram a remissão quase completa em 20 semanas. Neste momento, três respostas foram consideradas “boas” e uma “razoável”. As dosagens diárias finais ficaram na faixa de 17 a 33 mg / kg. O título de IgG anti-DSC-1 diminuiu drasticamente em um cão, foi indetectável em dois e não foi interpretável em um cão. Nenhum cão apresentou IgG detectável para DSG1. Um possível efeito adverso ocorreu em um cão. CONCLUSÕES E IMPORTÂNCIA CLÍNICA: A monoterapia com BTKi PRN1008 pode ter efeitos benéficos em alguns casos de cPF.

All categories of pleural effusion subjectively display as soft tissue opacity on computed tomography (CT). Quantitative measurement using Hounsfield units (HU) has the potential to bring additional information regarding the nature of the fluid in a noninvasive way. The purposes of this retrospective cross-sectional analytical study were to compare Hounsfield units of different pleural effusion categories in dogs and cats, assess association between specific cytologic parameters and Hounsfield units, and evaluate the effect of dependent vs. nondependent aspect of the effusion pool on Hounsfield unit. A total of 111 patients (74 dogs and 37 cats) with pleural effusion, that underwent thoracic CT and diagnostic thoracocentesis, were included in the study. Effusions were cytologically categorized as exudate, transudate, modified transudate, hemorrhage, or chyle. Significant differences existed in Hounsfield units between categories in dogs (P 0.0001) but not in cats (P = 0.334). Canine chylous effusion (6.1 ± 4.7 HU (mean ± standard deviation)) and transudate (5.6 ± 2.0) were significantly lower than exudate (20.3 ± 9.5) and hemorrhage (21.4 ± 9.2). No significant differences were found between modified transudate (13.6 ± 10.3) and other categories. Significant, weak linear correlation was identified in dogs between Hounsfield units and total protein (P = 0.018, R

Practical relevance:Stem cell therapy is an innovative field of scientific investigation with tremendous potential for clinical application in veterinary medicine. Based on the known desirable immunomodulatory properties of mesenchymal stem cells, this therapy holds promise for the treatment of a variety of inflammatory diseases in cats.Aims:This review details our current understanding of feline stem cell biology and proposed mechanism of action. Studies performed in feline clinical trials for diseases including gingivostomatitis, chronic enteropathy, asthma and kidney disease are summarized, with the goal of providing an overview of the current status of this treatment modality and its potential for the future.

Spinal cord injury (SCI) is a devastating disorder that affects humans and dogs. The prognosis of SCI depends on the severity of the injury and can include varying levels of motor and sensory deficits including devastating paraplegia and quadriplegia. Placental mesenchymal stromal cells (PMSCs) have been shown to improve wound healing and possess neuroprotective and immunomodulatory capabilities, but have not yet been clinically tested for the treatment of SCI. This study established a protocol to isolate fetal PMSCs from canine placentas and characterized their paracrine secretion profile and ability to stimulate neurons in vitro to assess their potential as a treatment option for neurological disorders in dogs. Canine PMSCs (cPMSCs) were plastic adherent and capable of trilineage differentiation. cPMSCs expressed typical MSC markers and did not express hematopoietic or endothelial cell markers. Genotyping of cPMSCs revealed fetal rather than maternal origin of the cells. cPMSCs were viable and mitotically expansive in a collagen hydrogel delivery vehicle, and they secreted the immunomodulatory and neurotrophic paracrine factors interleukin (IL)-6, IL-8, monocyte chemoattractant protein 1 (MCP-1), and vascular endothelial growth factor (VEGF). cPMSCs also stimulated the growth of complex neural networks when co-cultured with SH-SY5Y cells, a neuroblastoma cell line used to model neuron growth in vitro. cPMSCs are analogous to human PMSCs. They meet the criteria to be defined as MSCs and represent a potential regenerative therapy option for neurological disorders in dogs with their robust growth in collagen hydrogel, stimulation of neural network formation, and secretion of potent paracrine factors. © 2017 International Society for Advancement of Cytometry.

Endotoxemia is a leading cause of morbidity and mortality in the equine industry, with colic being the most common cause of endotoxemia in horses. The objective of this study was to evaluate the safety and potential efficacy of a single dose of allogeneic equine bone marrow derived mesenchymal stem cells (BM-MSCs) in horses after the IV administration of lipopolysaccharide (LPS). Six horses were administered an IV infusion of 30 ng/kg LPS (O55:B5 Escherichia coli) in 500 ml saline over 30 min. Immediately after infusion test horses (n = 3) were administered 100 × 106 allogeneic BM-MSCs diluted in saline IV and control horses (n = 3) were administered saline. Clinicopathological data, pro-inflammatory cytokine measurements and sCD14 concentrations were compared between groups. No adverse reactions were observed in horses administered BM-MSCs intravenously. There were no significant differences between test and control horses with regard to clinicopathological values or pro-inflammatory cytokine production. At no time point did concentrations of sCD14 exceed the reference range in any horse. Results suggest that administration of a single IV dose of freshly cultured MSCs is safe and well-tolerated in horses with induced endotoxemia. Further study to evaluate their efficacy as a potential therapeutic in a larger number of horses with clinical disease is required.

Background The ability of mesenchymal stem cells (MSCs) to modulate immune responses inspired a series of clinical trials addressing oral mucosal inflammation. We previously reported on the safety and efficacy of fresh, allogeneic and autologous, adipose-derived mesenchymal stem cells (ASCs) to treat feline gingivostomatitis (FCGS), an oral mucosal inflammatory disease that shares similarities with human oral lichen planus. Methods To meet clinical demand and goals for future commercialization, we determined the feasibility of shipping fresh ASCs to distant clinics and extended our pilot studies to expand safety and efficacy data for shipped and non-shipped ASCs in a cohort of 18 FCGS cats enrolled locally and at a few different locations within the USA. Results We found that ASCs retained their viability, phenotype, and function after shipment. ASCs administered systemically resulted in a 72% positive response rate, identical to that noted in our previous studies. Cats that responded to ASC therapy had a significant decrease in circulating globulin concentration and histological evidence of decreased CD3+ T cells and CD20+ B cells in the oral mucosa. Responder cats also had significantly decreased percentages of CD8lo cells in blood prior to and at 3 months post-ASC therapy. CD8lo cells may serve as a potential “predictor” for response to systemic ASC therapy. Conclusion Fresh feline ASCs can be successfully shipped and administered to cats with FCGS. ASCs modulate the immune response and demonstrate efficacy for chronic oral mucosal inflammatory lesions that are characterized by CD8+ T cell inflammation and T cell activation. FCGS is a potentially useful naturally occurring large animal model of human oral inflammatory diseases.

Equine recurrent uveitis (ERU) is an immune‐mediated disease causing repeated or persistent inflammatory episodes which can lead to blindness. Currently, there is no cure for horses with this disease. Mesenchymal stem cells (MSCs) are effective at reducing immune cell activation in vitro in many species, making them a potential therapeutic option for ERU. The objectives of this study were to define the lymphocyte phenotype of horses with ERU and to determine how MSCs alter T‐cell phenotype in vitro. Whole blood was taken from 7 horses with ERU and 10 healthy horses and peripheral blood mononuclear cells were isolated. The markers CD21, CD3, CD4, and CD8 were used to identify lymphocyte subsets while CD25, CD62L, Foxp3, IFNγ, and IL10 were used to identify T‐cell phenotype. Adipose‐derived MSCs were expanded, irradiated (to control proliferation), and incubated with CD4+ T‐cells from healthy horses, after which lymphocytes were collected and analyzed via flow cytometry. The percentages of T‐cells and B‐cells in horses with ERU were similar to normal horses. However, CD4+ T‐cells from horses with ERU expressed higher amounts of IFNγ indicating a pro‐inflammatory Th1 phenotype. When co‐incubated with MSCs, activated CD4+ T‐cells reduced expression of CD25, CD62L, Foxp3, and IFNγ. MSCs had a lesser ability to decrease activation when cell‐cell contact or prostaglandin signaling was blocked. MSCs continue to show promise as a treatment for ERU as they decreased the CD4+ T‐cell activation phenotype through a combination of cell‐cell contact and prostaglandin signaling.

Compared to the osteological record of herbivorous dinosaurs from the Late Cretaceous Prince Creek Formation of northern Alaska, there are relatively fewer remains of theropods. The theropod record from this unit is mostly comprised of isolated teeth, and the only non-dental remains known can be attributed to the troodontid cf. Troodon and the tyrannosaurid Nanuqsaurus. Thus far, the presence of members of Dromaeosauridae has been limited to isolated teeth. Here we describe a symphyseal portion of a small dentary with two ziphodont teeth. Based on tooth shape, denticle morphology, and the position of the Meckelian groove, we attribute this partial dentary to a saurornitholestine dromaeosaurid. The fibrous bone surface, small size, and higher number of mesial denticles compared to distal ones point to a juvenile growth stage for this individual. Multivariate comparison of theropod teeth morphospace by means of principal component analysis reveals an overlap between this dentary and Saurornitholestinae dromaeosaurid morphospace, a result supported by phylogenetic analyses. This is the first confirmed non-dental fossil specimen from a member of Dromaeosauridae in the Arctic, expanding on the role of Beringia as a dispersal route for this clade between Asia and North America. Furthermore, the juvenile nature of this individual adds to a growing body of data that suggests Cretaceous Arctic dinosaurs of Alaska did not undergo long-distance migration, but rather they were year-round residents of these paleopolar latitudes.

The potential of mesenchymal stem cells (MSCs) for tissue repair and regeneration has garnered great attention. While MSC interaction with microbes at sites of tissue damage and inflammation is likely, especially in the gut, the consequences of bacterial association have yet to be elucidated. This study investigated the effect ofSalmonella entericasspentericaserotype Typhimurium on MSC trilineage differentiation path and mechanism. Through examination of key markers of differentiation, immunomodulatory regulators, and inflammatory cytokines, we demonstrated thatSalmonellaaltered osteogenic and chondrogenic differentiation pathways in human and goat adipose-derived MSCs. Gene expression profiles defined signaling pathway alterations in response toSalmonellaassociation not observed in epithelial cells. We uncovered significant differential expression (P< 0.05) of genes associated with anti-apoptotic and pro-proliferative responses in MSCs duringSalmonellachallenge. These observations led us to conclude that bacteria, specificallySalmonella,induce pathways that influence functional differentiation trajectories in MSCs, thus implicating substantial microbial influence on MSC physiology and immune activity.

Background Feline adipose-derived mesenchymal stem cells (ASCs) have been successfully used in clinical trials for the treatment of immune-mediated diseases with T cell dysregulation. However, the immunomodulatory pathways utilized by feline ASCs to suppress T cell activation have not been fully determined. We investigated the mechanisms used by feline ASCs to inhibit T cell proliferation, including the soluble factors and the cell-cell contact ligands responsible for ASC-T cell interaction. Methods The immunomodulatory activity of feline ASCs was evaluated via cell cycle analysis and in vitro mixed leukocyte reaction using specific immunomodulatory inhibitors. Cell-cell interactions were assessed with static adhesion assays, also with inhibitors. Results Feline ASCs decrease T cell proliferation by causing cell cycle arrest in G0–G1. Blocking prostaglandin (PGE2), but not IDO, partially restored lymphocyte proliferation. Although PDL-1 and CD137L are both expressed on activated feline ASCs, only the interaction of intercellular adhesion molecule 1 (ICAM-1, CD54) with its ligand, lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), was responsible for ASC-T cell adhesion. Blocking this interaction reduced cell-cell adhesion and mediator (IFN-γ) secretion and signaling. Conclusions Feline ASCs utilize PGE2 and ICAM-1/LFA-1 ligand interaction to inhibit T cell proliferation with a resultant cell cycle arrest in G0–G1. These data further elucidate the mechanisms by which feline ASCs interact with T cells, help define appropriate T cell-mediated disease targets in cats that may be amenable to ASC therapy, and may also inform potential translational models for human diseases. Electronic supplementary material The online version of this article (10.1186/s13287-019-1300-3) contains supplementary material, which is available to authorized users.

BACKGROUND: A technique for intra‐arterial injection of mesenchymal stem cells (MSC) has been established for front limbs with the use of the median artery. This approach has been proposed for treatment of soft tissue injuries of the equine distal limb. A technique has not been validated yet for hindlimb injection. OBJECTIVES: To assess the feasibility of injection of the cranial tibial artery in horses, and to evaluate the distribution and persistence of MSC after injection. STUDY DESIGN: In vivo experiment. METHODS: In a first phase, the cranial tibial arteries of both hindlimbs of three research horses were catheterised with ultrasound guidance under general anaesthesia and injected with iodinated contrast. In the second phase, iodinated contrast was injected in three standing sedated horses with ultrasound guidance. In the final phase, ⁹⁹ᵐTechnetium‐HMPAO labelled allogenic bone marrow derived equine MSC were injected under standing sedation with the same technique in three other horses. Scintigraphy was used to assess MSC distribution and persistence for 24 h. Ultrasound was performed 24 h after injection to assess vessel impairment. RESULTS: Arterial injection was achieved in all 18 limbs without any significant complications. Mild partial periarterial injection was observed in four limbs. Scintigraphic images demonstrated diffuse MSC distribution from the tarsal area to the foot. Persistence decreased over time but signal was still present at 24 h. MAIN LIMITATIONS: Limited retention of the radiolabel in the MSC. CONCLUSIONS: Ultrasound‐guided injection of the cranial tibial artery can be performed both under general anaesthesia and standing sedation in horses. This technique could be used for MSC treatment of equine proximal suspensory desmopathy or other injuries in the distal hindlimb.

© 2018, Mary Ann Liebert, Inc. Joint injury is a common cause of premature retirement for the human and equine athlete alike. Implantation of engineered cartilage offers the potential to increase the success rate of surgical intervention and hasten recovery times. Mesenchymal stem cells (MSCs) are a particularly attractive cell source for cartilage engineering. While bone marrow-derived MSCs (BM-MSCs) have been most extensively characterized for musculoskeletal tissue engineering, studies suggest that cord blood MSCs (CB-MSCs) may elicit a more robust chondrogenic phenotype. The objective of this study was to determine a superior equine MSC source for cartilage engineering. MSCs derived from bone marrow or cord blood were stimulated to undergo chondrogenesis through aggregate redifferentiation and used to generate cartilage through the self-assembling process. The resulting neocartilage produced from either BM-MSCs or CB-MSCs was compared by measuring mechanical, biochemical, and histological properties. We found that while BM constructs possessed higher tensile properties and collagen content, CB constructs had superior compressive properties comparable to that of native tissue and higher GAG content. Moreover, CB constructs had alkaline phosphatase activity, collagen type X, and collagen type II on par with native tissue suggesting a more hyaline cartilage-like phenotype. In conclusion, while both BM-MSCs and CB-MSCs were able to form neocartilage, CB-MSCs resulted in tissue more closely resembling native equine articular cartilage as determined by a quantitative functionality index. Therefore, CB-MSCs are deemed a superior source for the purpose of articular cartilage self-assembly.

Made available in DSpace on 2018-12-11T17:36:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-04-10 Background: It is currently unknown if the intrathecal administration of a high dose of allogeneic mesenchymal stem cells (MSCs) is safe, how MSCs migrate throughout the vertebral canal after intrathecal administration, and whether MSCs are able to home to a site of injury. The aims of the study were: 1) to evaluate the safety of intrathecal injection of 100 million allogeneic adipose-derived MSCs (ASCs); 2) to assess the distribution of ASCs after atlanto-occipital (AO) and lumbosacral (LS) injection in healthy horses; and 3) to determine if ASCs homed to the site of injury in neurologically diseased horses. Methods: Six healthy horses received 100 × 106 allogeneic ASCs via AO (n = 3) or LS injection (n = 3). For two of these horses, ASCs were radiolabeled with technetium and injected AO (n = 1) or LS (n = 1). Neurological examinations were performed daily, and blood and cerebrospinal fluid (CSF) were evaluated prior to and at 30 days after injection. Scintigraphic images were obtained immediately postinjection and at 30 mins, 1 h, 5 h, and 24 h after injection. Three horses with cervical vertebral compressive myelopathy (CVCM) received 100 × 106 allogeneic ASCs labeled with green fluorescent protein (GFP) via AO injection and were euthanized 1-2 weeks after injection for a full nervous system necropsy. CSF parameters were compared using a paired student's t test. Results: There were no significant alterations in blood, CSF, or neurological examinations at any point after either AO or LS ASC injections into healthy horses. The radioactive signal could be identified all the way to the lumbar area after AO ASC injection. After LS injection, the signal extended caudally but only a minimal radioactive signal extended further cranially. GFP-labeled ASCs were not present at the site of disease at either 1 or 2 weeks following intrathecal administration. Conclusions: The intrathecal injection of allogeneic ASCs was safe and easy to perform in horses. The AO administration of ASCs resulted in better distribution within the entire subarachnoid space in healthy horses. ASCs could not be found after 7 or 15 days of injection at the site of injury in horses with CVCM. Veterinary Institute for Regenerative Cures Department of Pathology Microbiology and Immunology University of California Department of Medicine and Epidemiology University of California Department of Surgical and Radiological Sciences University of California Department of Veterinary Clinics São Paulo State University Julio de Mesquita Filho UNESP Department of Veterinary Clinics São Paulo State University Julio de Mesquita Filho UNESP

Distal extremity wounds are a significant clinical problem in horses and humans and may benefit from mesenchymal stem cell (MSC) therapy. This study evaluated the effects of direct wound treatment with allogeneic stem cells, in terms of gross, histologic, and transcriptional features of healing. Three full-thickness cutaneous wounds were created on each distal forelimb in six healthy horses, for a total of six wounds per horse. Umbilical cord-blood derived equine MSCs were applied to each wound 1 day after wound creation, in one of four forms: (a) normoxic- or (b) hypoxic-preconditioned cells injected into wound margins, or (c) normoxic- or (d) hypoxic-preconditioned cells embedded in an autologous fibrin gel and applied topically to the wound bed. Controls were one blank (saline) injected wound and one blank fibrin gel-treated wound per horse. Data were collected weekly for 6 weeks and included wound surface area, thermography, gene expression, and histologic scoring. Results indicated that MSC treatment by either delivery method was safe and improved histologic outcomes and wound area. Hypoxic-preconditioning did not offer an advantage. MSC treatment by injection resulted in statistically significant increases in transforming growth factor beta and cyclooxygenase-2 expression at week 1. Histologically, significantly more MSC-treated wounds were categorized as pro-healing than pro-inflammatory. Wound area was significantly affected by treatment: MSC-injected wounds were consistently smaller than gel-treated or control wounds. In conclusion, MSC therapy shows promise for distal extremity wounds in horses, particularly when applied by direct injection into the wound margin.

Mesenchymal stem cells have been proposed to treat liver disease in the dog. The objective of this study was to compare portal, systemic intravenous and splenic injections for administration of mesenchymal stem cells to target the liver in healthy beagle dogs. Four healthy beagle dogs were included in the study. Each dog received mesenchymal stem cells via all three delivery methods in randomized order, 1 week apart. Ten million fat-derived allogeneic mesenchymal stem cells labeled with Technetium-99m (99mTc)-hexamethyl-propylene amine oxime(HMPAO) were used for each injection. Right lateral, left lateral, ventral, and dorsal scintigraphic images were obtained with a gamma camera equipped with a low-energy all-purpose collimator immediately after injection and 1, 6, and 24 h later. Mesenchymal stem cells distribution was assessed subjectively using all four views. Pulmonary, hepatic, and splenic uptake was quantified from the right lateral view, at each time point. Portal injection resulted in diffuse homogeneous high uptake through the liver, whereas the systemic intravenous injection led to mesenchymal stem cell trapping in the lungs. After splenic injection, mild splenic retention and high homogeneous diffuse hepatic uptake were observed. Systemic injection of mesenchymal stem cells may not be a desirable technique for liver therapy due to pulmonary trapping. Splenic injection represents a good alternative to portal injection. Scintigraphic tracking with 99mTc-HMPAO is a valuable technique for assessing mesenchymal stem cells distribution and quantification shortly after administration. Data obtained at 24 h should be interpreted cautiously due to suboptimal labeling persistence.

Mesenchymal stem cells (MSCs) have potent immunomodulatory functions and are a promising therapy for immune-mediated inflammatory disorders. We previously demonstrated the efficacy of fresh, autologous, adipose-derived MSCs (ASCs) to treat feline chronic gingivostomatitis (FCGS), a chronic oral mucosal inflammatory disease similar to human oral lichen planus. Here, we investigate the use of fresh allogeneic ASCs for treatment of FCGS in seven cats. Radiolabeled ASCs were also tracked systemically. Each cat received two intravenous injections of 20 million ASCs, 1 month apart. Oral inflammation, blood lymphocyte subsets, anti-fetal bovine serum antibody levels, ASC crossmatching and serum proteins and cytokine concentrations were determined. Four of the 7 cats (57%) responded to treatment [complete clinical remission (n = 2) or substantial clinical improvement (n = 2)]. Three cats were nonresponders. Prior to therapy, most cats had increased circulating CD8+ T cells, decreased CD8lo cells, and a decreased CD4/CD8 ratio, however clinical resolution was not associated with normalization of these parameters. Nonresponders showed more severe systemic inflammation (neutrophilia, hyperglobulinemia and increased interferon gamma and tumor necrosis factor alpha concentration) prior to ASC therapy. Clinical remission took up to 20 months and no clinical relapse has occurred. A higher fraction of radiolabeled ASCs were identified in the oral cavity of FCGS affected cats than the control cat. The administration of fresh, allogenic ASCs appeared to have lower clinical efficacy with a delayed response as compared to the fresh, autologous ASCs. In addition, the mechanism(s) of action for autologous and allogenic ASCs may differ in this model of oral inflammation.

Bruton's tyrosine kinase (BTK) is important in B-cell signalling. Efficacy has been reported for BTK inhibitors (BTKi) in human autoimmune diseases. Canine pemphigus foliaceus (cPF) is the most common canine autoimmune skin disease.To determine the safety and efficacy of a BTKi in cPF treatment.Nine privately owned dogs.Nine dogs diagnosed with PF were administered BTKi PRN473. Initial dosages were ≈15 mg/kg once daily, increased to twice daily if inadequate response was seen. Treatment continued for a maximum of 20 weeks, attempting decrease to every other day. Dogs were monitored with complete blood counts, serum biochemistry panels, urinalyses and evaluated with a modified version of a validated human Pemphigus Disease Activity Index (cPDAI). Anti-desmocollin-1 (DSC-1) and desmoglein-1 (DSG-1) immunoglobulin G (IgG) titres were performed before and after the treatment period. Drug bound to target was measured in peripheral blood mononuclear cells.All nine dogs showed reduction in lesions and cPDAI score during the first two weeks of treatment. At the end of the study, four responses were considered "good", two "fair", two "poor" and one dog withdrawn due to recurrence of a previously excised mast cell tumour. Four dogs continued to improve by Week 4; three sustained near complete remission by study's end. The anti-DSC-1 IgG titre decreased in three dogs, increased in two, was undetected in three and was not performed in the withdrawn dog. No dogs had detectable IgG to DSG1. Possible adverse effects occurred in three dogs.Bruton's tyrosine kinase inhibitor monotherapy may have beneficial effects in some cases of cPF.La BTK (Bruton's tyrosine kinase) est une voie importante d'activation des cellules B. L'efficacité a été rapportée pour les inhibiteurs de BTK (BTKi) dans les maladies auto-immunes de l'homme. Le pemphigus foliacé du chien (cPF) est la dermatite auto-immune la plus fréquente du chien.Déterminer l'efficacité et l'innocuité d'un BTKi dans le traitement d'un cPF.Neufs chiens de propriétaires. MATÉRIELS ET MÉTHODES: Neuf chiens diagnostiqués avec PF ont reçu du BTKi PRN473. Les doses initiales étaient ≈15 mg/kg une fois par jour, augmenté à deux fois par jour si une réponse inadéquate était observée. Le traitement était poursuivi au maximum 20 semaines, tentant de réduire à jours alternés. Les chiens étaient monitorés par une formule sanguine, un profil biochimique, une analyse urinaire et évalués par une version modifiée de cPDAI (Pemphigus Disease Activity Index) validée chez l'homme. Les concentrations d'immunoglobuline G (IgG) anti-desmocolline-1 (DSC-1) et desmogléine-1 (DSG-1) ont été réalisées avant et après traitement. Le portage de la molécule à sa cible a été mesuré dans les cellules mononuclées sanguines périphériques. RÉSULTATS: Tous les neuf chiens ont montré une diminution des lésions et du score cPDAI au cours des deux premières semaines de traitement. A la fin de l’étude, quatre réponses ont été considérées comme « bonnes », deux « moyennes, deux « faibles » et un chien a été retiré en raison d'une récidive d'un mastocytome précédemment retiré. Quatre chiens ont continué à s'améliorer jusqu’à la semaine 4 ; trois étaient presque en rémission complète à la fin de l’étude. La concentration d'IgG anti-DSC-1 a diminué pour trois chiens, augmenté pour deux et était non détectable pour trois et n'a pas été réalisée pour le chien exclu. Aucun chien n'avait d'IgG détectable pour DSG1. Les effets secondaires se sont produits chez trois chiens.Le traitement unique par inhibiteur de tyrosine kinase de Bruton pourrait avoir des effets bénéfiques pour certains cas de cPF.INTRODUCCIÓN: la tirosina quinasa (BTK) de Bruton es importante en las señales de células B. Se ha informado de resultados beneficiosos de los inhibidores de BTK (BTKi) en enfermedades autoinmunes humanas. El pénfigo foliáceo canino (cPF) es la enfermedad cutánea autoinmune canina más común. OBJETIVOS: Determinar la seguridad y eficacia de un BTKi en el tratamiento de cPF. ANIMALES: Nueve perros de propietarios particulares. MÉTODOS Y MATERIALES: nueve perros diagnosticados con PF recibieron BTKi PRN473. Las dosis iniciales fueron de ≈ 15 mg/kg una vez al día, aumentadas a dos veces al día si se observaba una respuesta inadecuada. El tratamiento continuó durante un máximo de 20 semanas, intentando disminuirlo cada dos días. Los perros fueron evaluados con recuentos sanguíneos completos, paneles de bioquímica en suero, análisis de orina y una versión modificada de un índice de actividad validado de la enfermedad del pénfigo humano (cPDAI). Los títulos de inmunoglobulina G (IgG) de anti-desmocolina-1 (DSC-1) y desmogleína-1 (DSG-1) se analizaron antes y después del período de tratamiento. La cantidad de fármaco unido a su diana se midió en células mononucleares de sangre periférica. RESULTADOS: los nueve perros mostraron una reducción en las lesiones y de los valores de cPDAI durante las primeras dos semanas de tratamiento. Al final del estudio, cuatro respuestas se consideraron “buenas”, dos “aceptables”, dos “pobres” y un perro fue retirado debido a la recidiva de un mastocitoma previamente extirpado. Cuatro perros continuaron mejorando en la semana 4; y tres mostraron una remisión casi completa al final del estudio. El título de IgG anti-DSC-1 disminuyó en tres perros, aumentó en dos, no se detectó en tres y no se realizó en el perro retirado. Ningún perro tenía IgG detectable a DSG1. Posibles efectos adversos se observaron en tres perros. CONCLUSIONES E IMPORTANCIA CLÍNICA: la monoterapia con el inhibidor de la tirosina quinasa de Bruton puede tener efectos beneficiosos en algunos casos de cPF.Die Bruton Tyrosinkinase (BTK) spielt beim Signalweg der B-Zellen eine wichtige Rolle. Von der Wirksamkeit der BTK Inhibitoren (BTKi) bei Autoimmunerkrankungen des Menschen wurde bereits berichtet. Pemphigus foliaceus beim Hund (cPF) ist die häufigste autoimmune Hauterkrankung des Hundes.Feststellung der Sicherheit und Wirksamkeit eines BTKi bei der Behandlung des cPF.Neun Hunde in Privatbesitz.Neun Hunden, die mit PF diagnostiziert worden waren, wurde BTKi PRN473 verabreicht. Anfangsdosen waren ≈ 15 mg/kg einmal täglich, bei einer unzureichenden Antwort wurde eine Erhöhung auf zweimal täglich durchgeführt. Die Behandlung wurde für ein Maximum von 20 Wochen fortgeführt, wobei eine Dosisreduktion auf jeden zweiten Tag versucht wurde. Die Hunde wurden mit Blutbild, Serumbiochemie, Urinanalyse überwacht und wurden mit einer modifizerten Version eines validierten humanen Pemphigus Disease Activity Index (cPDAI) evaluiert. Es wurden Immunglobulin-G (IgG)-Titer auf Anti-Desmocollin-1 (DSC-1) und Desmoglein-1 (DSG-1) vor und nach einer jeden Behandlungsperiode bestimmt. Der Wirkstoff, welcher an das Target gebunden war, wurde in den peripheren Blutmononuklearzellen bestimmt.Alle neun Hunde zeigten eine Verminderung ihrer Läsionen und des cPDAI Werts während der ersten zwei Wochen der Behandlung. Am Ende der Studie wurden vier Hunde als „gut“, zwei als „moderat“, zwei als „geringgradig“ und zwei als „schlecht“ eingestuft, ein Hund wurde aufgrund eines wiederaufgetretenen, zuvor entfernten, Mastzelltumors aus der Studie genommen. Vier Hunde verbesserten sich weiterhin bis zur Woche 4; bei dreien bestand eine fast vollständige Remission zu Studienende. Der Anti-DSC-1 IgG Titer nahm bei drei Hunden ab, bei zwei Hunden zu, konnte bei drei weitere Hunden nicht bestimmt werden und wurde bei dem ausgefallenen Hund nicht bestimmt. Keiner der Hunde hatte feststellbare IgG gegenüber DSG1. Mögliche Nebenwirkungen traten bei drei Hunden auf.Eine Bruton Tyrosinkinase-Inhibitor Monotherapie könnte günstige Auswirkungen auf einige Fälle von cPF bei Hunden haben.背景: ブルトン型チロシンキナーゼ(BTK)はB細胞シグナル伝達において重要である。人の自己免疫性疾患においてBTK阻害剤(BTKi)の有効性が報告されている。犬の落葉状天疱瘡(cPF)は、最も一般的な犬の自己免疫性皮膚疾患である。 目的: 本研究の目的は、cPF治療におけるBTKiの安全性と有効性を決定することである。 供試動物: 9頭の飼育犬。 材料と方法: PFと診断した9頭の犬にBTKi PRN473を投与した。初期用量は1日1回≈15mg / kgであったが、反応が不十分な場合は1日2回に増量した。治療は最大20週間継続し、1日おきの減量を試みた。犬を、全血球計算、血清生化学パネル、尿検査でモニターし、検証済みの人天疱瘡疾患活動性指数(cPDAI)の修正版で評価した。治療期間の前後で、抗デスモコリン-1(DSC-1)およびデスモグレイン-1(DSG-1)免疫グロブリンG(IgG)力価を測定した。標的に結合した薬物を、末梢血単核細胞で測定した。 結果: 9頭すべての犬が、治療の最初の2週間で病変とcPDAIスコアの減少を認めた。研究終了時に4頭が「良」、2頭が「可」、2頭が「不良」とみなし、1頭が以前に切除されたマスト細胞腫瘍の再発により離脱した。 4頭の犬は4週目まで改善し続けた。 3頭は研究終了時までにほぼ完全寛解を維持した。抗DSC-1 IgG力価は3頭の犬で減少し、2頭で増加し、3頭で検出されず、離脱した犬では実施されなかった。 DSG1に対するIgGは検出されなかった。 3頭の犬で副作用の可能性が生じた。 結論と臨床的重要性: ブルトン型チロシンキナーゼ阻害剤の単独療法は、cPFのいくつかの症例で有益な効果をもたらす可能性がある。.背景: Bruton酪氨酸激酶 (BTK) 在B细胞信号转导中很重要。已报告BTK抑制剂(BTKi)在人类自身免疫性疾病中的疗效。犬落叶型天疱疮(cPF)是最常见的犬自身免疫性皮肤病。 目的: 确定BTKi治疗cPF 的安全性和疗效。 动物: 9只私家犬。 方法和材料: 对诊断为PF的9只犬给予BTKi PRN473。初始剂量约为15mg/kg,每日一次,如果观察到反应不足,则增加至每日两次。治疗最长持续20周,尝试降低到隔日一次。通过全血细胞计数、血清生化全项、尿分析对犬进行监测,并使用改良版本的经验证的人天疱疮疾病活动指数(cPDAI)进行评估。在治疗期前后进行抗桥粒糖蛋白-1(DSC-1)和桥粒芯蛋白-1(DSG-1)免疫球蛋白G(IgG)滴度检测。在外周血单核细胞中检测与靶点结合的药物。 结果: 在治疗前两周,所有9只犬均显示病变和cPDAI评分减少。研究结束时,认为4例缓解为“良好”,2例为“一般”,2例为“较差”,1只犬因既往切除的肥大细胞瘤复发而退出研究。4只犬在第4周时持续改善;3只犬在研究结束时持续接近完全缓解。3只犬的抗 DSC-1 IgG滴度降低,2只犬的抗 DSC-1 IgG滴度升高,3只犬中未检测到,退出犬中未进行检测。所有犬均未检出DSG1 IgG。3只犬可能发生不良反应。 结论和临床重要性: Bruton酪氨酸激酶抑制剂单药治疗可能对某些cPF病例有益。.A tirosina quinase de Bruton (BTK) é importante na sinalização de células B. Tem sido relatada em humanos a eficácia de inibidores da BTK (BTKi) em doenças autoimunes. O pênfigo foliáceo canino (cPF) é a doença de pele autoimune canina mais comum.Determinar a segurança e a eficácia de um BTKi no tratamento de cPF.Nove cães de propriedade privada. MÉTODOS E MATERIAIS: Nove cães diagnosticados com PF receberam BTKi PRN473. As doses iniciais foram de ≈ 15 mg / kg uma vez ao dia, e aumentadas para duas vezes ao dia caso fosse observada resposta inadequada. O tratamento foi mantido por no máximo 20 semanas, tentando reduzir para dias alternados. Os cães foram monitorados com hemograma, painéis de bioquímica sérica, urinálise e avaliados com uma versão modificada de um Índice de Atividade de Doença do Pênfigo Humano validado (cPDAI). Os títulos de imunoglobulina G (IgG) anti-desmocolina-1 (DSC-1) e desmogleina-1 (DSG-1) foram realizados antes e após o período de tratamento. O fármaco ligado ao alvo foi medido em células mononucleares do sangue periférico.Todos os nove cães apresentaram redução nas lesões e escore de cPDAI durante as duas primeiras semanas de tratamento. No final do estudo, quatro respostas foram consideradas “boas”, duas “justas”, duas “ruins” e um cão foi retirado devido à recorrência de um mastocitoma previamente extirpado. Quatro cães continuaram a melhorar na semana 4; três apresentaram remissão quase completa até o final do estudo. O título de IgG anti-DSC-1 diminuiu em três cães, aumentou em dois, não foi detectado em três e não foi realizado no cão retirado. Nenhum cão apresentou IgG detectável para DSG1. Possíveis efeitos adversos ocorreram em três cães. CONCLUSÕES E IMPORTÂNCIA CLÍNICA: A monoterapia com inibidores de tirosina quinase de Bruton pode ter efeitos benéficos em alguns casos de cPF.

Veterinary adult stem cell therapy is an emerging area of basic and clinical research. Like their human counterparts, veterinary mesenchymal stem cells (MSCs) offer many potential therapeutic benefits. The characterization of canine-derived MSCs, however, is poorly defined compared to human MSCs. Furthermore, little consensus exists regarding the expression of canine MSC cell surface markers. To address this issue, this study investigated characteristics of cultured canine MSCs derived from both adipose tissue and bone marrow. The canine MSCs were obtained from donors of various breeds and ages. A panel of cell surface markers for canine MSCs was selected based on current human and canine literature and the availability of canine-reactive antibodies. Using flow cytometry, canine MSCs were defined to be CD90(+)CD44(+)MHC I(+)CD14(-)CD29(-)CD34(-)MHC II(-). Canine MSCs were further characterized using real-time RT-PCR as CD105(+)CD73(+)CD14(+)CD29(+)MHC II(+)CD45(-) at the mRNA level. Among these markers, canine MSCs differed from canine peripheral blood mononuclear cells (PBMCs) by the absence of CD45 expression at the mRNA level. A novel high-throughput canine-specific PCR array was developed and used to identify changes in the gene expression profiles of canine MSCs. Genes including PTPRC, TNF, β2M, TGFβ1, and PDGFRβ, were identified as unique to canine MSCs as compared to canine PBMCs. Our findings will facilitate characterization of canine MSCs for use in research and clinical trials. Moreover, the high-throughput PCR array is a novel tool for characterizing canine MSCs isolated from different tissues and potentially from different laboratories.

The use of cell transplantation for spinal cord injury is a rapidly evolving field in regenerative medicine. Numerous animal models are currently being used. However, translation to human patients is still a challenging step. Dogs are of increasing importance as a translational model for human disease since there is a greater awareness of the need to increase the quality of preclinical data. The use of dogs ultimately brings benefit to both human and veterinary medicine. In this review we analyze experimental and clinical studies using cell transplantation for canine spinal cord injury. Overall, in experimental studies, transplantation groups showed improvement over control groups. Improvements were measured at the functional, electrophysiological, histological, RNA and protein levels. Most clinical studies support beneficial effects of cell transplantation despite the fact that methodological limitations preclude definitive conclusions. However, the mechanisms of action and underlying the behavior of transplanted cells in the injured spinal cord remain unclear. Overall, we conclude here that stem cell interventions are a promising avenue for the treatment of spinal cord injury. Canines are a promising model that may help bridge the gap between translational research and human clinical trials.

Summary Anaplasma phagocytophilum, which causes granulocytic anaplasmosis in humans and animals, is a tick-transmitted obligate intracellular bacterium that mediates its own uptake into neutrophils and non-phagocytic cells. Invasins of obligate intracellular pathogens are attractive targets for protecting against or curing infection because blocking the internalization step prevents survival of these organisms. The complement of A. phagocytophilum invasins is incompletely defined. Here, we report the significance of a novel A. phagocytophilum invasion protein, AipA. A. phagocytophilum induced aipA expression during transmission feeding of infected ticks on mice. The bacterium upregulated aipA transcription when it transitioned from its non-infectious reticulate cell morphotype to its infectious dense-cored morphotype during infection of HL-60 cells. AipA localized to the bacterial surface and was expressed during in vivo infection. Of the AipA regions predicted to be surface-exposed, only residues 1 to 87 (AipA1–87) were found to be essential for host cell invasion. Recombinant AipA1–87 protein bound to and competitively inhibited A. phagocytophilum infection of mammalian cells. Antiserum specific for AipA1–87, but not other AipA regions, antagonized infection. Additional blocking experiments using peptide-specific antisera narrowed down the AipA invasion domain to residues 9 to 21. An antisera combination targeting AipA1–87 together with two other A. phagocytophilum invasins, OmpA and Asp14, nearly abolished infection of host cells. This study identifies AipA as an A. phagocytophilum surface protein that is critical for infection, demarcates its invasion domain, and establishes a rationale for targeting multiple invasins to protect against granulocytic anaplasmosis.

Background & Aim The canine is increasingly recognized as a valuable pre-clinical large animal model for many human diseases including cancer, respiratory disease, and inflammatory disease. Like humans, canines live in similar conditions, receive long term medical care, and develop congenital diseases including spina bifida (SB). Canine SB clinically presents very similarly to human SB, and English bulldogs in particular have a high incidence of naturally-occurring SB. Placental mesenchymal stem cells (PMSCs) are being investigated as an adjunct to prenatal repair of SB; however, similar treatments have not been explored for postnatal repair. English bulldogs could serve as the first postnatal animal model of SB. The goal of this study is to evaluate canine PMSCs (cPMSCs) to test their efficacy as a postnatal therapy in a naturally-occurring large animal disease model. If successful, this approach may be a significant step toward translating this therapy to human patients, and may offer the first regenerative treatment for their canine companions born with SB. Methods, Results & Conclusion To date, we have enrolled four 9-week-old English bulldogs with SB defects confirmed by neurological evaluation and magnetic resonance imaging (MRI). Each dog underwent a multi-segment laminectomy, coupled with transplant of allogeneic cPMSCs embedded in hydrogel and extracellular matrix (ECM) scaffold. One-year follow-up has been completed on two study animals and the remaining two animals are currently being evaluated at 8 weeks post-treatment. The first two dogs enrolled were initially ambulatory with notable abnormal gaits and incontinence. MRI revealed L7-S1 defects of varying severity. One MRI also showed a large syringohydromyelnia involving the lumbar spinal cord. Electrophysiologic testing revealed low-normal conduction velocity for both motor and sensory hindlimb nerves. Both dogs recovered from posterior laminectomy and cPMSC implantation uneventfully. Following treatment both dogs showed improved ambulatory gaits. No significant adverse events occurred in either dog by 12 months. Two additional dogs have been enrolled and are currently being evaluated. Postnatal treatment of a naturally-occurring canine model of SB with allogeneic cPMSCs is clinically feasible and appears safe. Further studies are currently being performed to evaluate efficacy.The findings from this study suggest that naturally-occuring canine SB is a valuable translational model to evaluate PMSC postnatal therapy.

The discovery of multipotent neural crest-derived stem cells, named epidermal neural crest stem cells (EPI-NCSC), that persist postnatally in an easy-to-access location—the bulge of hair follicles—opens a spectrum of novel opportunities for patient-specific therapies. We present a detailed characterization of canine EPI-NCSC (cEPI-NCSC) from multiple dog breeds and protocols for their isolation and ex vivo expansion. Furthermore, we provide novel tools for research in canines, which currently are still scarce. In analogy to human and mouse EPI-NCSC, the neural crest origin of cEPI-NCSC is shown by their expression of the neural crest stem cell molecular signature and other neural crest-characteristic genes. Similar to human EPI-NCSC, cEPI-NCSC also expressed pluripotency genes. We demonstrated that cEPI-NCSC can generate all major neural crest derivatives. In vitro clonal analyses established multipotency and self-renewal ability of cEPI-NCSC, establishing cEPI-NCSC as multipotent somatic stem cells. A critical analysis of the literature on canine spinal cord injury (SCI) showed the need for novel treatments and suggested that cEPI-NCSC represent viable candidates for cell-based therapies in dog SCI, particularly for chondrodystrophic dogs. This notion is supported by the close ontological relationship between neural crest stem cells and spinal cord stem cells. Thus, cEPI-NCSC promise to offer not only a potential treatment for canines but also an attractive and realistic large animal model for human SCI. Taken together, we provide the groundwork for the development of a novel cell-based therapy for a condition with extremely poor prognosis and no available effective treatment.

Background aims. Mesenchymal stromal cells (MSCs) have been extensively studied as a cellular therapeutic for various pathologic conditions. However, there remains a paucity of data regarding regional and systemic safety of MSC transplantations, particularly with multiple deliveries of allogeneic cells. The purpose of this study was to investigate the safety and systemic immunomodulatory effects of repeated local delivery of allogeneic MSCs into the region of the lacrimal gland, the gland of the third eyelid and the knee joint in dogs. Methods. Allogeneic adipose tissue-derived canine MSCs were delivered to the regions of the lacrimal gland and the third eyelid gland as well as in the knee joints of six healthy laboratory beagles as follows: six times with 1-week intervals for delivery to the lacrimal gland and the third eyelid gland regions and three to four times with 1- to 2-week intervals for intra-articular transplantations. Dogs were sequentially evaluated by clinical examination. At the conclusion of the study, dogs were humanely euthanized, and a complete gross and histopathologic examination of all organ systems was performed. Mixed leukocyte reactions were also performed before the first transplantation and after the final transplantation. Results. Clinical and pathologic examinations found no severe consequences after repeated MSC transplantations. Results of mixed leukocyte reactions demonstrated suppression of T-cell proliferation after MSC transplantations. Conclusions. This is the first study to demonstrate regional and systemic safety and systemic immunomodulatory effects of repeated local delivery of allogeneic MSCs in vivo.

During bacterial infection, hematopoietic stem and progenitor cells (HSPCs) differentiate into polymorphonuclear leukocytes (PMNs) in the bone marrow. We reported that HSPCs recruited to Staphylococcus aureus-infected skin wounds in mice undergo granulopoiesis, whereas other authors have demonstrated their differentiation in vitro after Toll-like receptor 2 (TLR2)/MyD88 stimulation. Here, we examined this pathway in HSPC trafficking and granulopoiesis within S aureus-infected wounds. Lineage- HSPCs from TLR2- or MyD88-deficient mice injected into infected wounds of wild-type (WT) mice exhibited impaired granulopoiesis. However, HSPCs from WT mice produced similar numbers of PMNs whether transferred into wounds of TLR2-, MyD88-deficient, or WT mice. Prostaglandin E2 (PGE2), which stimulates HSPC survival and proliferation, was produced by HSPCs after TLR2 stimulation, suggesting that TLR2/MyD88 activation promotes granulopoiesis in part by production and autocrine activity of PGE2. Pretreatment of TLR2- or MyD88-deficient HSPCs with PGE2 rescued granulocytic differentiation in vivo. Finally, we demonstrate that bone marrow-derived lin-/Sca-1+/c-kit+ cells produced PGE2 and underwent granulopoiesis after TLR2 stimulation. lin-/Sca-1+/c-kit+ cells deficient in TLR2 or MyD88 produced PMNs after PGE2 treatment when transferred into uninfected wounds. We conclude that granulopoiesis in S aureus-infected wounds is induced by TLR2/MyD88 activation of HSPCs through a mechanism that involves autocrine production and activity of PGE2.

Canine hyperadrenocorticism (HAC) is a common endocrinopathy often associated with hypercoagulability, thrombosis and thromboembolism and it can contribute to increased morbidity and mortality. The condition results in increased, unregulated secretion of glucocorticoids (GCs). While prospective identification of hypercoagulability is challenging, thrombelastography (TEG) is a diagnostic tool that enables the detection of hypercoagulability in a clinical setting. The objective of this prospective cohort study was to serially assess coagulation in dogs with HAC using TEG to test the hypothesis that dogs with HAC have increased TEG maximal amplitude (MA) and that the MA would normalize once clinical control was achieved. Twenty-three dogs with naturally occurring HAC were enrolled and hemostatic (including TEG, platelet function, thrombin-antithrombin complexes and coagulation panel) and hematological variables were measured at presentation. TEG was serially monitored until clinical resolution of HAC was attained. At presentation, most dogs with HAC had increased MA values, increased thrombin-antithrombin complexes and many were hyperfibrinogenemic. Platelet function analyzer-100 (PFA-100) closure times were significantly prolonged. TEG tracings did not normalize in either medically- or surgically-managed dogs, but fibrinogen concentrations decreased. It seems that dogs with HAC have a complex coagulopathy in which hypercoagulability and platelet hyporeactivity or dysfunction might occur simultaneously. As TEG tracings did not normalize in well-controlled dogs, it is unlikely that increased blood GCs are solely responsible for TEG alterations seen in dogs with HAC.

Background aims Delivery of bone marrow–derived stem and progenitor cells to the site of injury is an effective strategy to enhance bone healing. An alternate approach is to mobilize endogenous, heterogeneous stem cells that will home to the site of injury. AMD3100 is an antagonist of the chemokine receptor 4 (CXCR4) that rapidly mobilizes stem cell populations into peripheral blood. Our hypothesis was that increasing circulating numbers of stem and progenitor cells using AMD3100 will improve bone fracture healing. Methods A transverse femoral fracture was induced in C57BL/6 mice, after which they were subcutaneously injected for 3 d with AMD3100 or saline control. Mesenchymal stromal cells, hematopoietic stem and progenitor cells and endothelial progenitor cells in the peripheral blood and bone marrow were evaluated by means of flow cytometry, automated hematology analysis and cell culture 24 h after injection and/or fracture. Healing was assessed up to 84 d after fracture by histomorphometry and micro–computed tomography. Results AMD3100 injection resulted in higher numbers of circulating mesenchymal stromal cells, hematopoietic stem cells and endothelial progenitor cells. Micro-computed tomography data demonstrated that the fracture callus was significantly larger compared with the saline controls at day 21 and significantly smaller (remodeled) at day 84. AMD3100-treated mice have a significantly higher bone mineral density than do saline-treated counterparts at day 84. Conclusions Our data demonstrate that early cell mobilization had significant positive effects on healing throughout the regenerative process. Rapid mobilization of endogenous stem cells could provide an effective alternative strategy to cell transplantation for enhancing tissue regeneration.

Lymphoma is the most common haematopoietic malignancy in dogs and it has been associated with hypercoagulability and subsequent thromboembolism. The objectives of this study were to serially characterize the haemostatic status of dogs with multicentric lymphoma. Thromboelastography, thrombin-antithrombin complex concentration and routine haematology and coagulation panels were measured. Twenty-seven dogs were included in the study and 15 completed the study in remission. At presentation, 81% (22/27) of dogs with multicentric lymphoma had altered haemostatic profiles consistent with hypercoagulability. Laboratory evidence of hypercoagulability did not resolve during treatment or for up to 1 month following attainment of clinical remission. Accelerated rate of clot formation at the time of chemotherapeutic protocol completion was associated with decreased survival time. We concluded that dogs with multicentric lymphoma were frequently hypercoagulable from presentation through 4 weeks after the completion of chemotherapy. Increased angle and shortened K in dogs that have successfully completed their chemotherapeutic protocol may be associated with shorter survival times.

Reasons for performing studyIntralesional (i.l.) injection is currently the most commonly used technique for stem cell therapy in equine tendon injury. A comparison of different techniques of injection of mesenchymal stem cells for the treatment of tendon lesions is required.ObjectivesWe hypothesised that vascular perfusion of the equine distal limb with mesenchymal stem cells (MSCs) would result in preferential distribution of MSCs to acute tendon injuries.Study designIn vivo experimental study.MethodsLesions were surgically induced in forelimb superficial digital flexor tendons of 8 horses. Three or 10 days after lesion induction, technetium-99 hexamethyl propylene amine oxime-labelled MSCs were injected via i.v. or intra-arterial (i.a.) regional limb perfusion (RLP) at the level of the distal antebrachium and compared to i.l. injection. Mesenchymal stem cell persistence and distribution within the forelimb and tendon lesions was assessed with scintigraphy for 24h.ResultsLesion uptake was higher with i.l. injection than with RLP, but MSC persistence decreased similarly over time in all 3 techniques. Intra-arterial RLP resulted in a better distribution of MSCs and a higher uptake at the lesion site than i.v. RLP. Limbs perfused i.a. on Day 10 showed greater accumulation of MSCs in the lesion than limbs perfused on Day 3. Arterial thrombosis occurred in 50% of the i.v. RLP limbs and in 100% of the i.a. RLP limbs, which led to clinical complications in one horse.Conclusions and potential relevanceCompared with i.l. injection, RLP results in lower uptake but similar persistence of MSCs at the site of tendon lesions. A time dependent accumulation of MSCs was identified with i.a. RLP. The i.a. RLP appears more advantageous than the i.v. RLP in terms of distribution and uptake. However, the described i.a. technique produced arterial thrombosis and thus cannot currently be recommended for clinical use. The Summary is available in Chinese - see Supporting information.

Summary Reasons for performing study Joint inflammation and septic arthritis are both potential complications of intra-articular injections of bone marrow-derived mesenchymal stem cells (BM-MSCs). Clinicians may prophylactically co-inject BM-MSCs admixed with either antimicrobials or hyaluronic acid; however, the effect of these agents on cultured BM-MSCs is unknown. Objective To determine the effects of therapeutic levels of gentamicin, amikacin and hyaluronic acid on cultured equine BM-MSCs in vitro. Study design In vitro experimental study. Methods Equine BM-MSCs from 4 healthy mature horses were isolated. Cultured BM-MSCs from each donor were incubated with gentamicin (150 mg), amikacin (250 mg), hyaluronic acid (22 mg) or 1% penicillin/streptomycin (control) under sterile conditions. Mesenchymal stem cells viability, proliferation, mediator secretion and culture media pH were measured. Results Incubation of BM-MSCs with gentamicin resulted in >95% MSC death after 45 min, and incubation of BM-MSCs with amikacin resulted in >95% MSC death after 2 h. Incubation of BM-MSCs with hyaluronic acid or penicillin/streptomycin (control) for up to 6 h resulted in sustained BM-MSC viability of 80% and >93%, respectively. All additives resulted in decreased media pH in the first minute; however, the pH then remained constant over the 6 h incubation period. No significant differences in BM-MSC proliferation or mediator secretion between the penicillin/streptomycin (control) and cells treated with hyaluronic acid were observed. Conclusion Therapeutic concentrations of aminoglycoside antimicrobials are toxic to cultured equine BM-MSCs. The effects of hyaluronic acid on cultured MSC viability, proliferation and mediator secretion are minimal. Potential relevance Based on these findings, the mixing of aminoglycoside antimicrobials and cultured equine BM-MSCs prior to therapeutic use is not recommended.

Anaplasma phagocytophilum , a member of the family Anaplasmataceae , is the tick-transmitted obligate intracellular bacterium that causes human granulocytic anaplasmosis. The life cycle of A. phagocytophilum is biphasic, transitioning between the noninfectious reticulate cell (RC) and infectious dense-cored (DC) forms. We analyzed the bacterium's DC surface proteome by selective biotinylation of surface proteins, NeutrAvidin affinity purification, and mass spectrometry. Transcriptional profiling of selected outer membrane protein candidates over the course of infection revealed that aph_0248 (designated asp14 [14-kDa A. phagocytophilum surface protein]) expression was upregulated the most during A. phagocytophilum cellular invasion. asp14 transcription was induced during transmission feeding of A. phagocytophilum -infected ticks on mice and was upregulated when the bacterium engaged its receptor, P-selectin glycoprotein ligand 1. Asp14 localized to the A. phagocytophilum surface and was expressed during in vivo infection. Treating DC organisms with Asp14 antiserum or preincubating mammalian host cells with glutathione S -transferase (GST)–Asp14 significantly inhibited infection of host cells. Moreover, preincubating host cells with GST-tagged forms of both Asp14 and outer membrane protein A, another A. phagocytophilum invasin, pronouncedly reduced infection relative to treatment with either protein alone. The Asp14 domain that is sufficient for cellular adherence and invasion lies within the C-terminal 12 to 24 amino acids and is conserved among other Anaplasma and Ehrlichia species. These results identify Asp14 as an A. phagocytophilum surface protein that is critical for infection, delineate its invasion domain, and demonstrate the potential of targeting Asp14 in concert with OmpA for protecting against infection by A. phagocytophilum and other Anaplasmataceae pathogens.

© 2016 Oki, Huang, Beyer, May, Truchan, Walker, Galloway, Borjesson and Carlyon. Anaplasma phagocytophilum, a member of the family Anaplasmataceae and the obligate intracellular bacterium that causes granulocytic anaplasmosis, resides in a host cell-derived vacuole. Bacterial proteins that localize to the A. phagocytophilum-occupied vacuole membrane (AVM) are critical host-pathogen interfaces. Of the few bacterial AVM proteins that have been identified, the domains responsible for AVM localization and the host cell pathways that they co-opt are poorly defined. APH0032 is an effector that is expressed and localizes to the AVM late during the infection cycle. Herein, the APH0032 domain that is essential for associating with host cell membranes was mapped. Immunofluorescent labeling of infected cells that had been differentially permeabilized confirmed that APH0032 is exposed on the AVM's cytosolic face, signifying its potential to interface with host cell processes. SUMOylation is the covalent attachment of a member of the small ubiquitin-like modifier (SUMO) family of proteins to lysines in target substrates. Previous work from our laboratory determined that SUMOylation is important for A. phagocytophilum survival and that SUMOylated proteins decorate the AVM. Algorithmic prediction analyses identified APH0032 as a candidate for SUMOylation. Endogenous APH0032 was precipitated from infected cells using a SUMO affinity matrix, confirming that the effector co-opts SUMOylation during infection. APH0032 pronouncedly colocalized with SUMO1, but not SUMO2/3 moieties on the AVM. Ectopic expression of APH0032 in A. phagocytophilum infected host cells significantly boosted the bacterial load. This study delineates the first domain of any Anaplasmataceae protein that is essential for associating with the pathogen-occupied vacuole membrane, demonstrates the importance of APH0032 to infection, and identifies it as the second A. phagocytophilum effector that co-opts SUMOylation, thus underscoring the relevance of this post-translational modification to infection.

Summary 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).

Immune-mediated diseases affect millions of people worldwide with an economic impact measured in the billions of dollars. Mesenchymal stem cells (MSCs) are being investigated in the treatment of certain immune mediated diseases, but their application in the treatment of the majority of these disorders remains largely unexplored. Keratoconjunctivitis sicca can occur as a result of progressive immune-mediated destruction of lacrimal tissue in dogs and humans, and immune-mediated joint disease is common to both species. In dogs, allogeneic MSC engraftment and migration have yet to be investigated in vivo in the context of repeated injections.With these aims in mind, the engraftment of allogeneic canine MSCs after an injection into the periocular and intra-articular regions was followed in vivo using magnetic resonance and fluorescent imaging.The cells were shown to be resident near the site of the injection for a minimum of 2 weeks. Analysis of 61 tissues demonstrated preferential migration and subsequent engraftment of MSCs in the thymus as well as the gastrointestinal tract. These results also detail a novel in vivo imaging technique and demonstrate the differential spatial distribution of MSCs after migration away from the sites of local delivery.The active engraftment of the MSCs in combination with their previously documented immunomodulatory capabilities suggests the potential for therapeutic benefit in using MSCs for the treatment of periocular and joint diseases with immune involvement.

Mesenchymal stem cells (MSCs) from adult and neonatal tissues are intensively investigated for their use in regenerative medicine. The purpose of this study was to compare the onset of replicative senescence in MSCs isolated from equine bone marrow (BMSC), adipose tissue (ASC), and umbilical cord tissue (UCMSC). MSC proliferation (cell doubling), senescence-associated β-galactosidase staining, telomere length, Sox-2, and lineage-specific marker expression were assessed for MSCs harvested from tissues of 4 different donors. The results show that before senescence ensued, all cell types proliferated at ∼1 day/cell doubling. BMSCs significantly increased population doubling rate by passage 10 and ceased proliferation after a little30 total population doublings, whereas UCMSCs and ASCs achieved about 60 to 80 total population doublings. UCMSC and ASCs showed marked β-galactosidase staining after ∼70 population doublings, whereas BMSCs stained positive by ∼30 population doublings. The onset of senescence was associated with a significant reduction in telomere length averaging 10.2 kbp at passage 3 and 4.5 kbp in senescent cultures. MSCs stained intensively for osteonectin at senescence compared with earlier passages, whereas vimentin and low levels of smooth muscle actin were consistently expressed. Sox-2 gene expression was consistently noted in all 3 MSC types. In conclusion, equine BMSCs appear to senesce much earlier than ASCs and UCMSCs. These results demonstrate the limited passage numbers of subcultured BMSCs available for use in research and tissue engineering and suggest that adipose tissue and umbilical cord tissue may be preferable for tissue banking purposes.

Reasons for performing study: Mesenchymal stem cells (MSCs) are commonly injected intralesionally for treatment of soft tissue injuries in the horse. Alternative routes of administration would be beneficial for treatment of lesions that cannot be accessed directly or to limit needle-induced iatrogenic damage to the surrounding tissue. Objectives: The purpose of our study was to evaluate MSC distribution after intra-arterial (IA) and intravenous (IV) regional limb perfusions (RLP) using scintigraphy. We hypothesised that MSCs would persist in the distal limb after tourniquet removal and that both techniques would lead to diffuse MSC distribution. Methods: Six horses were used in the study. MSCs were labelled with hexamethyl propylene amine oxime (HMPAO) and technetium-99m. RLP was performed through the median artery of one forelimb and the cephalic vein of the opposite limb under general anaesthesia. The tourniquet was left in place for 45 min. Scintigraphic images were obtained at 0, 45, 75 min, 6 h and 24 h post injection. Results: Distribution of labelled MSCs through the entire distal limb was achieved with all 6 IA RLP, but 3 out of 6 IV RLP showed poor or absent uptake distal to the metacarpus. Mesenchymal stem cell persistence was 39% (3060%) and 28% (1450%) (median [minimummaximum]) at 6 h for IA and IV RLP, respectively. Severe arterial thrombosis occurred in one horse after IA RLP. Conclusions: Both IA and IV RLP of the distal limb result in MSC persistence in perfused tissues. The IA perfusion resulted in more reliable cell distribution to the pastern and foot area. Potential relevance: Regional limb perfusion of MSCs might be used in cases where intralesional injection is not possible or in order to avoid iatrogenic needle damage. Further work is needed to assess the safety of IA RLP before its clinical use.

Background The therapeutic use of bone marrow-derived mononuclear cells (MNCs) and mesenchymal stem cells for the treatment of soft tissue and orthopedic injuries in equine patients is expanding. After collection, bone marrow must be reduced in volume and depleted of RBCs for immediate therapeutic use or to prepare cells for culture or cryopreservation and storage. The MarrowXpress (MXP) System is an automated, closed, sterile system designed to process human bone marrow samples. Objectives The purpose of this study was to evaluate the capacity of the MXP System to process equine bone marrow to reduce volume, deplete RBCs, and enhance recovery of MNCs. Methods Bone marrow was collected from 47 horses into 2 60-mL syringes containing heparin and processed using the MXP System. HCT, total nucleated cell (TNC) count, and MNC count were obtained for each sample before and after processing using an Advia 120 hematology analyzer. Volume reduction, RBC depletion, and recovery of TNCs and MNCs were calculated. Results For equine bone marrow samples, mean values were 73.2% for RBC depletion and 78.0% for volume reduction. TNC count before processing was 2.5 ± 1.2 × 107 and after processing was significantly higher at 7.8 ± 3.3 × 107 (P

The development of an allogeneic mesenchymal stem cell (MSC) product to treat equine disorders would be useful; however, there are limited in vivo safety data for horses. We hypothesized that the injection of self (autologous) and non-self (related allogeneic or allogeneic) MSC would not elicit significant alterations in physical examination, gait or synovial fluid parameters when injected into the joints of healthy horses.Sixteen healthy horses were used in this study. Group 1 consisted of foals (n = 6), group 2 consisted of their dams (n = 5) and group 3 consisted of half-siblings (n = 5) to group 1 foals. Prior to injection, MSC were phenotyped. Placentally derived MSC were injected into contralateral joints and MSC diluent was injected into a separate joint (control). An examination, including lameness evaluation and synovial fluid analysis, was performed at 0, 24, 48 and 72 h post-injection.MSC were major histocompatibility complex (MHC) I positive, MHC II negative and CD86 negative. Injection of allogeneic MSC did not elicit a systemic response. Local responses such as joint swelling or lameness were minimal and variable. Intra-articular MSC injection elicited marked inflammation within the synovial fluid (as measured by nucleated cell count, neutrophil number and total protein concentration). However, there were no significant differences between the degree and type of inflammation elicited by self and non-self-MSC.The healthy equine joint responds similarly to a single intra-articular injection of autologous and allogeneic MSC. This pre-clinical safety study is an important first step in the development of equine allogeneic stem cell therapies.

This article focuses on the emerging field of equine regenerative medicine with an emphasis on the use of mesenchymal stem cells (MSCs) for orthopedic diseases. We detail laboratory procedures and protocols for tissue handling and MSC isolation, characterization, expansion, and cryopreservation from bone marrow, fat, and placental tissues. We provide an overview of current clinical uses for equine MSCs and how MSCs function to heal tissues. Current laboratory practices in equine regenerative medicine mirror those in the human field. However, the translational use of autologous and allogeneic MSCs for patient therapy far exceeds what is currently permitted in human medicine. © 2011 Elsevier Inc.

Background: Recognition of different cytologic patterns in lymph nodes (LNs) from dogs with canine monocytic ehrlichiosis (CME) and noninfectious causes of lymphoid reactivity may have diagnostic utility. Objectives: The aims of the present study were to compare cytologic patterns in LNs of dogs with different phases of CME, to investigate the association of cytologic pattern and presence of Ehrlichia spp. morulae, and to compare patterns of lymphoid reactivity between dogs with CME and those with noninfectious causes of lymphoid hyperplasia. Methods: Cytologic preparations of LNs from 35 dogs with nonmyelosuppressive CME (group A), 16 dogs with myelosuppressive CME (group B), 26 dogs with noninfectious diseases (group C), and 15 healthy dogs (group D) were evaluated. Percentages of lymphocyte types, plasma cells, macrophages, neutrophils, and eosinophils were determined. Samples from dogs in groups A and B were evaluated for the presence of morulae. Results: Cytologic abnormalities in LNs were recorded in 54% of dogs in group A, 88% in group B, 39% in group C, and 0% in group D and were more frequent (P=.02) in dogs with myelosuppressive CME than those with nonmyelosuppressive CME. Plasma cell hyperplasia was more frequent in CME than in noninfectious diseases (P=.03). An association between the presence of cytologic abnormalities and morulae in group A dogs was not found. Conclusions: Dogs with myelosuppressive CME have more lymphoid cytologic abnormalities than dogs with nonmyelosuppressive CME. LN plasmacytosis is the major pattern of lymphadenopathy in dogs with CME and is found more frequently in dogs with CME than in dogs with noninfectious causes of lymphadenopathy.

Thrombelastograph analyzers are point-of-care hemostatic analyzers that provide global assessment of the hemostatic process. Thrombelastography (TEG) detects and provides a continuous recording of the changes in the viscoelastic properties of whole blood from initial clot formation through fibinolysis. TEG has been validated for use in dogs, horses, and cats. Hemostasis research using TEG has focused on test validation, alterations of TEG tracings in animals with naturally occurring diseases, and the use of TEG for monitoring various therapeutic modalities. This article reviews TEG methodology and terminology, including potential sources of preanalytical and analytical errors, the correlation between TEG and other routine hemostatic assays, and current clinical applications of TEG, with emphasis on veterinary medical practice. Data suggest that TEG may be a sensitive and useful adjunctive tool for evaluating an animal with an underlying coagulopathy, including hypercoagulability and hypocoagulability. Additional prospective studies are needed to (1) correlate TEG tracing patterns with a clinical predisposition for bleeding or thrombosis in various disease states and (2) determine whether monitoring and treating hemostatic disorders based on TEG tracings improve clinical outcome.

Objective—To evaluate N-hydroxysuccinimide (NHS)-biotin labeling of equine RBCs and determine posttransfusion survival of autologous equine RBCs stored in citrate phosphate dextrose adenine-1 (CPDA-1) for 0, 1, 14, and 28 days. Animals—13 healthy adult Thoroughbreds. Procedures—Serial dilutions of biotin and streptavidin-phycoerythrin (PE) were evaluated in vitro in blood collected from 3 horses. One horse was used to determine RBC distribution and recovery. Twelve horses were allocated to 4 groups for in vivo experiments in which blood was collected into CPDA-1. Blood was labeled with biotin and reinfused or stored at 4°C for 1, 14, or 28 days prior to labeling with NHS-biotin and reinfusion. Posttransfusion blood samples were collected 15 minutes and 1, 2, 3, 5, 7, 14, 21, 28, and 35 days after reinfusion. Biotin-labeled RBCs were detected via flow cytometry by use of streptavidin-PE. Posttransfusion lifespan of RBCs and RBC half-life were determined. Results—Optimal biotin concentration was 0.04 pg of biotin/RBC, and the optimal streptavidin-PE ratio was 1.2 μg of streptavidin-PE/1 × 106 RBCs. Posttransfusion lifespan of autologous RBCs was 99, 89, 66, and 59 days after storage for 0, 1, 14, and 28 days, respectively. Storage did not result in significant alterations in RBC lifespan. Mean posttransfusion RBC half-life was 50, 45, 33, and 29 days for 0, 1, 14, and 28 days of storage, respectively. Conclusions and Clinical Relevance—Biotin can be used to label equine RBCs for RBC survival studies. Posttransfusion survival of equine autologous RBCs was greater than previously reported.