Intravenous injection of allogenic canine mesenchymal stem cells in 40 client-owned dogs: a safety assessment in veterinary clinical trials.

BACKGROUND: The aim of this study was to evaluate the adverse effects of allogeneic mesenchymal stem cells (MSCs) transplanted via intravenous infusion in dogs and examine their safety. We performed a retrospective analysis of various clinical assessments, including physical examination, blood tests, and radiographs, and monitored the formation of neoplasms during a 6-month follow-up period in 40 client-owned dogs that received intravenous infusion of adipose tissue-derived MSCs (AT-MSCs) for the treatment of various underlying diseases between 2012 and 2018. RESULTS: No significant adverse effects of MSC therapy were detected by clinical assessment, blood tests, or radiographic examination in the 6-month follow-up period after the first MSC treatment. Additionally no new neoplasms were observed during this period. CONCLUSIONS: To our knowledge, this study is the first to evaluate the safety aspects (≥ 6 months) associated with intravenous allogeneic AT-MSC infusion. These results suggest that allogenic AT-MSC infusion could be a useful and relatively safe therapeutic approach in canines.

Deferoxamine preconditioning of canine stem cell derived extracellular vesicles alleviates inflammation in an EAE mouse model through STAT3 regulation.

Extracellular vesicles (EVs) from mesenchymal stem cells (MSCs), specifically those preconditioned with deferoxamine (DFO) in canine adipose tissue-derived MSCs (cAT-MSCs), were explored for treating autoimmune diseases. This study assessed the effects of DFO-preconditioned EVs (EVDFO) in an experimental autoimmune encephalomyelitis (EAE) mouse model. cAT-MSCs were treated with DFO for 48 h, after which EVs were isolated. EAE mice received intranasal EV or EVDFO treatments and were euthanized following histopathologic analysis; RNA and protein expression levels were measured. Histologically, EV and EVDFO groups showed a significant reduction in inflammatory cell infiltration and demyelination. Immunofluorescence revealed increased CD206 and Foxp3 expression, indicating elevated M2 macrophages and regulatory T (Treg) cells, particularly in the EVDFO group. Treg cells also notably increased in the spleen of EVDFO -treated mice. STAT3 and pSTAT3 proteins were upregulated in the EAE groups compared to the naïve group. However, following EV treatment, STAT3 expression decreased compared to the EAE group, whereas pSTAT3 expression was similar in both the EV and EAE groups. In conclusion, EVDFO treatment resulted in reduced STAT3 expression, suggesting its role in T cell regulation and the potential of EVDFO in modulating the STAT3 pathway for reducing inflammation more effectively than non-preconditioned EVs.

Romiplostim for treatment of thrombocytopenia in dogs: A retrospective assessment and clinical outcomes.

BACKGROUND: Romiplostim, a thrombopoietin analog, is commonly used to treat immune-mediated thrombocytopenia (ITP) in humans, but its use in dogs remains limited. OBJECTIVES: Evaluate the effects and adverse events of romiplostim administration in dogs with thrombocytopenia caused by various underlying diseases. ANIMALS: Forty-two client-owned dogs with naturally occurring thrombocytopenia at 2 referral animal hospitals. METHODS: Retrospective, multi-institutional analysis to evaluate the outcomes of romiplostim treatment in dogs. RESULTS: Among the dogs treated with romiplostim, 27 experienced an increase in platelet count and 26 maintained a platelet count within the reference range. Platelet count improvement was observed in various conditions: primary ITP (90%, n = 18/20), pancytopenia of unknown etiology (42.9%, n = 3/7), chemotherapy-induced thrombocytopenia (50%, n = 3/6), babesiosis (100%, n = 1/1), radiotherapy-induced thrombocytopenia (0%, n = 0/1), and disseminated intravascular coagulopathy (33.3%, n = 2/6). The median time for platelet recovery (>50 000/µL) after romiplostim administration was 4 days, and the median time for platelet count normalization was 7 days. Median hospitalization time for the improvement group (I) was 5 days. The survival-to-discharge rates were 85%, 40%, and 28.6% for dogs with primary ITP, secondary thrombocytopenia, and thrombocytopenia of unknown etiology, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Romiplostim is a well-tolerated and promising treatment for primary ITP in dogs, suggesting its potential as a valuable therapeutic option for dogs with thrombocytopenia caused by various underlying conditions. These findings emphasize the need for further research to optimize romiplostim dosing and understand its role in treating secondary thrombocytopenia and pancytopenia of unknown etiology.

Analyzing small RNA sequences from canine stem cell-derived extracellular vesicles primed with TNF-α and IFN-γ and exploring their potential in lung repair.

Acute lung injury is an acute inflammation disorder that disrupts the lung endothelial and epithelial barriers. In this study, we investigated the extracellular vesicles (EVs) obtained via priming inflammatory cytokines such as tumor necrosis factor (TNF)-α and interferon (IFN)-γ on canine adipose mesenchymal stem cells in improving their anti-inflammatory and/or immunosuppressive potential, and/or their ability to alleviate lipopolysaccharide-induced lung injury in vitro. We also explored the correlation between epithelial-to-mesenchymal transition and the inflammatory repressive effect of primed EVs. Using small RNA-Seq, we confirmed that miR-16 and miR-502 significantly increased in EVs from TNF-α and IFN-γ-primed canine adipose mesenchymal stem cells. The pro and anti-inflammatory cytokines were analyzed in a lipopolysaccharide-induced lung injury model and we found that the EV anti-inflammatory effect improved on priming with inflammatory cytokines. EVs obtained from primed stem cells effectively suppress endothelial-to-mesenchymal transition in a lung injury model. Our results suggest a potential therapeutic approach utilizing EVs obtained from adipose mesenchymal stem cells primed with TNF-α and IFN-γ against lung inflammation and endothelial to mesenchymal transition.

The Role of Natural Killer Cells in Oncolytic Virotherapy: Friends or Foes?

Oncolytic virotherapy (OVT) has emerged as a promising cancer immunotherapy, and is capable of potentiating other immunotherapies due to its capacity to increase tumor immunogenicity and to boost host antitumor immunity. Natural killer (NK) cells are a critical cellular component for mediating the antitumor response, but hold a mixed reputation for their role in mediating the therapeutic efficacy of OVT. This review will discuss the pros and cons of how NK cells impact OVT, and how to harness this knowledge for the development of effective strategies that could modulate NK cells to improve OVT-based therapeutic outcomes.

Safety of Gonadal Tissue-Derived Mesenchymal Stem Cell Therapy in Geriatric Dogs with Chronic Disease.

Ensuring the safety of mesenchymal stem cell (MSC) therapy is a fundamental requirement in clinical practice. This study aimed to assess the safety of using gonadal tissue-derived MSCs (n = 10) compared to the commonly utilized adipose tissue-derived MSCs (n = 9) in geriatric dogs with chronic diseases. All participants received allogeneic MSC therapy, and no allergic reactions due to allogeneic cell immunogenicity were noted. Both groups showed no adverse changes in physical exams or hematological parameters before and after therapy. Importantly, there were no instances of tumor formation or growth post-treatment in either group. The findings demonstrated that dogs treated with gonadal tissue-derived MSCs experienced no clinical adverse effects. However, clinical adverse effects were reported in one case of adipose tissue-derived MSC therapy. Despite limitations in monitoring beyond one year and constraints due to a small and diverse patient group, this pioneering study validates the safe use of gonadal tissue-derived MSCs in aged companion animals. It underscores the potential of utilizing tissues from neutering procedures to advance regenerative medicine and expand cell banks and therapy options for companion animals.

Effect of Oligo-Fucoidan, Fucoxanthin, and L-Carnitine on Chronic Kidney Disease in Dogs: A Retrospective Study.

Chronic kidney disease (CKD) commonly occurs in old dogs and cats. Oligo-fucoidan, fucoxanthin, and L-carnitine (OFL) compounds have a variety of reno-protective properties, including anti-inflammatory, anti-oxidative, and anti-fibrotic effects. Because their effects have not been investigated in naturally occurring canine CKD, we examined their reno-protective activities in dog patients with CKD. A total of 50 patients (OFL, n = 28; control, n = 22) were included in the analysis. A significant difference was identified in serum blood urea nitrogen and creatinine concentrations between the control and OFL groups at 6 months. No significant difference in electrolytes was found between the groups. A significant difference was identified in serum creatinine concentration between the control and OFL groups in azotemic (CKD IRIS stage 2-4) at 6 months. The OFL compounds showed a reno-protective effect, consistent with previous animal studies. The OFL combination can potentially delay the progression of canine CKD and be used as an adjuvant therapy.

Pamidronate-induced irreversible symptomatic hypocalcemia in a dog with hypercalcemia after glucocorticoid withdrawal: a case report.

BACKGROUND: Pamidronate is used for the treatment of hypercalcemia. However, a rare but potential adverse event of pamidronate treatment is hypocalcemia. This report describes an unusual case of severe, irreversible hypocalcemia after a single injection of pamidronate for the treatment of hypercalcemia due to glucocorticoid withdrawal in a dog. CASE PRESENTATION: An 11-year-old castrated male Maltese dog presented with anorexia, vomiting, and diarrhea (day 0). The patient had calcinosis cutis throughout the body, calcification of intraabdominal organs, mild azotemia, and severe hypercalcemia. The severe calcification was attributed to long-term glucocorticoid administration, which was discontinued 1 month before presentation. Fluid therapy, diuretics, calcitonin, and a single intravenous injection of pamidronate were used for the treatment of hypercalcemia. On day 14, normocalcemia was achieved, but renal failure occurred. On day 20, severe and irreversible hypocalcemia occurred, and on day 42, the patient was euthanized at the owner's request because of worsened hypocalcemia and renal failure. CONCLUSIONS: Although hypocalcemia is an extremely rare adverse event of bisphosphonate treatment, bisphosphonates like pamidronate can result in potentially life-threatening conditions according to the patient's underlying conditions. Therefore, the patient's condition should be closely monitored and any underlying conditions should be carefully evaluated before initiating the treatment for hypercalcemia using pamidronate.

Blood vessel organoids generated by base editing and harboring single nucleotide variation in Notch3 effectively recapitulate CADASIL-related pathogenesis.

Human blood vessel organoids (hBVOs) offer a promising platform for investigating vascular diseases and identifying therapeutic targets. In this study, we focused on in vitro modeling and therapeutic target finding of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common form of hereditary stroke disorder caused by mutations in the NOTCH3 gene. Despite the identification of these mutations, the underlying pathological mechanism is elusive, and effective therapeutic approaches are lacking. CADASIL primarily affects the blood vessels in the brain, leading to ischemic strokes, migraines, and dementia. By employing CRISPR/Cas9 base-editing technology, we generated human induced pluripotent stem cells (hiPSCs) carrying Notch3 mutations. These mutant hiPSCs were differentiated into hBVOs. The NOTCH3 mutated hBVOs exhibited CADASIL-like pathology, characterized by a reduced vessel diameter and degeneration of mural cells. Furthermore, we observed an accumulation of Notch3 extracellular domain (Notch3ECD), increased apoptosis, and cytoskeletal alterations in the NOTCH3 mutant hBVOs. Notably, treatment with ROCK inhibitors partially restored the disconnection between endothelial cells and mural cells in the mutant hBVOs. These findings shed light on the pathogenesis of CADASIL and highlight the potential of hBVOs for studying and developing therapeutic interventions for this debilitating human vascular disorder.

Effect of the sodium-glucose cotransporter-2 inhibitor, DWP16001, as an add-on therapy to insulin for diabetic dogs: A pilot study.

BACKGROUND: Sodium-glucose cotransporter-2 (SGLT2) inhibitors are a novel class of anti-hyperglycaemic agents. OBJECTIVE: This study aimed to evaluate the safety and the adjuvant glycaemic control effect of an SGLT2 inhibitor, DWP16001, in diabetic dogs receiving insulin treatment. METHODS: Nineteen diabetic dogs receiving insulin treatment (NPH, porcine lente and glargine insulin) were divided into two groups according to dosing frequency: DWP TOD group (n = 10) and DWP SID group (n = 9). In the DWP TOD group, 0.025 mg/kg of DWP16001 was administered once every 3 days, whereas, in the DWP SID group, 0.025 mg/kg of DWP16001 was administered once a day. Food intake was maintained during the trial period. Hypoglycaemia, ketoacidosis or unexpected life-threatening reactions were assessed as adverse effects before and after DWP16001 administration. We compared insulin requirement reduction and blood glucose level control between two groups. RESULTS: No specific adverse effects were observed during the clinical trial, and haematological parameter remained unchanged. Moreover, the fasting glucose levels and daily insulin dose in the DWP TOD group were lower than the pre-administration values, but not significantly different for 8 weeks. Systolic blood pressure, fructosamine and insulin dose decreased significantly in the DWP SID group compared to the DWP TOD group at 8 weeks (p < 0.05) without affecting food consumption. Among these patients, 10 patients were monitored while receiving DWP16001 for 12 months (DWP TOD group n = 5, DWP SID group n = 5). The fasting glucose and fructosamine levels and daily insulin dose were reduced in both groups at 12 months compared with those before receiving DWP16001. CONCLUSION: When DWP16001, an SGLT2 inhibitor, was supplied to dogs with type 1 diabetes, no adverse effects were observed, and it was confirmed that the administered insulin dose can be reduced in controlling blood glucose.

In vitro evaluation of the antitumor activity of axitinib in canine mammary gland tumor cell lines.

BACKGROUND: Axitinib, a potent and selective inhibitor of vascular endothelial growth factor (VEGF) receptor (VEGFR) tyrosine kinase 1,2 and 3, is used in chemotherapy because it inhibits tumor angiogenesis by blocking the VEGF/VEGFR pathway. In veterinary medicine, attempts have been made to apply tyrosine kinase inhibitors with anti-angiogenic effects to tumor patients, but there are no studies on axitinib in canine mammary gland tumors (MGTs). OBJECTIVES: This study aimed to confirm the antitumor activity of axitinib in canine mammary gland cell lines. METHODS: We treated canine MGT cell lines (CIPp and CIPm) with axitinib and conducted CCK, wound healing, apoptosis, and cell cycle assays. Additionally, we evaluated the expression levels of angiogenesis-associated factors, including VEGFs, PDGF-A, FGF-2, and TGF-ß1, using quantitative real-time polymerase chain reaction. Furthermore, we collected canine peripheral blood mononuclear cells (PBMCs), activated them with concanavalin A (ConA) and lipopolysaccharide (LPS), and then treated them with axitinib to investigate changes in viability. RESULTS: When axitinib was administered to CIPp and CIPm, cell viability significantly decreased at 24, 48, and 72 h (p < 0.001), and migration was markedly reduced (6 h, p < 0.05; 12 h, p < 0.005). The apoptosis rate significantly increased (p < 0.01), and the G2/M phase ratio showed a significant increase (p < 0.001). Additionally, there was no significant change in the viability of canine PBMCs treated with LPS and ConA. CONCLUSION: In this study, we confirmed the antitumor activity of axitinib against canine MGT cell lines. Accordingly, we suggest that axitinib can be applied as a new treatment for patients with canine MGTs.

Anti-cancer effects of DHP107 on canine mammary gland cancer examined through in-vitro and in-vivo mouse xenograft models.

BACKGROUND: Canine mammary gland cancer (CMGC) is a common neoplasm in intact bitches. However, the benefit of adjuvant chemotherapy is unclear. The aim of this study was to investigate the anti-proliferative effects of paclitaxel on CMGC in in-vitro and in-vivo settings. RESULTS: Paclitaxel dose-dependently inhibited viability and induced G2/M phase cell cycle arrest and apoptosis in both primary and metastatic CMGC cell lines (CIPp and CIPm). In animal experiments, the average tumour volume decreased significantly in proportion to the administered oral paclitaxel dose. By examining tumour tissue using a TUNEL assay and immunohistochemical staining with anti-CD31 as a marker of endothelial differentiation, respectively, it was confirmed that oral paclitaxel induced apoptosis and exerted an anti-angiogenetic effect in tumour tissues. Further, downregulation of cyclin D1 in tumour tissues suggested that oral paclitaxel induced cell cycle arrest in tumour tissues in-vivo. CONCLUSIONS: Our results suggest that paclitaxel may have anti-cancer effects on CMGC through cell cycle arrest, induction of apoptosis, and anti-angiogenesis. This study could provide a novel approach to treat CMGC.

Changes in Urinary Exosomal Nuclear Matrix Protein-22 in Dogs With Urothelial Carcinoma: A Pilot Study.

BACKGROUND/AIM: Nuclear matrix protein-22 (NMP-22) is widely used in human medicine as a prognostic and diagnostic tool for urothelial carcinoma (UC). In addition, the use of urinary exosomes as a liquid biopsy tool is emerging for the diagnosis of certain types of cancer in human medicine. This study aimed to investigate the change in urinary exosomal NMP-22 for the diagnosis of UC in dogs. PATIENTS AND METHODS: Among canine patients who visited the veterinary hospital, urine was collected from those whose owners provided consent. A total of 23 dogs (UC group, n=6; control group, n=17) were included in the analysis. After exosomes were isolated from the urine, NMP-22 was measured using enzyme-linked immunosorbent assay. RESULTS: In the UC group, the expression of NMP-22 in urinary exosomes was significantly higher than that in non-UC groups (p<0.0001). CONCLUSION: NMP-22 is significantly increased in exosomes in the urine of dogs diagnosed with UC, suggesting that urinary exosome NMP-22 can be considered as one of the liquid biopsy tools for diagnosing UC in dogs.

Canine adipose tissue-derived mesenchymal stem cell therapy in a dog with renal Fanconi syndrome.

Renal Fanconi syndrome (RFS) affects the proximal tubular resorption in the nephrons. This causes excessive loss of key solutes through the urine. In a canine patient, we successfully managed the renal tubular acidosis and proteinuria caused by RFS via transplantation of canine adipose tissue-derived mesenchymal stem cells (cAT-MSCs). cAT-MSCs were administered ten times at intervals of 2-4 weeks. The post-therapy check-up revealed that the cAT-MSC treatment improved the renal tubular acidosis and proteinuria. Hence, a cAT-MSC transplant may be considered as an adjuvant therapy in veterinary medicine to initiate and maintain relief of RFS-induced acidosis and proteinuria.