Treatment of solid tumors in dogs using veterinary high-intensity focused ultrasound: A retrospective clinical study.

High-intensity focused ultrasound (HIFU) is a cancer treatment tool that focuses ultrasound energy on tumor tissues, which initiates necrosis via heat and mechanical effects. The efficacy of veterinary HIFU (vHIFU) was evaluated for the treatment of solid tumors in dogs. Data from 11 client-owned dogs with various solid tumors treated by vHIFU between 2013 and 2017 were retrospectively evaluated. Ten of the 11 dogs were followed up; clinical signs were alleviated in five. Four dogs exhibited a decrease in tumor size, and bleeding stopped in all four dogs with hemorrhagic tumors. Side effects included hyperthermia or erythema on the application site, enteritis, and skin ulcerations. These results suggest that vHIFU could be used as an alternative cancer treatment for dogs with solid tumors.

Long-Term Management with Adipose Tissue-Derived Mesenchymal Stem Cells and Conventional Treatment in a Dog with Hepatocutaneous Syndrome.

Hepatocutaneous syndrome (HS) is an uncommon skin disorder that occurs in conjunction with liver disease and is diagnosed based on decreased plasma concentrations of amino acids and the histopathology of skin lesions. The survival period generally is <6 months. A 10-year-old castrated male Maltese dog was presented for evaluation of lethargy, polyuria, polydipsia, and skin lesions including alopecia, erythema, and crusts. Based on increased liver enzyme activity, low plasma amino acid concentrations, and findings from liver cytology and skin biopsy, the dog was diagnosed with HS. In addition to administration of antioxidants, hepatoprotective agents, and amino acids IV, allogenic adipose tissue-derived mesenchymal stem cells were infused 46 times over a 30-month period: 8 times directly into the liver parenchyma guided by ultrasonography and the remainder of the times into peripheral veins. After commencing stem cell therapy, the dog's hair re-grew and the skin lesions disappeared or became smaller. During ongoing management, the patient suddenly presented with anorexia and uncontrolled vomiting, and severe azotemia was observed. The dog died despite intensive care. On necropsy, severe liver fibrosis and superficial necrolytic dermatitis were observed. The dog survived for 32 months after diagnosis. A combination of amino acid and stem cell therapy may be beneficial for patients with HS.

Characteristics of extracellular cyclic AMP-dependent protein kinase as a biomarker of cancer in dogs.

OBJECTIVE: Early and proper diagnosis of cancer is the most critical factor for the survival and treatment of veterinary cancer patients. In this study, we evaluated extracellular cyclic AMP-dependent protein kinase A (ECPKA) level in serum as a useful cancer biomarker in dogs. METHODS: ECPKA levels were detected in sera from dogs with cancers (n = 48), benign tumours (n = 18), and non-tumour diseases (n = 102) as well as healthy control dogs (n = 54) utilizing enzyme-linked immunosorbent assay (ELISA). RESULTS: Sera from dogs bearing various types of cancer exhibited markedly increased levels of ECPKA by up to 7.1-, 8.8-, and 10.9-fold compared with those from dogs harbouring benign tumours, dogs with non-tumour diseases, and healthy control dogs, respectively (P < .0001). In addition, serum ECPKA level did not show statistically significant correlation with gender, breed, or age of dogs or their non-cancerous disease conditions. CONCLUSION: Our data strongly propose that detection of serum ECPKA level is a potential and specific diagnostic tool for cancer in dogs.

Clinical Relationship between Cholestatic Disease and Pituitary-Dependent Hyperadrenocorticism in Dogs: A Retrospective Case Series.

BACKGROUND: A high prevalence of cholestatic disease, including gallbladder mucocele (GBM), has been reported in dogs with naturally occurring pituitary-dependent hyperadrenocorticism (PDH). HYPOTHESIS/OBJECTIVES: Differences exist in the clinical features of dogs with PDH and concurrent cholestatic disease, and also is the management of these dogs with trilostane. ANIMALS: Sixty-five client-owned dogs with naturally occurring PDH. METHODS: This was a retrospective, observational case series. Each dog was treated with trilostane for at least 3 months before the study, and had a good clinical response, as determined by owners. Statistical comparisons of clinical signs, results of routine blood tests, basal and post-ACTH cortisol concentration, and optimal trilostane dosage were made after dogs were separated into the following 3 groups by ultrasonographic imaging: normal on ultrasound (NOU) group, cholestasis group, and GBM group. RESULTS: The GBM group had more severe clinical signs and significantly different total serum cholesterol concentration and post-ACTH stimulation cortisol concentration at the time of diagnosis. Dogs that weighed <6 kg had a significantly higher prevalence of cholestatic disease than did the other dogs (P = .003). The optimal trilostane dosages for the GBM and cholestasis groups were 2.5 and 1.5 times the dosage of the NOU group, respectively (P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Gallbladder disease associated with cholestatic disease is correlated with PDH in dogs, in both its clinical features and drug management. These findings may be associated with hypercholesterolemia, unidentified genetic factors, and the hydrophobic nature of trilostane.

Pharmacokinetic study of meropenem in healthy beagle dogs receiving intermittent hemodialysis.

Meropenem, a second carbapenem antimicrobial agent with a broad spectrum of activity, is used to treat sepsis and resistant-bacterial infections in veterinary medicine. The objective of this study was to identify the pharmacokinetics of meropenem in dogs receiving intermittent hemodialysis (IHD) and to determine the proper dosing in renal failure patients receiving IHD. Five healthy beagle dogs were given a single i.v. dose of 24 mg/kg of meropenem and received IHD. The blood flow rate, dialysate flow, and ultrafiltration rate were maintained at 40 mL/min, 300 mL/min, and 40 mL/h, respectively. Blood samples were collected for 24 h from the jugular vein and from the extracorporeal arterial and venous line. Urine samples and dialysate were also collected. The concentrations of meropenem were assayed using HPLC/MS/MS determination. The peak plasma concentration was 116 ± 37 µg/mL at 15 min. The systemic clearance was 347 ± 117 mL/h/kg, and the steady-state volume of distribution was 223 ± 67 mL/kg. Dialysis clearance was 71.1 ± 34.3 mL/h/kg, and the extraction ratio by hemodialysis was 0.455 ± 0.150. The half-life (T1/2 ) in dogs with IHD decreased compared with those without IHD, and the reduction in T1/2 was greater in renal failure patients than in normal patients. Sixty-nine percent and 21% of the administered drug were recovered by urine and dialysate in the unchanged form, respectively. In conclusion, additional dosing of 24 mg/kg of meropenem after dialysis could be necessary according to the residual renal function of the patient based on the simulated data.

Prevalence, Isolation and Molecular Characterization of Bartonella Species in Republic of Korea.

To determine the prevalence of Bartonella species and identify which species of Bartonella naturally infects the striped field mouse (Apodemus agrarius) in the Republic of Korea (ROK), spleens from 200 mice were assayed by nested polymerase chain reaction (nPCR) targeting the RNA polymerase subunit beta (rpoB) gene and the 16S-23S internal transcribed spacer (ITS) region for members of the genus Bartonella. Utilizing PCR techniques, the prevalence of Bartonella spp. ranged from 31.5% (63/200) to 62.0% (124/200) for the rpoB and ITS gene fragments, respectively. The most prevalent species, Bartonella grahamii, was assigned to 17 genotypes and closely related to the zoonotic pathogens, B. taylorii, B. tribocorum, B. phoceensis and B. henselae, which also were detected. Two Bartonella isolates (KRBG28 and KRBG32) were recovered from blood of A. agrarius captured in Gyeonggi Province, ROK. Comparison of the 16S rRNA, hemin-binding protein E (hbpE), glutamate dehydrogenase 1 (gdh1), invasion-associated protein B (ialB), cell division protein (ftsZ), citrate synthase (gltA), 60 kDa heat shock protein (groEL), rpoB gene fragments and the ITS region sequences from the isolates with GenBank was confirmed as B. grahamii. Phylogenetic analysis based on the alignment of concatenated sequences (4933 bp) of KRBG28 and KRBG32 clustered with B. grahamii, forming an independent clade between Asian and American/European B. grahamii genogroups.

Netrin-1 protects hypoxia-induced mitochondrial apoptosis through HSP27 expression via DCC- and integrin α6ß4-dependent Akt, GSK-3ß, and HSF-1 in mesenchymal stem cells.

Netrin (Ntn) has the potential to be successfully applied as an anti-apoptotic agent with a high affinity for tissue, for therapeutic strategies of umbilical cord blood-derived mesenchymal stem cells (UCB-MSC), although the mechanism by which Ntn-1 protects hypoxic injury has yet to be identified. Therefore, the present study examined the effect of Ntn-1 on hypoxia-induced UCB-MSC apoptosis, as well as the potential underlying mechanisms of its protective effect. Hypoxia (72 h) reduced cell viability (MTT reduction, and [(3)H]-thymidine incorporation) and cell number, and induced apoptosis (annexin and/or PI positive), which were reversed by Ntn-1 (10 ng/ml). Moreover, Ntn-1 decreased the increase of hypoxia-induced Bax, cleaved caspase-9, and -3, but blocked the decrease of hypoxia-reduced Bcl-2. Next, in order to examine the Ntn-1-related signaling cascade in the protection of hypoxic injury, we analyzed six Ntn receptors in UCB-MSC. We identified deleted in colorectal cancer (DCC) and integrin (IN) α6ß4, except uncoordinated family member (UNC) 5A-C, and neogenin. Among them, IN α6ß4 only was detected in lipid raft fractions. In addition, Ntn-1 induced the dissociation of DCC and APPL-1 complex, thereby stimulating the formation of APPL-1 and Akt2 complex. Ntn-1 also reversed the hypoxia-induced decrease of Akt and glycogen synthase kinase 3ß (GSK-3ß) phosphorylation, which is involved in heat shock factor-1 (HSF-1) expression. Ntn-1-induced phospho-Akt and -GSK-3ß were inhibited by DCC function-blocking antibody, IN a6b4 function-blocking antibody, and the Akt inhibitor. Hypoxia and/or Ntn-1 stimulated heat shock protein (HSP)27 expression, which was blocked by HSF-1-specific small interfering RNA (siRNA). Furthermore, HSP27-specific siRNA reversed the Ntn-1-induced increase of phospho-Akt. Additionally, HSP27-specific siRNA attenuated the Ntn-1-reduced loss of mitochondrial membrane injury via the inhibition of cytochrome c (cyt c) release and formation of cyt c and HSP27 complex. Moreover, the inhibition of each signaling protein attenuated Ntn-1-induced blockage of apoptosis. In conclusion, Ntn-1-induced HSP27 protected hypoxic injury-related UCB-MSC apoptosis through DCC- and IN α6ß4-dependent Akt, GSK-3ß, and HSF-1 signaling pathways.

Zonisamide monotherapy for idiopathic epilepsy in dogs.

AIM: To evaluate the efficacy of zonisamide as a monotherapy in dogs with idiopathic epileptic seizure. METHODS: The experiment was conducted on 10 dogs with idiopathic epilepsy that were treated at the Seoul National University Hospital for Animals. A diagnosis was conducted based on physical and neurologic examination, complete blood count and chemical analysis, magnetic resonance imaging and cerebrospinal fluid analyses. Idiopathic epilepsy was diagnosed when all of these examinations were normal. Oral zonisamide was administrated to 10 dogs with idiopathic epilepsy at 5-15 mg/kg per os every 12 h to achieve a concentration of zonisamide in serum of 10-40 µg/mL. The frequency of seizures before and after the administration of zonisamide therapy was recorded and the concentrations of zonisamide in serum were measured. RESULTS: Six (60%) of the dogs were favourable responders to treatment, showing a ≥50% reduction in monthly frequency of seizures. Of the remaining four, two dogs did not show a reduction and the other two showed an increase in frequency of seizures. The mean dosage of zonisamide for favourable responders was 7.92 (SD 3.79) mg/kg, which was administered orally twice a day. Only one dog, which was one of the unfavourable responders in the whole study, experienced mild side effects. CONCLUSIONS: Among the dogs treated with oral zonisamide, 60% responded favourably. The effect of zonisamide as an anticonvulsant drug was demonstrated in this study. CLINICAL RELEVANCE: Based on these results, zonisamide monotherapy is effective in some dogs with idiopathic epilepsy.

Malignant peripheral nerve sheath tumour in the liver of a dog.

A 14-year-old male mixed breed dog was presented for abdominal distension and abdominal pain. Radiographical examination identified a large space-occupying mass in the abdomen. Necropsy examination revealed the presence of a 12cm hepatic mass that occupied almost half of the abdominal cavity. Microscopically, this mass consisted of spindle-shaped neoplastic cells that were arranged in short streams and interlacing bundles. Immunohistochemically, the neoplastic cells expressed vimentin, S-100, protein gene product 9.5 and neuron specific enolase, but were negative for cytokeratin, smooth muscle actin, melan A and von Willebrand Factor. These findings indicated that the hepatic mass was a primary hepatic peripheral nerve sheath tumour. To our knowledge, this is the first documentation of a primary hepatic malignant peripheral nerve sheath tumour in a dog.

A rapid molecular method for diagnosing epidemic dermatophytosis in a racehorse facility.

REASONS FOR PERFORMING STUDY: Identification of the species and strain of dermatophyte can play an effective role in control of disease outbreaks by establishing the source of infection. Current methods of identification are based on cultural and microscopic methods, often involving weeks before a positive identification are made. A rapid molecular diagnostic method would therefore be an important laboratory technique, but requires confirmation in equine clinical practice. OBJECTIVES: To test the sensitivity and specificity of molecular diagnostic methods applied to a racehorse herd from the Korean Racehorse Authority (KRA). METHODS: A total of 57 DNA samples were collected from hairs and crusts of skin lesions in KRA racehorses with histories and clinical signs suggestive of dermatophytosis, which was confirmed by dermatophyte-specific PCR amplification analysis using the primer pair for the chitin synthase 1 (CHS1) gene. RESULTS: Thirty-eight racehorses were definitively diagnosed with dermatophytosis using molecular and traditional diagnostic methods. PCR fingerprinting profiles using simple repetitive (GACA)4 primers showed that all diagnosed horses had the same pattern profile. Oligonucleotide sequencing of CHS1 gene PCR products confirmed Trichophyton mentagrophytes as the infectious agent. CONCLUSIONS: This study demonstrates that the PCR-based molecular diagnostic method is sensitive and specific and offers fast precise diagnosis of dermatophytosis in horses.

Hormonal change and cytokine mRNA expression in peripheral blood mononuclear cells during the development of canine autoimmune thyroiditis.

To elucidate the hormonal change and alteration in cytokine expression in peripheral blood mononuclear cells (PBMC) during the early stage of autoimmune thyroiditis, we have developed a canine model of this disease, in which normal dogs were immunized with bovine thyroglobulin (Tg) and/or canine thyroid extract. Serum samples were collected weekly, anti-canine Tg antibody was measured by enzyme-linked immunosorbent assay (ELISA) and thyroid stimulating hormone (TSH) and total T4 levels by radioimmunoassay. We also assayed T lymphocyte proliferation in response to Tg, as well as measuring cytokine mRNA by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). All six dogs immunized with bovine Tg had both canine Tg autoantibody and anti-T4 antibody. When the sample from the highest TgAA titre time-point was compared with baseline the expression of mRNA encoding the Th1-type cytokine such as interferon (IFN)-gamma, interleukin (IL)-18 and IL-15 was increased during the development of autoimmune thyroiditis. Expression of the Th2-type cytokine, IL-6 showed minimal change and IL-4 expression was not detected in any of the samples. Expression of the T suppressive cytokine, IL-10 and transforming growth factor (TGF)-beta was increased in the presence of antigen stimulation. These findings suggest that, although autoimmune thyroiditis is an organ-specific autoimmune disease, systemic cytokine mRNA expression is also changed.

Effects of SKI 306X, a new herbal agent, on proteoglycan degradation in cartilage explant culture and collagenase-induced rabbit osteoarthritis model.

OBJECTIVE: Protective effects of SKI 306X, a natural herbal product extracted from three herbs Clematis mandshurica, Trichosanthes kirilowii, and Prunella vulgaris, on articular cartilage was examined and compared with other osteoarthritis (OA) drugs using in vitro and in vivo models. METHODS: In vitro culture of rabbit articular cartilage explants was used as a model to measure the effects of drugs on the matrix degradation. The recombinant human interleukin-1alpha (rhIL-1alpha, 5 ng/ml) was added to induce proteoglycan (PG) degradation and the degree of PG degradation was assessed by measuring the amount of glycosaminoglycan (GAG) released into the culture medium. In in vivo experiment, collagenase was intraarticularly injected twice into the right knee joint of rabbits to induce OA-like change, and test agents were orally administered once a day for 28 days. The degrees of OA-like changes were evaluated through a histological examination. RESULTS: In vitro study revealed SKI 306X inhibited the degradation of PG in a concentration-dependent manner. Trichosanthes kirilowii, which is one of the major components of SKI 306X, also significantly inhibited the GAG release in cartilage explant culture at 0.3 and 0.1 mg/ml. Dexamethasone and NSAIDs, such as diclofenac and rofecoxib, had no significant effects on the suppression of PG degradation. In in vivo studies, OA-like degeneration of the articular cartilage and synovial tissue was induced by injecting collagenase into the right knee joint of mature rabbits. At a dose of 200 mg/kg, SKI 306X reduced the OA-like histological changes, whereas diclofenac had no effect at 10 mg/kg. CONCLUSION: These results indicate that SKI 306X inhibited PG degradation in cartilage explant culture, and its prophylactic administration significantly protected the knee joint of rabbit from OA-like change in collagenase-induced experimental OA model. This strongly suggests that SKI 306X can be a good OA agent with some cartilage protection activity.

Studies of cocktail therapy with multiple cytokines for neoplasia or infectious disease of the dog I. cDNA cloning of canine IL-3 and IL-6.

This paper describes the cloning and sequence analysis of the cDNAs encoding the canine homologues of interleukin-3 (IL-3) and interleukin-6 (IL-6). The coding sequences for canine IL-3 and IL-6 were obtained by using the reverse transcription polymerase chain reaction (RT-PCR) with RNA harvested from canine peripheral blood mononuclear cells (PBMCs). Canine IL-3 cDNA includes a single open reading frame of 432 nucleotides, which encodes a 143 amino acid polypeptide and has 44.7, 42.4, 37 and 23.7% homology with the cow, sheep, human and rat IL-3 sequences, respectively. Canine IL-6 cDNA (GenBank accession number; AF275796) encodes a putative 20-amino acid signal peptide followed by a 187-amino acid mature protein. The predicted amino acid sequence of canine IL-6 shares 60.4, 77.2, 71.0, 55.8 and 42.0% sequence identity with those of human, feline, porcine, sheep and rat IL-6, respectively.

Detection of canine distemper virus (CDV) through one step RT-PCR combined with nested PCR.

A one step reverse transcription PCR (RT-PCR) combined nested PCR was set up to increase efficiency in the diagnosis of canine distemper virus (CDV) infection after developement of nested PCR. Two PCR primer sets were designed based on the sequence of nucleocapsid gene of CDV Onderstepoort strain. One-step RT-PCR with the outer primer pair was revealed to detect 10(2) PFU/ml. The sensitivity was increased hundredfold using the one-step RT-PCR combined with the nested PCR. Specificity of the PCR was also confirmed using other related canine virus and peripheral blood mononuclear cells (PBMC) and body secretes of healthy dogs. Of the 51 blood samples from dogs clinically suspected of CD, 45 samples were revealed as positive by one-step RT-PCR combined with nested PCR. However, only 15 samples were identified as positive with a single one step RT-PCR. Therefore approximately 60% increase in the efficiency of the diagnosis was observed by the combined method. These results suggested that one step RT-PCR combined with nested PCR could be a sensitive, specific, and practical method for diagnosis of CDV infection.

Cloning of canine GM-CSF and SCF genes.

Cytokines have pleiotropic regulatory effects on hematopoietic cells and many other cell types that participate in host defence and repair processes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates the growth and differentiation of granulocytes and macrophages and regulates the biological functions expressed by mature cells of these lineages. Stem cell factor (SCF) is a multifunctional cytokine involved in hematopoiesis, melanogenesis and gametogenesis. In order to determine the complementary DNA (cDNA) of canine GM-CSF and canine SCF, cDNA clones were generated from lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells (PBMCs) and bone marrow cells by reverse transcription PCR amplification. The canine GM-CSF cDNA obtained in this study contains an open reading frame encoding 144 amino acid residues and has 53-75% homology with those of human, cat, sheep, pig, cow and mouse, Canine SCF cDNA consist of an open reading frame encoding 274 amino acid residues and shares 81-92% homology with those of human, cat, pig, cow and mouse.

Cloning and mapping of cat (Felis catus) immunoglobulin and T-cell receptor genes.

Molecular cloning and chromosomal mapping of the cat immunoglobulin (Ig) and T-cell receptor (TcR) genes were carried out to provide basic information for genetic analysis of immunologic diseases including leukemias and lymphomas in cats. We cloned two Ig constant genes, IGHM and IGHG and three TcR constant genes, TRAC, TRGC, and TRDC, by polymerase chain reaction (PCR) amplification of cDNA from cat peripheral blood mononuclear cells. For chromosomal mapping of the Ig and TcR loci including the IGK, IGL, and TRB on the cat genome, we performed PCR screening of DNAs from 37 cat x rodent somatic cell hybrids by using specific primers for the given genes. Consequently, three loci for IGH, TRA, and TRD, and two loci for TRB and TRG were found to be syntenic and assigned to cat chromosomes (FCA) B3 and A2, respectively. Further, IGK and IGL loci were mapped on FCA A3 and D3, respectively. These findings support the notion that the genetic linkages between the Ig and TcR genes are extensively conserved between humans and cats.

Genomic structure of the bovine mb-1 gene encoding the Ig-alpha subunit of the B cell antigen receptor complex.

The B cell antigen receptor, (BCR) comprises surface immunoglobulin and disulfide-bonded heterodimer of Ig-alpha and Ig-beta chains, which are the products of the mb-1 and B29 genes, respectively. In this study, we describe the isolation and analysis of a 6.2-kb genomic DNA clone containing bovine mb-1 gene encoding Ig-alpha. Sequence data revealed that the bovine mb-1 gene is composed of five exons and four introns, and that its overall structure is very similar to those of murine and human genes. The 5' upstream region of the bovine mb-1 gene contained potential protein binding motifs of transcription factors including EBF, Sp1, NF-kappa B, MUF/Ets-1 and AP 2. As with the murine and human mb-1 genes, the 5' region of the bovine mb-1 gene lacked a TATA box. The present study will be useful for understanding the regulated expression of the bovine mb-1 gene at different stages of development and activation as well as in bovine leukemia virus infection.

A proposed nomenclature of the domestic cat karyotype.

High-resolution G- and Q-band patterns of cat (Felis catus) prometaphase chromosomes with more than 450 numbered bands are presented. This number represents approximately twice the number of bands per haploid set exhibited by feline chromosomes at mid-metaphase. A diagrammatic representation of G-banded cat chromosomes has already been described (O'Brien and Nash, 1982); however, precise numbering of bands and landmarks, as in the human karyotype and in the karyotypes of other domestic and laboratory animals, has not yet been available for the domestic cat karyotype, except for the RBG-banded ideograms constructed by Shibasaki et al. (1987) and Rønne and Storm (1995). In this report, we propose a numbering system for the G-banded ideogram reported previously (O'Brien and Nash, 1982) and an extended ideogram at the high-resolution level (473 numbered bands) as a contribution to the future standardization of the feline karyotype.