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.

Neuroprotective and immunomodulatory effects of superoxide dismutase on SH-SY5Y neuroblastoma cells and RAW264.7 macrophages.

Superoxide dismutase (SOD) is an antioxidant enzyme that protects the body from free radicals. It has both antioxidant and immunomodulatory properties, inducing macrophage polarization from M1 to M2. Macrophages, key mediators of the innate immune response, are divided into the M1 (pro-inflammatory) and M2 (anti-inflammatory) subtypes. In this study, we aimed to assess the antioxidant and neuroprotective effects of SOD on nerve cells and its immunomodulatory effects on macrophages. We observed that SOD inhibited the accumulation of reactive oxygen species and enhanced the viability of H2O2-treated nerve cells. Furthermore, SOD reduced the degree of necrosis in nerve cells treated with the conditioned medium from macrophages, which induced inflammation. In addition, SOD promoted the M1 to M2 transition of macrophages. Our findings suggest that SOD protects nerve cells and regulates immune responses.

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.

Assessment of MMP-9 and clinical characteristics in dogs with tracheal collapse based on cough severity and fluoroscopic findings: a cross-sectional study.

BACKGROUND: Tracheal collapse (TC), a common disease in dogs, is characterized by cough; however, little is known about the serum biomarkers that can objectively evaluate the severity of cough in canine TC. Furthermore, studies elucidating the relationship of fluoroscopic characteristics with the severity of cough are lacking. Therefore, this study aimed to evaluate the relationship between cough severity and clinical characteristics, fluoroscopic images, and new serum biomarkers in canine TC. RESULTS: Fifty-one client-owned dogs diagnosed with TC based on fluoroscopic and clinical signs were enrolled in this study and divided into three groups according to the severity of cough (grade of cough: 0, 1, and 2). Signalments, comorbidities, and fluoroscopic characteristics were compared among the groups retrospectively. The serum matrix metalloproteinase-9 (MMP-9), interleukin-6 (IL-6), surfactant protein-A (SP-A), and syndecan-1 (SDC-1) levels were measured in all groups. No significant differences in age, breed, sex, or clinical history were observed among the groups. Concomitant pharyngeal collapse increased significantly with the severity of cough (p = .031). Based on the fluoroscopic characteristics, the TC grade of the carinal region increased significantly and consistently with the grade of cough (p = .03). The serum MMP-9 level was significantly higher in the grade 2 group than that in the grade 0 group (p = .014). The serum IL-6 level was significantly lower in the grade 1 group than that in the grade 0 group (p = .020). The serum SP-A and SDC-1 levels did not differ significantly among the groups. CONCLUSIONS: The severity of cough with the progression of TC can be predicted with the fluoroscopic TC grade at the carinal region. MMP-9 may be used as an objective serum biomarker that represents cough severity to understand the pathogenesis.

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.

Anti-tumor Effect of Activated Canine B Cells With Interleukin-21 and Anti-B Cell Receptor.

BACKGROUND/AIM: Recently, novel studies on the pivotal role of B cells in the tumor-microenvironment and anti-tumor immunity have been conducted. Additionally, Interleukin-21 (IL-21) and anti-B cell receptor (BCR) have been reported to stimulate B cells to secrete granzyme B, which exhibits cytotoxic effects on tumor cells. However, the direct anti-tumor effect of B cells is not yet fully understood in the veterinary field. This study is the first attempt in veterinary medicine to identify the immediate effect of B cells on tumor suppression and the underlying mechanisms involved. MATERIALS AND METHODS: Canine B cells were isolated from peripheral blood and activated with IL-21 and anti-B cell receptor (BCR). The canine leukemia cell line GL-1 was co-cultured with B cells, and the anti-tumor effect was confirmed by assessing the changes in cell viability and apoptotic ratio. RESULTS: When B cells were activated with IL-21 and anti-BCR, the secretion of granzyme B and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increased. Simultaneously, the viability of GL-1 cells decreased, and the apoptotic ratio increased, particularly when co-cultured with activated B cells. CONCLUSION: The results demonstrated the direct anti-tumor effect of granzyme B-and TRAIL and its enhanced potential of B cells to inhibit tumor cell growth after activation with IL-21 and anti-BCR. This study is the first study dealing with immunomodulation in the canine tumor micro-environment.

Macrophage induces anti-cancer drug resistance in canine mammary gland tumor spheroid.

Tumor-associated macrophages (TAMs) play an important role in the tumor microenvironment by producing cytokines and growth factors. Furthermore, TAMs play multifunctional roles in tumor progression, immune regulation, metastasis, angiogenesis, and chemoresistance. Hypoxia in the tumor microenvironment induces tumor-supporting transformation of TAMs, which enhances tumor malignancy through developing anti-cancer resistance, for example. In this study, a hybrid spheroid model of canine mammary gland tumor (MGT) cell lines (CIPp and CIPm) and canine macrophages (DH82) was established. The effects of hypoxia induced by the spheroid culture system on the anti-cancer drug resistance of canine MGT cells were investigated. A hybrid spheroid was created using an ultralow-adhesion plate. The interactions between canine MGT cells and DH82 were investigated using a co-culture method. When co-cultured with DH82, cell viability and expression levels of tumor growth factors and multi-drug resistance genes were increased in canine MGT cells under doxorubicin. Additionally, doxorubicin-induced apoptosis and G2/M cell cycle arrest were attenuated in canine MGT cells co-cultured with DH82. In conclusion, the hybrid spheroid model established in this study reflects the hypoxic TME, allowing DH82 to induce anti-cancer drug resistance in canine MGT cells.

Canine adipose tissue-derived MSCs engineered with mRNA to overexpress TSG-6 and enhance the anti-inflammatory effects in canine macrophages.

Background: Mesenchymal stem cells (MSCs) are useful agents in the treatment of various inflammatory diseases. The immunomodulatory effects of MSCs are largely related to their secretory properties. mRNA engineering emerged as a safe alternative to enhance the secretory function of MSCs. Optimization of the untranslated region (UTR) sequence is important for enhancing the translational efficiency of exogenous mRNAs. However, research on the optimization of UTR in canine MSCs has not yet been conducted. Objectives: We aimed to identify the UTR sequence related to the expression efficiency of in vitro transcription (IVT) mRNA in canine MSCs and investigate whether mRNA-engineered MSCs that overexpress TSG-6 exhibit enhanced anti-inflammatory effects. Methods: Canine adipose tissue-derived (cAT)-MSCs were transfected with green fluorescence protein (GFP) mRNA with three different UTRs: canine hemoglobin subunit alpha-like 1 (HBA1), HBA2, and hemoglobin subunit beta-like (HBB). The translation efficacy of each mRNA was evaluated using relative fluorescence. TSG-6 mRNA was produced with the UTR optimized according to relative fluorescence results. cAT-MSCs were transfected with TSG-6 mRNA (MSCTSG-6), and TSG-6 expression was analyzed using real-time quantitative PCR, ELISA, and western blotting. To evaluate the anti-inflammatory effects of MSCsTSG-6, DH82 cells were co-cultured with MSCsTSG-6 or treated with dexamethasone, and changes in the expression of inflammatory cytokines were analyzed using qPCR. Results: The highest fluorescence level was observed in the HBA1 UTR at 24 h post-transfection. TSG-6 mRNA transfection yielded high levels of TSG-6 in the cAT-MSCs. In DH82 cells co-cultured with MSCsTSG-6, the expression of inflammatory cytokines decreased compared to that in co-culturing with naïve MSCs and dexamethasone treatment. Conclusions: Optimization of the HBA1 UTR improved the translation efficiency of IVT mRNA in canine MSCs. cAT-MSCs engineered with TSG-6 mRNA effectively enhanced the anti-inflammatory effects of the MSCs when co-cultured with LPS-activated DH82 cells.

Changes in Lactate-related Fecal Microbiome in Hyperlactatemia Diabetic Dogs.

BACKGROUND/AIM: The correlation between the intestinal microbiome and endocrine disorders has recently been drawing attention as an important key for determining their pathology and clinical assessment. In this study, we evaluated the microbiome of dogs with insulin-dependent diabetes mellitus (IDDM) with respect to blood lactate. MATERIALS AND METHODS: Fecal samples were obtained from 17 subjects and real-time quantitative polymerase chain reaction determinations were performed to quantify the gene expression levels of lactate-producing and dysbiosis index-related bacteria. RESULTS: Expression levels of the lactate-producing bacteria Lactobacillus spp., Enterococcus spp., and Bifidobacterium spp., were confirmed in patients with high concentrations of lactate in the blood. The abundance of Enterococcus and Bifidobacterium was higher in diabetic dogs compared to that of non-diabetic dogs. When blood lactate concentrations were high, the abundance of Bifidobacterium also increased. CONCLUSION: Blood lactate levels influence the gut microbiome in dogs with IDDM. This study will help understand the gut microbiota in the context of diabetes in human and veterinary medicine.