Serum thymidine kinase 1 protein concentrations and presence of its autoantibody as biomarkers for screening dogs with malignant tumors.

BACKGROUND: An accurate and easily accessible method for diagnosing malignancies in local veterinary clinics has not yet been established. OBJECTIVES: To investigate the usefulness of serum thymidine kinase 1 (TK1) protein and its autoantibody as tumor biomarkers in dogs. ANIMALS: Serum samples from 1702 dogs were collected from local animal hospitals and referral animal medical centers in South Korea. METHODS: TK1 protein OD value and TK1 autoantibody ratio (TK1 autoantibody OD/total IgG OD) in serum samples of dogs classified into healthy controls, group with nontumor disease, group with benign and group with malignant tumors were measured using lateral flow immunochromatographic assay methods. RESULTS: TK1 autoantibody levels were significantly higher in malignant tumor group (median 0.71) than in healthy controls (median 0.34), group with nontumor disease (median 0.34), and group with benign tumor (median 0.32, Welch t test, P < .0001). They were also significantly different among dogs with carcinomas (median 0.77), hematopoietic tumors (median 0.71), and sarcomas (median 0.56) than in healthy controls (median 0.34, post hoc Games-Howell test, P < .0001). In the receiver operating characteristic curve of TK1 protein, AUC was 0.633 (95% CI: 0.592-0.675, P < .0001). The AUC of TK1 autoantibody ratio was 0.758 (95% CI: 0.723-0.793, P < .0001). CONCLUSIONS AND CLINICAL IMPORTANCE: TK1 autoantibody is a potentially useful biomarker for differentiating between healthy and tumor-bearing dogs, better than TK1 protein measurement. However, both were inadequate when used as single biomarkers for screening dogs to discover occult malignant tumors.

AniScan Using Extracellular Cyclic AMP-Dependent Protein Kinase A as a Serum Biomarker Assay for the Diagnosis of Malignant Tumors in Dogs.

The early detection of tumors improves chances of decreased morbidity and prolonged survival. Serum biomarkers are convenient to use and have several advantages over other approaches, such as accuracy and straightforward protocols. Reliable biomarkers from easily accessible sources are warranted for the development of cost-effective assays for routine screening, particularly in veterinary medicine. Extracellular c-AMP-dependent protein kinase A (ECPKA) is a cytosolic leakage enzyme. The diagnostic accuracy of detecting autoantibodies against ECPKA was found to be higher than that of ECPKA activity from enzymatic assays, which use a complicated method. Here, we investigated the diagnostic significance of measuring serum ECPKA autoantibody levels using an in-house kit (AniScan cancer detection kit; Biattic, Anyang, Korea). We used sera from 550 dogs, including healthy dogs and those with malignant and benign tumors. Serum ECPKA and immunoglobulin G were determined using the AniScan cancer detection kit. ECPKA autoantibody levels were significantly higher (p < 0.01) in malignant tumors than in benign tumors, non-tumor diseases, and healthy controls. On the basis of sensitivity and specificity values, AniScan ECPKA is a rapid and easy-to-use assay that can be applied to screen malignant tumors from benign tumors or other diseases in dogs.

Author Correction: TNF-α and INF-γ primed canine stem cell-derived extracellular vesicles alleviate experimental murine colitis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

TNF-α and INF-γ primed canine stem cell-derived extracellular vesicles alleviate experimental murine colitis.

The inflammatory bowel diseases (IBD) are characterized by relapsing inflammation and immune activation diseases of the gastrointestinal tract. Extracellular vesicles, which elicit similar biological activity to the stem cell themselves, have been used experimentally to treat dextran sulfate sodium (DSS)-induced colitis in murine models though immunosuppressive potential. In this study, we investigated whether the Extracellular vesicles (EVs) obtained by stimulating inflammatory cytokine on canine adipose mesenchymal stem cells (cASC) improved anti-inflammatory and/or immunosuppressive potential of EVs, and/or their ability to alleviate inflammation in colitis. We also explored the correlation between immune cells and the inflammatory repressive effect of primed EVs. Pro-inflammatory cytokines such as TNF-α and IFN-γ increased immunosuppressive protein such as HGF, TSG-6, PGE2 and TGF-ß in EVs. Moreover, the anti-inflammatory effect of EVs was improved through pretreatment with inflammatory cytokines. Importantly, EVs obtained from primed stem cells effectively induced macrophage polarization toward an anti-inflammatory M2 phenotype and suppressed activated immunity by enhancing regulatory T cells in inflamed colon in mice. Our results provide a new and effective therapy for the EVs obtained from ASC stimulated with TNF-α and IFN-γ against not only IBD, but also immune-mediated disease.

TSG-6 in extracellular vesicles from canine mesenchymal stem/stromal is a major factor in relieving DSS-induced colitis.

Adipose tissue derived mesenchymal stem/stromal cell (ASC)-derived extracellular vesicles (EV) have been reported to be beneficial against dextran sulfate sodium (DSS)-induced colitis in mice. However, the underlying mechanisms have not been fully elucidated. We hypothesize that the tumor necrosis factor-α-stimulated gene/protein 6 (TSG-6) in EVs is a key factor influencing the alleviation of colitis symptoms. DSS-induced colitis mice (C57BL/6, male, Naïve = 6, Sham = 8, PBS = 8 EV = 8, CTL-EV = 8, TSG-6 depleted EV = 8) were intraperitoneally administered EVs (100 ug/mice) on day 1, 3, and 5; colon tissues were collected on day 10 for histopathological, RT-qPCR, western blot and immunofluorescence analyses. In mice injected with EV, inflammation was alleviated. Indeed, EVs regulated the levels of pro- and anti-inflammatory cytokines, such as TNF-α, IL-1ß, IFN-γ, IL-6, and IL-10 in inflamed colons. However, when injected with TSG-6 depleted EV, the degree of inflammatory relief was reduced. Furthermore, TSG-6 in EVs plays a key role in increasing regulatory T cells (Tregs) and polarizing macrophage from M1 to M2 in the colon. In conclusion, this study shows that TSG-6 in EVs is a major factor in the relief of DSS-induced colitis, by increasing the number of Tregs and macrophage polarization from M1 to M2 in the colon.

Extracellular cyclic adenosine monophosphate-dependent protein kinase A autoantibody and C-reactive protein as serum biomarkers for diagnosis of cancer in dogs.

Protein kinase A, a cyclic adenosine monophosphate (AMP)-dependent enzyme, normally exists within mammalian cells; however, in cancer cells, it can leak out and be found in the serum. Extracellular cyclic AMP-dependent protein kinase A (ECPKA) has been determined to increase in the serum of cancer-bearing dogs. However, there have been no reports in the veterinary literature on serum ECPKA autoantibody (ECPKA-Ab) expression in dogs with cancer. The aim of this study was to evaluate ECPKA-Ab and C-reactive protein (CRP) as serum biomarkers for cancer in dogs. ECPKA-Ab and CRP levels were detected by an enzyme-linked immunosorbent assay in serum samples from dogs with malignant tumours (n = 167), benign tumours (n = 42), or non-tumour disease (n = 155) and from healthy control dogs (n = 123). ECPKA-Ab and CRP levels were significantly higher in the dogs with malignant tumours than in those with benign tumours or non-tumour diseases, as well as in the healthy controls (P < 0.001, Kruskal-Wallis test). There was a significant positive correlation between the neoplastic index, which was developed using ECPKA-Ab and CRP levels, and the presence of cancer in dogs (P < 0.001); the area under the receiver-operating characteristic curve was estimated to be >0.85 (P < 0.001). In conclusion, ECPKA-Ab is a potential serum biomarker for a broad spectrum of cancers. Combined measurement of CRP and ECPKA-Ab levels in serum improves the sensitivity and accuracy of a diagnosis of cancer in dogs.

Mesenchymal Stem Cells Contribute to Improvement of Renal Function in a Canine Kidney Injury Model.

BACKGROUND/AIM: The kidney excretes waste materials and regulates important metabolic functions, and renal disorders constitute a significant medical problem and can result in fatalities. In the present study, mesenchymal stem cells derived from canine umbilical cord blood (cUCB-MSCs) were isolated and evaluated for their ability to improve renal function in a canine model of acute kidney injury (AKI). MATERIALS AND METHODS: The canine AKI model was developed by i.v. injection of cisplatin and gentamycin into 14 male beagle dogs. cUCB-MSCs were administered into the renal corticomedullary junction following AKI induction. Survival time, clinical signs, blood analysis and histological parameters were analyzed. RESULTS: The group treated with AKI plus cUCB-MSCs had decreased blood urea nitrogen and creatinine levels, and showed an extended life-span and improved histological manifestations. MSCs were detected around the tubules of these kidneys at the histological level. CONCLUSION: Taken together, our findings suggest that cUCB-MSCs could be an alternative therapeutic agent for canine AKI.

Therapeutic effects of hepatocyte growth factor-overexpressing human umbilical cord blood-derived mesenchymal stem cells on liver fibrosis in rats.

Fibrosis is a common end stage for a variety of liver diseases, including most chronic liver diseases, and results from an imbalance between collagen deposition and degradation. Mesenchymal stem cells (MSCs) have the ability to migrate into fibrotic livers and differentiate into hepatocytes. Hepatocyte growth factor (HGF) has potent anti-apoptotic and mitogenic effects on hepatocytes during liver injury and plays an essential role in the development and regeneration of the liver. In this study, human HGF-overexpressing human umbilical cord blood-derived MSCs (hHGF-HUCB-MSCs) were prepared using the pMEX Expression System, and the upregulation of hHGF expression was confirmed by RT-PCR and ELISA. HGF expressed by hHGF-HUCB-MSCs exerted a stimulatory effect on hepatocyte proliferation in vitro. hHGF-HUCB-MSCs were transplanted to investigate the therapeutic effects of these cells on carbon tetrachloride (CCL4)-induced liver fibrosis in a rat model. After 4 weeks of cell treatment once per week with 2 × 10(6) cells, biochemical analysis of the serum and histopathological analysis of the liver tissue were performed. The results of the biochemical analysis of the serum show that the hHGF-HUCB-MSC-treated group had higher levels of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase, indicating the improvement of liver function. Histopathology showed that the hHGF-HUCB-MSC-treated group had reduction in the density of collagen fibres. Thus hHGF-HUCB-MSCs can enhance liver regeneration and could be useful for the treatment of patients with liver fibrosis or cirrhosis.

Thrombospondin-1 mediates oncogenic Ras-induced senescence in premalignant lung tumors.

Progression of premalignant lesions is restrained by oncogene-induced senescence. Oncogenic Ras triggers senescence in many organs, including the lung, which exhibits high levels of the angiogenesis inhibitor thrombospondin-1 (TSP-1). The contribution of TSP-1 upregulation to the modulation of tumorigenesis in the lung is unclear. Using a mouse model of lung cancer, we have shown that TSP-1 plays a critical and cell-autonomous role in suppressing Kras-induced lung tumorigenesis independent of its antiangiogenic function. Overall survival was decreased in a Kras-driven mouse model of lung cancer on a Tsp-1-/- background. We found that oncogenic Kras-induced TSP-1 upregulation in a p53-dependent manner. TSP-1 functioned in a positive feedback loop to stabilize p53 by interacting directly with activated ERK. TSP-1 tethering of ERK in the cytoplasm promoted a level of MAPK signaling that was sufficient to sustain p53 expression and a senescence response. Our data identify TSP-1 as a p53 target that contributes to maintaining Ras-induced senescence in the lung.

Selective targeting of brain tumors with gold nanoparticle-induced radiosensitization.

Successful treatment of brain tumors such as glioblastoma multiforme (GBM) is limited in large part by the cumulative dose of Radiation Therapy (RT) that can be safely given and the blood-brain barrier (BBB), which limits the delivery of systemic anticancer agents into tumor tissue. Consequently, the overall prognosis remains grim. Herein, we report our pilot studies in cell culture experiments and in an animal model of GBM in which RT is complemented by PEGylated-gold nanoparticles (GNPs). GNPs significantly increased cellular DNA damage inflicted by ionizing radiation in human GBM-derived cell lines and resulted in reduced clonogenic survival (with dose-enhancement ratio of ~1.3). Intriguingly, combined GNP and RT also resulted in markedly increased DNA damage to brain blood vessels. Follow-up in vitro experiments confirmed that the combination of GNP and RT resulted in considerably increased DNA damage in brain-derived endothelial cells. Finally, the combination of GNP and RT increased survival of mice with orthotopic GBM tumors. Prior treatment of mice with brain tumors resulted in increased extravasation and in-tumor deposition of GNP, suggesting that RT-induced BBB disruption can be leveraged to improve the tumor-tissue targeting of GNP and thus further optimize the radiosensitization of brain tumors by GNP. These exciting results together suggest that GNP may be usefully integrated into the RT treatment of brain tumors, with potential benefits resulting from increased tumor cell radiosensitization to preferential targeting of tumor-associated vasculature.

Canine mesenchymal stem cells are effectively labeled with silica nanoparticles and unambiguously visualized in highly autofluorescent tissues.

BACKGROUND: Development of a method for long-term labeling of cells is critical to elucidate transplanted cell fate and migration as well as the contribution to tissue regeneration. Silica nanoparticles have been recently developed and demonstrated to be biocompatible with a high labeling capacity. Thus, our study was designed to assess the suitability of silica nanoparticles for labeling canine mesenchymal stem cells (MSCs) and the fluorescence afficiency in highly autofluorescent tissue. RESULTS: We examined the effect of silica nanoparticle labeling on stem cell morphology, viability and differentiation as compared with those of unlabeled control cells. After 4 h of incubation with silica nanoparticles, they were internalized by canine MSCs without a change in the morphology of cells compared with that of control cells. The viability and proliferation of MSCs labeled with silica nanoparticles were evaluated by a WST-1 assay and trypan blue exclusion. No effects on cell viability were observed, and the proliferation of canine MSCs was not inhibited during culture with silica nanoparticles. Furthermore, adipogenic and osteogenic differentiation of silica nanoparticle-labeled canine MSCs was at a similar level compared with that of unlabeled cells, indicating that silica nanoparticle labeling did not alter the differentiation capacity of canine MSCs. Silica nanoparticle-labeled canine MSCs were injected into the kidneys of BALB/c mice after celiotomy, and then the mice were sacrificed after 2 or 3 weeks. The localization of injected MSCs was closely examined in highly autofluorescent renal tissues. Histologically, canine MSCs were uniformly and completely labeled with silica nanoparticles, and were unambiguously imaged in histological sections. CONCLUSIONS: The results of the current study showed that silica nanoparticles are useful as an effective labeling marker for MSCs, which can elucidate the distribution and fate of transplanted MSCs.

Topical 0.03% tacrolimus for treatment of pemphigus erythematosus in a Korea Jindo dog.

Topical 0.03% tacrolimus was used for treatment of a Korea Jindo dog diagnosed with pemphigus erythematosus. The dog was slowly improved following application of tacrolimus but did not achieve complete remission until end of this study. No adverse effects on clinical or laboratory parameters were noted during the topical tacrolimus therapy period.

Allergens causing atopic diseases in canine.

Canine atopic skin disease is seasonal or sometimes non-seasonal immune-mediated skin disease which occurs commonly in Korea. The definite clinical sign is systemic pruritus, especially on periocular parts, external ear, interdigit spaces and lateral flank. For diagnosis of this dermatitis, complete history taking followed by intradermal skin test and serum in vitro IgE test needs to be performed. Allergen selection for the diagnosis and treatment of atopic dermatitis should be varied geographically. In this study, with intradermal skin test(IDST) the prevalence of atopic disease and what allergens are involved in are researched. Allergens used for IDST included 26 allergen extracts from six allergen groups: grasses, trees, weeds, molds, epidermal allergens and environmental allergens. The number of allergens was 42 in which the positive and negative controls are included. The most common positive allergen reaction was the house dust mites on IDST(22/35, 63%). The other positive allergen reactions were to flea(3/35, 9%), molds(1/35, 3%), house dusts(2/35, 6%), feathers (1/35, 3%), cedar/juniper(1/35, 3%), timothy grass(1/35, 3%) and dandelion(1/35, 3%). In this study, the most prevalent allergen causing atopic dermatitis in dogs in Korea was the house dust mites followed by the flea.