COAST Development Group's international consensus guidelines for the treatment of canine osteoarthritis.

This report describes consensus guidelines and recommendations for the treatment of canine osteoarthritis (OA) according to the "Canine OsteoArthritis Staging Tool excluding radiography" (COASTeR) stage of OA, by the COAST Development Group. The recommendations are based on evidence-based medicine and clinical experience and are proposed with international relevance in mind. The aim is to provide veterinarians with a practical reference to consolidated information and to support the development of patient-specific OA management protocols and informed treatment choices based on the stage of OA.

Pentosan polysulfate sodium promotes redifferentiation to the original phenotype in micromass-cultured canine articular chondrocytes and exerts molecular weight-dependent effects.

Pentosan polysulfate sodium (PPS) is a heparin-like polysaccharide that is applied as a therapeutic treatment for osteoarthritis (OA) in animals. This study investigated the efficacy of different molecular weights PPS (1,500-7,000 Da) on the phenotype regulatory and chondrogenic properties of canine articular chondrocytes. The cytotoxicity of PPS on chondrocytes was assessed using flow cytometry and 3-(4,5-dimehylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. After 72 hr of exposure, PPS did not induce chondrocyte apoptosis, regardless of molecular weight. In addition, chondrogenic properties were determined according to the mRNA and protein levels in micromass-cultured chondrocytes. Quantitative polymerase chain reaction analysis confirmed that PPS promotes a chondrogenic phenotype in chondrocytes in a molecular weight-dependent manner, with significant upregulation of collagen type II alpha 1 chain, aggrecan, and SRY-box transcription factor 9 (SOX9) mRNA levels relative to those in the control. However, the collagen type I alpha 2 chain mRNA level simultaneously increased after 7,000 Da PPS treatment. PPS exposure also increased collagen type II and SOX9 protein production in a molecular weight-dependent manner and inhibited Akt phosphorylation in chondrocytes. Alcian blue staining indicated that PPS treatment enhanced proteoglycan deposition in micromass cultures, with stronger effects observed in 5,000 and 7,000 Da groups. Overall, these results indicate that PPS exerts protective effects on the chondrocyte phenotype and may represent a potential therapeutic target for OA treatment. Increasing the molecular weight of PPS could enhance these anabolic effects.

In vitro chondrotoxicity of bupivacaine, levobupivacaine and ropivacaine and their effects on caspase activity in cultured canine articular chondrocytes.

Bupivacaine, levobupivacaine and ropivacaine are potent, long acting, amide-type local anesthetics that have several clinical applications including intra-articular administration. The objectives of this study were to evaluate their in vitro effects on cell viability and caspase activity to elucidate whether they activate the extrinsic or intrinsic pathways of apoptosis in canine articular chondrocytes. Chondrocytes in monolayer culture were treated with culture medium as the control, or with 0.062% (0.62 mg/mL) bupivacaine, 0.062% levobupivacaine, and 0.062% ropivacaine for 24 hr. Cell viability was evaluated using the live/dead, 3-(4,5-dimehylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), and Cell Counting Kit-8 (CCK-8) assays. Evaluation of caspase-3, caspase-8, and caspase-9 activity was performed using colorimetric assays. The MTT and CCK-8 assays were used to evaluate the effect of caspase inhibitors on local anesthetic chondrotoxicity. All three local anesthetics decreased chondrocyte viability after 24 hr (P<0.001). Apoptosis was induced through both the extrinsic and intrinsic pathways. Bupivacaine increased caspase-3, caspase-8, and caspase-9 activity (P<0.001). Levobupivacaine increased caspase-3 (P=0.03) while ropivacaine did not significantly upregulate activity for all three caspases. Caspase inhibition did not suppress bupivacaine chondrotoxicity whereas inhibition of caspase-8 and caspase-9 decreased ropivacaine chondrotoxicity and mildly attenuated levobupivacaine chondrotoxicity. In summary, the level of chondrotoxicity, the type of caspase activated, the level of caspase activation, and the response to caspase inhibitors was dependent on the type of local anesthetic. Therefore, ropivacaine may be a safer choice for intra-articular administration compared to levobupivacaine and bupivacaine.

Regulation of programmed death ligand 1 expression by interferon-γ and tumour necrosis factor-α in canine tumour cell lines.

Expression of programmed death ligand 1 (PD-L1) on tumour cells provides an immune evasion mechanism by inducing suppression of cytotoxic T cells. Various regulatory mechanisms of PD-L1 expression have been described in human tumours, however, little is known in canine tumours. To investigate whether inflammatory signalling is involved in PD-L1 regulation in canine tumours, the effects of interferon (IFN)-γ and tumour necrosis factor (TNF)-α treatment were examined in canine malignant melanoma cell lines (CMeC and LMeC) and an osteosarcoma cell line (HMPOS). The protein level of PD-L1 expression was upregulated by IFN-γ and TNF-α stimulation. Upon IFN-γ stimulation, all cell lines showed an increase in expression of PD-L1, signal transducer and activator of transcription (STAT)1, STAT3 and genes regulated by STAT activation. Upregulated expression of these genes was suppressed by the addition of a JAK inhibitor, oclacitinib. Contrastingly, upon TNF-α stimulation, all cell lines exhibited higher gene expression of the nuclear factor kappa B (NF-κB) gene RELA and genes regulated by NF-κB activation, whereas expression of PD-L1 was upregulated in LMeC only. Upregulated expression of these genes was suppressed by the addition of an NF-κB inhibitor, BAY 11-7082. The expression level of cell surface PD-L1 induced by IFN-γ and TNF-α treatment was reduced by oclacitinib and BAY 11-7082, respectively, indicating that upregulation of PD-L1 expression by IFN-γ and TNF-α stimulation is regulated via the JAK-STAT and NF-κB signalling pathways, respectively. These results provide insights into the role of inflammatory signalling in PD-L1 regulation in canine tumours.

Pentosan polysulfate regulates hepcidin expression in native Mongolian horses.

The aim of this study was to investigate the anti-hepcidin effect of pentosan polysulfate (PPS) in Mongolian horses. Twenty-six healthy horses were randomly allocated in to two-groups; one group was treated with a PPS once a week for 4-weeks while another group keeping as placebo. Blood samples at day 0 (D0), before race (BR; day 28) and after race (AR; day 28) were analyzed for serum biochemistry, hepcidin and iron concentrations. Significant reduction of hepcidin was observed at AR in PPS group when compared with BR placebo (P<0.05) and AR placebo (P<0.01). Mean hepcidin concentration difference of D0-BR and BR-AR in PPS was greater than the placebo whereas the iron concentration difference is reduced compared to placebo. Results indicate a novel therapeutic application of PPS as an anti-hepcidin compound to control hepcidin in horses while emphasizing further molecular studies.

Pentosan polysulfate regulates hepcidin 1-facilitated formation and function of osteoclast derived from canine bone marrow.

Hepcidin which is the crucial regulator of iron homeostasis, produced in the liver in response to anemia, hypoxia, or inflammation. Recent studies have suggested that hepcidin and iron metabolism are involved in osteoporosis by inhibiting osteoblast function and promoting osteoclastogenesis. Pentosan polysulfate (PPS) is a heparin analogue and promising novel therapeutic for osteoarthritis (OA). This study was undertaken to determine whether PPS inhibits hepcidin-facilitated osteoclast (OC) differentiation and iron overload. Canine (n = 3) bone marrow mononuclear cells were differentiated to OC by macrophage colony-stimulating factor and receptor-activator of nuclear factor kappaB ligand with the treatment of hepcidin1 (200, 400, 800, 1200 nmol/L) and PPS (1, 5, 10, 20, 40 µg/mL). Differentiation and function of OC were accessed using tartrate-resistant acid phosphate staining and bone resorption assay while monitoring ferroportin1 (FPN1) and iron concentration by immunocytochemistry. Gene expression of OC for cathepsin K (CTK), matrix metallopeptidase-9, nuclear factor of activated-T-cells cytoplasmic 1 and FPN1 was examined. Hepcidin1 showed significant enhancement of OC number at 800 nmol/L (p<0.01). PPS impeded hepcidin-facilitated OC at 1, 5 and 10 µg/mL and reduction of resorption pits at 5 and 10 µg/mL (p< 0.01). All OC specific genes were downregulated with PPS, specifically in significant manner with CTK at higher concentrations. However, heparin induced FPN1 internalization and degradation was inhibited at higher concentrations of PPS while restoring iron-releasing capability of OC. We demonstrate for the first time that PPS is a novel-inhibitor of hepcidin-facilitated OC formation/function which might be beneficial for treatment of OA and osteoporosis.

Antitumor activity of the dual PI3K/mTOR inhibitor gedatolisib and the involvement of ABCB1 in gedatolisib resistance in canine tumor cells.

The phosphatidylinositol 3­kinase/mammalian target of rapamycin (PI3K/mTOR) signaling pathway is a therapeutic target for various types of human tumors, and dual PI3K/mTOR inhibitors demonstrate antitumor activities in both preclinical and clinical studies. However, resistance mechanisms limit their abilities. As the molecular mechanisms involved in the cellular resistance are not clear in any canine tumors, an understanding of resistance mechanisms would support the potential use of dual PI3K/mTOR inhibitors in canine tumors. The antitumor activity of gedatolisib on cell viability, protein phosphorylation, and cell cycle distribution was assessed using 12 canine tumor cell lines from 6 types of tumors. In addition, the molecular determinants involved in the cellular sensitivity to gedatolisib were explored by investigating the involvement of serum­and­glucocorticoid­induced kinase 1 (SGK1), PIK3CA, and ATP­binding cassette, subfamily B, member 1 (ABCB1). The results demonstrated that gedatolisib decreased cell viability in all cell lines, with IC50 values <1 µM in 10 of the 12 lines. Gedatolisib inhibited Akt and mTOR complex 1 substrate phosphorylation and induced G0/G1 cell cycle arrest. However, certain cell lines with higher IC50 values were more resistant to these effects. These cell lines exhibited higher ABCB1 activity and the ABCB1 inhibitor cyclosporin A enhanced the decrease of cell viability caused by gedatolisib. SGK1 overexpression did not confer resistance to gedatolisib. The mutations of E545K and H1047R in PIK3CA were not observed. The present results indicated that gedatolisib decreased cell viability in canine tumor cell lines and ABCB1 played an important role in gedatolisib resistance, supporting the potential use of gedatolisib for canine tumors.

Visualizing the cancer stem-like properties of canine tumour cells with low proteasome activity.

Cancer stem-like cells (CSCs) cause treatment failure in various tumours; however, establishing CSC-targeted therapies has been hampered by difficulties in the identification and isolation of this small sub-population of cells. Recent studies have revealed that tumour cells with low proteasome activity display a CSC phenotype that can be utilized to image CSCs in canines. This study visualizes and reveals the CSC-like properties of tumour cells with low proteasome activity in HMPOS (osteosarcoma) and MegTCC (transitional cell carcinoma), which are canine cell lines. The parent cells were genetically engineered to express ZsGreen1, a fluorescent protein connected to the carboxyl-terminal degron of canine ornithine decarboxylase that accumulates with low proteasome activity (ZsG+ cells). ZsG+ cells were imaged and the mode of action of this system was confirmed using a proteasome inhibitor (MG-132), which increased the ZsGreen1 fluorescence intensity. The CSC-like properties of ZsG+ cells were evaluated on the basis of cell divisions, cell cycle, the expression of CSC markers and tumourigenicity. ZsG+ cells underwent asymmetric divisions and had a low percentage of G0/G1 phase cells; moreover, ZsG+ cells expressed CSC markers such as CD133 and showed a large tumourigenic capability. In histopathological analysis, ZsG+ cells were widely distributed in the tumour samples derived from ZsG+ cells and in the proliferative regions of the tumours. The results of this study indicate that visualized canine tumour cells with low proteasome activity have a CSC-like phenotype and that this visualization system can be utilized to identify and isolate canine CSCs.

Postoperative improvement of megaesophagus and esophageal motility in a 19-day-old puppy with gastroesophageal intussusception.

Canine gastroesophageal intussusception (GEI) is a rare and life-threatening condition that requires prompt diagnosis and treatment. A 19-day-old Siberian Husky with a 4-day history of regurgitation was diagnosed with GEI based on the findings of computed tomography (CT) performed without anesthesia. Endoscopic reduction of intussusception was impossible; thus, surgical reduction by traction of the duodenum was performed. CT revealed improvement of megaesophagus 82 days postoperatively. Eleven months postoperatively, fluoroscopy showed recovery to nearly normal esophageal motility. Two years postoperatively, no clinical signs were reported. CT is useful to diagnose GEI in neonate puppies with poor abdominal fat and to assess the gastric edema and the anatomical association of stomach with other organs. Fluoroscopy is helpful for evaluating postoperative esophageal motility.

Safety and efficacy of a nonmyeloablative pretransplant conditioning regimen using total lymphoid irradiation with volumetric modulated arc therapy in healthy dogs: A pilot study.

Allogeneic hematopoietic cell transplantation (HCT) has been an effective treatment for human patients with haematological malignancies (Baron & Storb, 2006; Bair et al., 2020; Copelan et al., 2019). However, the optimal pretransplant conditioning treatment is unclear in canine allogeneic HCT. This pilot study aimed to evaluate the safety and efficacy of total lymphoid irradiation (TLI) with volumetric modulated arc therapy (VMAT) for a nonmyeloablative HCT conditioning. Six healthy dogs were treated with 8 or 12 Gy TLI using VMAT. Haematological and physical changes were recorded over 8 weeks. To assess the effect of peripheral lymphocyte condition, lymphocyte subset and proliferative ability were examined. At the end of the experiment, necropsy was performed. All dogs showed mild-to-moderate neutropenia and thrombocytopenia, and these haematological changes resolved spontaneously. One dog treated with 8 Gy TLI developed transient cutaneous infection. No major complication was seen in the other seven dogs. Myelocytes and erythroblast cytopenia of bone marrow were detected in two dogs treated with 12 Gy TLI. This study is the first report of TLI using VMAT in dogs, and results suggest that this regimen is a feasible nonmyeloablative treatment.

Unrecognized difficult airway management during anesthesia in two brachycephalic dogs with narrow cricoid cartilage.

Difficulty in airway management during anesthesia was noted in a 10-year-old, castrated, male Pekingese dog and a 13-year-old male French Bulldog. They showed strong resistance during tracheal tube insertion through the subglottic lumen. Therefore, the airway was secured by using a small endotracheal tube or supraglottic airway device. Computed tomography scan revealed a markedly narrower vertical dimension of the cricoid cartilage compared to that seen in common brachycephalic breeds. Posterior glottis was relatively more accessible for translaryngeal intubation in the present cases. Our findings showed that brachycephalic airway syndrome may be associated with narrow cricoid cartilage. To the best of our knowledge, this is the first clinical case report of airway management during anesthesia in dogs with narrow cricoid cartilage.

An Insight into the Role of Apoptosis and Autophagy in Nitric Oxide-Induced Articular Chondrocyte Cell Death.

OBJECTIVE: To investigate the role and characterize the molecular mechanisms regulating apoptosis and autophagy in nitric oxide (NO)-induced chondrocyte cell death. DESIGN: Cell apoptosis and autophagy were evaluated in chondrocytes treated with sodium nitroprusside (SNP) combined with the presence or absence of interleukin-1 beta (IL-1ß) and nutrient-deprived conditions. The concentration of nitrite was determined by Griess reaction. Activation of apoptosis and autophagy were determined by immunocytochemistry, Western blot, and quantitative real-time polymerase chain reaction (qPCR) analysis. Flow cytometry and MTT assay were used to assess cell viability. RESULTS: Cotreatment of chondrocytes with SNP and IL-1ß under nutrient-deprived condition potentially enhanced the effect of NO-induced cell death. Immunocytochemistry, Western blot, and qPCR analysis indicated that treatment of chondrocytes with SNP significantly reduced autophagic activity, autophagic flux, and multiple autophagy-related (Atg) genes expression. These findings were associated with an increase in ERK, Akt, and mTOR phosphorylation, whereas autophagy induction through mTOR/p70S6K inhibition by rapamycin significantly suppressed NO-induced cell apoptosis. Furthermore, the cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-3 activation in response to apoptosis was weakly detected. These results corresponded with a significant increase in apoptosis-inducing factor (AIF) expression, suggesting the involvement of the caspase-independent pathway. CONCLUSIONS: These results demonstrate that in chondrocyte cultures with cells induced into an osteoarthritis state, NO inhibits autophagy and induces chondrocyte apoptosis mainly, but not completely through the caspase-independent pathway. Our data suggest that autophagy is a protective mechanism in the pathogenesis of osteoarthritis and could be proposed as a therapeutic target for degenerative joint diseases.

Low­dose trametinib and Bcl­xL antagonist have a specific antitumor effect in KRAS­mutated colorectal cancer cells.

KRAS­mutant colorectal cancer (CRC) is a highly malignant cancer with a poor prognosis, however specific therapies targeting KRAS mutations do not yet exist. Anti­epidermal growth factor receptor (EGFR) agents, including cetuximab and panitumumab, are effective for the treatment of certain patients with CRC. However, these anti­EGFR treatments have no effect on KRAS­mutant CRC. Therefore, new therapeutic strategies targeting KRAS­mutant CRC are urgently needed. To clarify the direct effect of KRAS gene mutations, the present study transduced mutant forms of the KRAS gene (G12D, G12V and G13D) into CACO­2 cells. A drug­screening system (Mix Culture assay) was then applied, revealing that the cells were most sensitive to the MEK inhibitor trametinib among tested drugs, Cetuximab, Panitumumab, Regorafenib, Vemurafenib, BEZ­235 and Palbociclib. Trametinib suppressed phosphorylated ERK (p­ERK) expression and inhibited the proliferation of KRAS­mutant CACO­2 cells. However, low­dose treatment with trametinib also increased the expression of the anti­apoptotic protein Bcl­xL in a dose­dependent manner, leading to drug resistance. To overcome the resistance of KRAS­mutant CRC to apoptosis, the combination of trametinib and the Bcl­xL antagonist ABT263 was assessed by in vitro and in vivo experiments. Compared with the effects of low­dose trametinib monotherapy, combination treatment with ABT263 had a synergistic effect on apoptosis in mutant KRAS transductants in vitro. Furthermore, in vivo combination therapy using low­dose trametinib and ABT263 against a KRAS­mutant (G12V) xenograft synergistically suppressed growth, with an increase in apoptosis compared with the effects of trametinib monotherapy. These data suggest that a low dose of trametinib (10 nM), rather than the usual dose of 100 nM, in combination with ABT263 can overcome the resistance to apoptosis induced by Bcl­xL expression, which occurs concurrently with p­ERK suppression in KRAS­mutant cells. This strategy may represent a promising new approach for treating KRAS­mutant CRC.

Effects of pentosan polysulfate on cell proliferation, cell cycle progression and cyclin-dependent kinases expression in canine articular chondrocytes.

Pentosan polysulfate (PPS) is a semi-synthetic sulfated polysaccharide compound which has been shown the benefits on therapeutic treatment for osteoarthritis (OA) and has been proposed as a disease modifying osteoarthritis drugs (DMOADs). This study investigated the effects of PPS on cell proliferation, particularly in cell cycle modulation and phenotype promotion of canine articular chondrocytes (AC). Canine AC were treated with PPS (0-80 µg/ml) for 24, 48 and 72 hr. The effect of PPS on cell viability, cell proliferation and cell cycle distribution were analyzed by MTT assay, DNA quantification and flow cytometry. Chondrocyte phenotype was analyzed by quantitative real-time PCR (qPCR) and glycosaminoglycan (GAG) quantification. PPS significantly reduced AC proliferation through cell cycle modulation particularly by maintaining a significantly higher proportion of chondrocytes in the G1 phase and a significantly lower proportion in the S phase of the cell cycle in a concentration- and time-dependent manner. While the proportion of chondrocytes in G1 phase corresponded with the significant downregulation of cyclin-dependent kinase (CDK) 1 and 4. Furthermore, the study confirms that PPS promotes a chondrogenic phenotype of AC through significant upregulation of collagen type II (Col2A1) mRNA and GAG synthesis. The effect of PPS on the inhibition of chondrocyte proliferation while promoting a chondrocyte phenotype could be beneficial in the early stages of OA treatment, which transient increase in proliferative activity of chondrocytes with subsequent phenotypic shift and less productive in an essential component of extracellular matrix (ECM) is observed.

Alterations in characteristics of canine articular chondrocytes in non-passaged long-term monolayer culture: Matter of differentiation, dedifferentiation and redifferentiation.

This study investigated the effects of culture time on phenotype stability of canine articular chondrocytes (CACs) in non-passaged long-term monolayer culture. Third passage (P3) CACs isolated from four cartilage samples were seeded at three different initial seeding densities (0.2 × 104, 1.0 × 104 and 5.0 × 104 cells/cm2) and maintained in monolayer condition up to 8 weeks without undergoing subculture after confluence. The characteristic changes of chondrocytes during the culture period were evaluated based on the cell morphology, cell proliferation, glycosaminoglycans (GAGs) content, DNA quantification, mRNA expression and ultrastructure of chondrocytes. Chondrocytes maintained under post-confluence condition exhibited a capability to grow and proliferate up to 4 weeks. Alcian blue staining and Dimethylmethylene blue (DMMB) assay revealed that the extracellular matrix (ECM) synthesis was increased in a time-dependent manner from 2 to 8 weeks. The chondrocyte mRNA expression profile was dramatically affected by prolonged culture time, with a significant downregulation of collagen type I, whereas the expression of collagen type II, aggrecan, Sox9 and matrix metalloproteinase 13 (MMP-13) were significantly upregulated. In addition, transmission electron microscopy (TEM) result indicated dilation of rough endoplasmic reticulum (RER) in these long-term monolayer cultured chondrocytes. These findings demonstrate that the chondrocytes phenotype could be partially redifferentiated through the spontaneous redifferentiation process in long-term cultures using standard culture medium without the addition of chondrogenic supplements or tissue-culture scaffolds.

Outcomes following balloon dilation for management of urethral obstruction secondary to urothelial carcinoma in dogs: 12 cases (2010-2015).

OBJECTIVE: To describe outcomes for dogs that underwent balloon dilation for palliative treatment of urethral obstruction caused by urothelial carcinoma. ANIMALS: 12 client-owned dogs. PROCEDURES: Medical records were searched to identify dogs with urothelial (bladder, urethra, or prostate) carcinoma that underwent balloon dilation for treatment of urethral obstruction between April 2010 and December 2015. Information regarding history, signalment, clinical signs, diagnostic imaging findings, balloon dilation technique, clinical outcomes, complications, and additional treatments was obtained by review of medical records. RESULTS: Improvement in clinical signs of urethral obstruction was observed after the initial dilation procedure for 9 of 12 dogs. Urethral obstruction was known to recur in 5 dogs 48 to 296 days after the initial procedure. Three of these dogs underwent a second dilation procedure, with clinical improvement in all 3 dogs for 41 to 70 days. One of 2 dogs that had a third procedure after the second reobstruction had clinical improvement in urinary tract signs until subsequent death from metastatic disease 22 days later. Complications included hematuria, urinary incontinence, and dysuria; these resolved within a few days after treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Urethral balloon dilation was a minimally invasive procedure that provided relief of urethral obstruction from urothelial carcinoma in most dogs of the study population. Prospective studies are needed to identify optimal techniques for balloon dilation in dogs with neoplastic urethral obstructions and to identify patients that are likely to benefit most from the treatment.

Anti-arthritic effect of pentosan polysulfate in rats with collagen-induced arthritis.

Pentosan polysulfate (PPS) is currently under investigation as a potential disease-modifying antiarthritic agent. In the present study the effects of PPS on arthritic profiles based on clinical score, ankle size, histological changes, and activity of inflammatory mediators using collagen-induced arthritic rat are reported. Model of arthritis was developed in Sprague Dawley rats by intradermal injection of bovine type II collagen emulsified with incomplete Freund's adjuvant. The rats were randomly divided into four groups: normal control, arthritic control, arthritic rats treated with PPS (at dose level 20 µg/g) and arthritic rats treated with meloxicam (2 µg/g). The treatment was continued daily until the day 30. Arthritic biomarkers (cartilage oligomeric matrix protein and tartrate-resistant acid phosphatase 5b) in synovial fluid, expression of inflammatory mediators (interleukin-1ß, and tumor necrosis factor-α) and osteoclast marker genes (cathepsin K, tartrate-resistant acid phosphatase) in synovial membrane were measured. Daily administration of PPS to the arthritic rats significantly decreased the severity of arthritis by effectively suppressing the symptoms of arthritis and improving the functional recovery based on clinical score and histopathological evidence. Intriguingly, identical downregulation pattern of arthritis profiles, biological markers as well as relative mRNA levels of osteoclast markers and cytokines were monitored in arthritic rats treated with PPS. In conclusion, PPS exerted protective effects against collagen-induced arthritis in rats. The results suggest that PPS acts as an anti-inflammatory and anti-arthritic agent in decreasing the arthritic effects in collagen-induced arthritic rats.

Analysis of radiosensitivity of cancer stem-like cells derived from canine cancer cell lines.

Cancer stem-like cells (CSCs) are self-renewing cells comprising a small subpopulation in tumours, and generate differentiated progeny through asymmetric division. It has been shown that CSCs are resistant to ionizing radiation, and this feature could be one of the mechanisms of tumour recurrence after radiation therapy. Much attention has been focused on to target CSCs; however, difficult of isolating CSCs and lack of knowledge on their radiosensitivity have limited this kind of research in veterinary medicine. In the present study, sphere-forming cells (SC), cultured using sphere formation method, were isolated from four type of canine tumour cell lines and evaluated if they have CSCs-like properties by expression of CSCs markers (real-time polymerase chain reaction) and capacity of tumorigenesis (xenograft transplantation in nude mice), and were assessed radiosensitivity (clonogenic survival assay) and DNA repair kinetics (immunofluorescence staining for p53-binding protein 1) after X-ray irradiation in comparison with the corresponding normal adherent culture cells (AC). All SCs were isolated using sphere formation and showed high gene expression of CD133 and tumorigenic ability as compared with AC. All SCs were significantly resistant against X-ray irradiation as compared with AC. In addition, the amount of DNA double-strand breaks after X-ray irradiation were significantly lower in SC compared with the corresponding AC. These results indicate that SC isolated through sphere formation possess CSCs-like characteristics and CSCs are important factor that affect radiosensitivity in canine tumours. In addition, radioresistance of CSCs may depend on reaction of DNA double-strand break after X-ray exposure.

Comparison of three mobilization protocols for peripheral blood stem cell apheresis with Spectra Optia continuous mononuclear cell protocol in healthy dogs.

Peripheral blood stem cell (PBSC) transplantation following consolidation therapy is a feasible treatment option for canine haematological malignancies. In veterinary medicine, haematopoietic stem cells are generally mobilized into peripheral circulation using a granulocyte colony-stimulating factor (G-CSF). This pilot study aimed to evaluate the haematopoietic stem cell mobilization effect of three different regimens for PBSC apheresis with Spectra Optia continuous mononuclear cell (CMNC) protocol in healthy dogs. Stem cell mobilization was performed using high-dose plerixafor (CXCR-4 inhibitor) alone, a G-CSF alone, or a combination of the low-dose plerixafor and G-CSF. Three dogs were assigned to each mobilization protocol. Regardless of the mobilization protocol, the total blood volume processed was uniformly set as 270 mL/kg and many PBSCs, defined as CD34+/CD45dim cells, within the apheresis product were compared. Changes in complete blood count, PBSC counts, and blood chemistry analysis were monitored before, during, and after apheresis. All dogs tolerated the apheresis procedure using the Spectra Optia system with minimal adverse effects. The mean PBSC counts of the apheresis products for plerixafor, G-CSF, and the combination groups were 1.3 ± 0.24, 4.2 ± 0.47, and 6.4 ± 0.9 × 106 cells/kg, respectively. The apheresis procedure using Spectra Optia CMNC protocol in dogs is safe and feasible. Furthermore, PBSC mobilization with a combination of G-CSF and plerixafor appeared more effective than either compound alone in mobilizing PBSC to the peripheral blood in dogs.

Independent chondrogenic potential of canine bone marrow-derived mesenchymal stem cells in monolayer expansion cultures decreases in a passage-dependent pattern.

Although chondroinductive growth factors are considered necessary for chondrogenesis of bone marrow-derived mesenchymal stem cells (BMSC), independent and spontaneous chondrogenesis has been previously demonstrated in adult horses, bovine calves and adult human BMSC. Surprisingly, adult canine BMSC under similar culture conditions previously failed to demonstrate chondrogenesis. The present study evaluated independent chondrogenic potential of BMSC sourced from three young dogs in the absence of known chondroinductive factors. BMSC were culture expanded in 10% DMEM up to third passage (P3). At each passage, the phenotype of BMSC was evaluated by RT-PCR gel electrophoresis and qPCR. BMSC exhibited a chondrogenic phenotype in the absence of dexamethasone and TGF-ß1 as verified by the expression of Sox-9, type II collagen and aggrecan. Sox-9 was significantly downregulated (P<0.05) from P1-P3 compared to P0 while type II and X collagen, and aggrecan were significantly downregulated at P3 compared to P0. There was a significant (P<0.01) negative correlation between passaging and Sox-9, type II collagen and aggrecan gene expression. These results indicate that independent chondrogenic potential and phenotype retention of BMSC decreases in a passage-dependent pattern. Therefore, caution should be exercised for future experiments evaluating the chondrogenic potential of BMSC after extensive expansion cultures in 10% DMEM.