Comparative Studies of Bioactivities and Chemical Components in Fresh and Black Garlics.

To investigate the bioactivities of fresh garlic and its processed product, black garlic, we conducted comparative analyses of antioxidant, anti-inflammatory, innate immune activation, and anti-cancer activities in addition to the chemical composition (sugar, amino acid, and polyphenol contents) of these materials. Simultaneous assay using neutrophil-like cells showed that fresh garlic exhibited antioxidant and innate immunostimulatory activities, whereas black garlic displayed a potent anti-inflammatory effect. The antioxidant activity index was correlated with phenol and flavonoid contents, while the innate immunostimulatory activity was correlated with fructan content. Furthermore, some black garlics with low fructose content were found to inhibit the proliferation of UM-UC-3 cancer cells, while other black garlics rich in fructose increased UM-UC-3 cell proliferation. It was shown that the processing of fresh garlic could change the composition of sugars, antioxidants, and amino acids, which have different effects on neutrophil-like cells and UM-UC-3 cells, as well as on bioactivities.

Histopathological Characterization of Cases of Spontaneous Fatal Feline Severe Fever with Thrombocytopenia Syndrome, Japan.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tickborne infectious disease caused by SFTS virus (SFTSV). We report 7 cases of spontaneous fatal SFTS in felines. Necropsies revealed characteristic lesions, including necrotizing lymphadenitis in 5 cases and necrotizing splenitis and SFTSV-positive blastic lymphocytes in all cases. We detected hemorrhagic lesions in the gastrointestinal tract in 6 cases and lungs in 3 cases, suggesting a more severe clinical course of SFTS in felids than in humans. We noted necrotic or ulcerative foci in the gastrointestinal tract in 3 cases, the lung in 2 cases, and the liver in 4 cases. We clarified that blastic lymphocytes are predominant targets of SFTSV and involved in induction of necrotic foci. We also found that thymic epithelial cells were additional targets of SFTSV. These results provide insights for diagnosing feline SFTS during pathological examination and demonstrate the similarity of feline and human SFTS cases.

Reovirus changes the expression of anti-apoptotic and proapoptotic proteins with the c-kit downregulation in canine mast cell tumor cell lines.

Although reovirus has reached phase II and III clinical trials in human cancers, the exact mechanism of reovirus oncolysis is still not completely understood. Previously, we have shown that canine mast cell tumor (MCT) cell lines were highly susceptible to reovirus, as compared with other kinds of canine cancer cell lines. In this study, we showed that reovirus infection not only led to the dephosphorylation but also downregulation of c-kit in four canine MCT cell lines, where c-kit activation is required for proliferation. Consistent with c-kit dysregulation, downstream signaling of c-kit, the level of Ras-GTP and phosphorylation of all the downstream effectors of Ras (Raf, MEK, and ERK) and Akt decreased in all the cell lines after reovirus infection, except for Akt in one of cell lines. Pro-apoptotic and anti-apoptotic proteins such as Bim, Bad and Mcl-1 were also altered by reovirus infection in these cell lines. In short, reovirus infection degraded c-kit in all the canine MCT cell lines, leading to the downregulation of downstream signaling of c-kit, which may relate to the cell death induced by reovirus.

Functional characterization of canine wild type glucocorticoid receptor and an insertional mutation in a dog.

BACKGROUND: Glucocorticoids, among the most widely utilized drugs in veterinary medicine, are employed to treat a wide variety of diseases; however, their use often induces adverse events in dogs. The efficacy of glucocorticoids usually depends on dosage, although differences in sensitivity to glucocorticoids in individual animals have been reported. Glucocorticoids bind to the cytoplasmic glucocorticoid receptor (GR), which is expressed in almost all cells. These receptors are key factors in determining individual sensitivity to glucocorticoids. This study examined individual differences in glucocorticoid sensitivity in dogs, focusing on reactivity of the GR to prednisolone. RESULTS: We first molecularly cloned the GR gene from a healthy dog. We discovered a mutant GR in a dog suspected to have iatrogenic Cushing syndrome. The mutant GR had extra nucleotides between exons 6 and 7, resulting in a truncated form of GR that was 98 amino acids shorter than the wild-type dog GR. The truncated GR exhibited very low reactivity to prednisolone, irrespective of concentration. CONCLUSIONS: We have identified the truncated form of canine GR in a dog with iatrogenic Cushing syndrome. This truncated form showed the very less sensitivity to glucocorticoid in vitro, unfortunately, we could not elucidate its clinical significance. However, our data is a first report about the function of canine GR, and will facilitate the analysis of canine glucocorticoid sensitivity.

The first nationwide surveillance of antibacterial susceptibility patterns of pathogens isolated from skin and soft-tissue infections in dermatology departments in Japan.

To investigate the trends of antimicrobial resistance in pathogens isolated from skin and soft-tissue infections (SSTI) at dermatology departments in Japan, a Japanese surveillance committee conducted the first nationwide survey in 2013. Three main organisms were collected from SSTI at 30 dermatology departments in medical centers and 10 dermatology clinics. A total of 860 strains - 579 of Staphylococcus aureus, 240 of coagulase-negative Staphylococci, and 41 of Streptococcus pyogenes - were collected and shipped to a central laboratory for antimicrobial susceptibility testing. The patient profiles were also studied. Among all 579 strains of S. aureus, 141 (24.4%) were methicillin-resistant (MRSA). Among 97 Staphylococcus epidermidis strains, 54 (55.7%) were methicillin-resistant (MRSE). MRSA and MRSE were more frequently isolated from inpatients than from outpatients. Furthermore, these methicillin-resistant strains were also isolated more frequently from patients with histories of taking antibiotics within 4 weeks and hospitalization within 1 year compared to those without. However, there were no significant differences in MIC values and susceptibility patterns of the MRSA strains between patients with a history of hospitalization within 1 year and those without. Therefore, most of the isolated MRSA cases at dermatology departments are not healthcare-acquired, but community-acquired MRSA. S. pyogenes strains were susceptible to most antibiotics except macrolides. The information in this study is not only important in terms of local public health but will also contribute to an understanding of epidemic clones of pathogens from SSTI.

The first identification and retrospective study of Severe Fever with Thrombocytopenia Syndrome in Japan.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a novel bunyavirus reported to be endemic in central and northeastern China. This article describes the first identified patient with SFTS and a retrospective study on SFTS in Japan. METHODS: Virologic and pathologic examinations were performed on the patient's samples. Laboratory diagnosis of SFTS was made by isolation/genome amplification and/or the detection of anti-SFTSV immunoglobulin G antibody in sera. Physicians were alerted to the initial diagnosis and asked whether they had previously treated patients with symptoms similar to those of SFTS. RESULTS: A female patient who died in 2012 received a diagnosis of SFTS. Ten additional patients with SFTS were then retrospectively identified. All patients were aged ≥50 years and lived in western Japan. Six cases were fatal. The ratio of males to females was 8:3. SFTSV was isolated from 8 patients. Phylogenetic analyses indicated that all of the Japanese SFTSV isolates formed a genotype independent to those from China. Most patients showed symptoms due to hemorrhage, possibly because of disseminated intravascular coagulation and/or hemophagocytosis. CONCLUSIONS: SFTS has been endemic to Japan, and SFTSV has been circulating naturally within the country.

Blockade of isoprenoids biosynthesis by simvastatin induces autophagy-mediated cell death via downstream c-Jun N-terminal kinase activation and cell cycle dysregulation in canine T-cell lymphoma cells.

Statins are inhibitors of the mevalonic acid pathway that mediates cellular metabolism by producing cholesterol and isoprenoids and are widely used in treating hypercholesterolaemia in humans. Lipophilic statins, including simvastatin, induce death in various tumour cells. However, the cytotoxic mechanisms of statins in tumour cells remain largely unexplored. This study aimed to elucidate the cytotoxic mechanisms of simvastatin in canine lymphoma cells. Simvastatin induced cell death via c-Jun N-terminal kinase (JNK) activation and autophagy in canine T-cell lymphoma cell lines Ema and UL-1, but not in B-cell lines. Cell death was mediated by induction of caspase-dependent apoptosis in UL-1 cells, but not in Ema cells. Blockade of autophagy by lysosomal inhibitors attenuated simvastatin-induced JNK activation and cell death. Isoprenoids, including farnesyl pyrophosphate and geranylgeranyl pyrophosphate, attenuated simvastatin-induced autophagy, JNK activation, and cell death. In UL-1 cells, simvastatin treatment resulted in the cell cycle arrest at the G2/M phase, which was altered to G0/1 phase cell cycle arrest by treatment with lysosomal inhibitors. These findings demonstrate that depletion of isoprenoids by simvastatin induces autophagy-mediated cell death via downstream JNK activation and cell cycle dysregulation in canine T-cell lymphoma cells.

Plasma cell-free DNA in canine lymphoma patients as a novel material for genotyping.

Canine lymphoma is a disease with high morbidity and poor long-term prognosis, despite a high response rate to chemotherapy. In this study, we focused on liquid biopsy, in which small amounts of substances from body fluids were analysed, to determine whether cell-free DNA (cfDNA) in the plasma can be used as a biomarker for lymphoma in dogs. We found that 23 patients with lymphoma had significantly higher cfDNA concentrations than the 12 healthy dogs (median 2360 ng/mL versus 299 ng/mL, p < .0001). Polymerase chain reaction for antigen receptor rearrangement (PARR) was also employed using cfDNA from the lymphoma group to investigate whether cfDNA could be used for the detection of genetic clonality of lymphomas, as well as the genomic DNA (gDNA) extracted from an original lesion in each case. The correlation of the PARR results between cfDNA and gDNA was observed in 100% of B-cell lymphomas (10/10), 77.8% of T-cell lymphomas (7/9), and 100% of other types of lymphomas (4/4), respectively. These results indicate that plasma cfDNA levels are increasing in canine lymphoma patients, that cfDNA concentration can be a novel diagnostic tool, and that it can be used as a diagnostic tool for PARR.

Proof-of-concept study of the caninized anti-canine programmed death 1 antibody in dogs with advanced non-oral malignant melanoma solid tumors.

BACKGROUND: The anti-programmed death 1 (PD-1) antibody has led to durable clinical responses in a wide variety of human tumors. We have previously developed the caninized anti-canine PD-1 antibody (ca-4F12-E6) and evaluated its therapeutic properties in dogs with advance-staged oral malignant melanoma (OMM), however, their therapeutic effects on other types of canine tumors remain unclear. OBJECTIVE: The present clinical study was carried out to evaluate the safety profile and clinical efficacy of ca-4F12-E6 in dogs with advanced solid tumors except for OMM. METHODS: Thirty-eight dogs with non-OMM solid tumors were enrolled prospectively and treated with ca-4F12-E6 at 3 mg/kg every 2 weeks of each 10-week treatment cycle. Adverse events (AEs) and treatment efficacy were graded based on the criteria established by the Veterinary Cooperative Oncology Group. RESULTS: One dog was withdrawn, and thirty-seven dogs were evaluated for the safety and efficacy of ca-4F12-E6. Treatment-related AEs of any grade occurred in 13 out of 37 cases (35.1%). Two dogs with sterile nodular panniculitis and one with myasthenia gravis and hypothyroidism were suspected of immune-related AEs. In 30 out of 37 dogs that had target tumor lesions, the overall response and clinical benefit rates were 6.9% and 27.6%, respectively. The median progression-free survival and overall survival time were 70 days and 215 days, respectively. CONCLUSIONS: The present study demonstrated that ca-4F12-E6 was well-tolerated in non-OMM dogs, with a small number of cases showing objective responses. This provides evidence supporting large-scale clinical trials of anti-PD-1 antibody therapy in dogs.

Anti-adhesive property of P-selectin glycoprotein ligand-1 (PSGL-1) due to steric hindrance effect.

P-selectin glycoprotein ligand-1 (PSGL-1) is an adhesive molecule that is known to be a ligand for P-selectin. An anti-adhesive property of PSGL-1 has not been previously reported. In this study, we show that PSGL-1 expression is anti-adhesive for adherent cells and we have elucidated the underlying mechanism. Overexpression of PSGL-1 induced cell rounding and floating in HEK293T cells. Similar phenomena were demonstrated in other adherent cell lines with overexpression of PSGL-1. PSGL-1 overexpression inhibits access of antibodies to cell surface molecules such as integrins, HLA and CD25. Cells transfected with PSGL-1 deletion mutants that lack a large part of the extracellular domain and chimeric construct expressing extracellular CD86 and intracellular PSGL-1 only showed rounded morphology, but there are no floating cells. These results indicated that PSGL-1 causes steric hindrance due to the extended structure of its extracellular domain that is highly O-glycosylated, but intracellular domain also has some effect on cell rounding. This study implies that PSGL-1 has Janus-faced functions, being both adhesive and anti-adhesive.

Decreased sensitivity of cyclin-dependent kinase 4/6 inhibitors, palbociclib and abemaciclib to canine lymphoma cells with high p16 protein expression and low retinoblastoma protein phosphorylation.

Canine lymphoma/leukemia cell lines with p16 protein expressions: high (17-71 and GL-1) and low (CLBL-1, CLC, Nody-1, and UL-1) were treated in vitro with cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, palbociclib or abemaciclib. Cell proliferation decreased as a result, with higher IC50 levels observed in the high p16 (17-71 and GL-1) and one low p16 (UL-1) cell lines compared with the low p16 cells (CLBL-1, CLC, and Nody-1). As expected, palbociclib and abemaciclib treatment reduced pRb phosphorylation in a dose-dependent manner, especially in cells with low p16. These results suggest that CDK4/6 inhibitors have potential as new chemotherapeutic agents for canine lymphoma and high p16 protein expression may be used as a biomarker for resistance to CDK4/6 inhibitor therapy.

Effect of tolvaptan on hyponatremia in a dog with syndrome of inappropriate secretion of antidiuretic hormone.

A 1-year-old spayed female Miniature Schnauzer had chronic hyponatremia, accompanied by polyuria and polydipsia. Blood tests and urinalysis revealed severe hyponatremia, low plasma osmolality with euvolemia, and increased sodium excretion in urine. Hypothyroidism and hypoadrenocorticism were ruled out as causes. These findings led to the diagnosis of syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Magnetic resonance imaging (MRI) showed dilation of the lateral ventricles, indicating severe hydrocephalus. Tolvaptan, a vasopressin V2 receptor antagonist commonly used in human SIADH, was administered along with water restriction. This treatment resulted in a consistent increase in plasma sodium levels without any adverse effects. This case report represents the first documented evidence of the therapeutic efficacy of tolvaptan in treating SIADH in a dog.

MUC5AC and MUC5B expression in canine gallbladder mucocele epithelial cells.

Gallbladder mucocele (GBM) is one of the most common gallbladder diseases in dogs. Its pathogenesis has not yet been clarified, but excessive accumulation of a secretory gel-forming mucin, MUC5AC in the gallbladder has been reported. This study aimed to ascertain if MUC5AC overproduction resulted in mucus accumulation in the gallbladder during GBM development. Eleven dogs undergoing cholecystectomy who were pathologically diagnosed with GBM were included, and the expression level of mucins, particularly MUC5AC and MUC5B, in their gallbladder epithelial cells was compared with those in normal gallbladder epithelial cells. On reverse transcription-quantitative polymerase chain reaction screening, there was a significant difference (P<0.05) in the mRNA expression level of MUC1, but not of other mucins including MUC5AC and MUC5B, between mucocele and normal gallbladder epithelial cells. Protein expression levels were also evaluated for MUC5AC and MUC5B using immunohistochemistry. There was little immunoreactivity for MUC5AC, whereas MUC5B showed definitive staining in gallbladder epithelial cells. There was no difference in MUC5AC and MUC5B protein expression levels between mucocele and normal gallbladder epithelial cells. These data suggest that excessive production of mucin, especially MUC5AC and MUC5B, does not occur in canine GBM, and that abnormal mucus excretion, rather than excessive mucus production, may be the cause of GBM development.

Feline infectious peritonitis virus with a large deletion in the 5'-terminal region of the spike gene retains its virulence for cats.

In this study, the Japanese strain of type I feline infectious peritonitis virus (FIPV), C3663, was found to have a large deletion of 735 bp within the gene encoding the spike (S) protein, with a deduced loss of 245 aa of the N-terminal region of the S protein. This deletion is similar to that observed in porcine respiratory coronavirus (PRCoV) when compared to transmissible gastroenteritis virus, which correlates with reduced virulence. By analogy to PRCoV, we expected that the pathogenicity of C3663 may be attenuated in cats. However, two of four cats inoculated with C3663 died of FIP, and a third C3663-inoculated cat showed FIP lesions at 91 days after challenge. These results indicate that the 5'-terminal region of the S gene is not essential for the development of FIP.

Simultaneous Analysis of the p16 Gene and Protein in Canine Lymphoma Cells and Their Correlation with pRb Phosphorylation.

Cyclin-dependent kinase inhibitor p16 (CDKN2A) primarily functions as a negative regulator of the retinoblastoma protein (pRb) pathway to prevent pRb phosphorylation, thus playing a critical role in cell cycle arrest. In canine lymphoma cells, methylation due to inactivation of the p16 gene has been reported. However, its protein expression has not been examined in previous studies. In our in vitro study, the gene and protein expression of p16 and phosphorylated pRb were examined simultaneously in eight canine lymphoma and leukemia cell lines (17-71, CLBL-1, GL-1, CLC, CLGL-90, Ema, Nody-1, and UL-1). Methylation of the p16 gene was also explored using the demethylation drug 5-Aza-2'-deoxycytidine (5-Aza). After 5-Aza treatment, p16 gene and protein expression increased and pRb phosphorylation decreased, suggesting that both hypermethylation of the p16 gene and pRb hyperphosphorylation occurred in four out of eight cell lines (CLBL-1, CLC, Nody-1, and UL-1). Moreover, the estimation of p16's protein expression was better than that of p16's mRNA expression because the expression of the protein was more stable than those of the gene, and highly related to the phosphorylation of pRb. These results revealed that p16's protein expression could be a promising biomarker for canine lymphoma cells.

Improvement of anemia in five dogs with nonregenerative anemia treated with allogeneic adipose-derived stem cells.

Background: Nonregenerative anemia is occasionally seen in dogs and can be caused by many factors, among which nonregenerative immune-mediated anemia (NRIMA) and pure red cell anemia are relatively common causes. These are thought to be caused by immune-mediated destruction of the erythroid lineage and are treated with immunosuppressive drugs, but some of them are refractory or recurrent, so new treatments are needed. Objectives: To examine the efficacy of allogeneic adipose-derived stem cells (ADSCs) for the treatment of nonregerative anemia in dogs. Methods: ADSCs were administered to total five nonregenerative anemia cases; two NRIMA cases and two suspected NRIMA cases that were refractory to immunosuppressive agents, and one NRIMA case that has not been treated with immunosuppressive agents. Results: In all cases, anemia was improved, and blood transfusion was no longer necessary. Conclusions: This study suggests that allogeneic ADSCs may be one of the rescue therapies for the refractory immune-mediated anemia in dogs.

Intratumoral heterogeneity of c-KIT mutations in a feline splenic mast cell tumor and their functional effects on cell proliferation.

A cat was presented with mast cell tumors (MCTs) of the skin and spleen. During the initial diagnosis, the exon 8 mutation of c-KIT was detected in the masses from skin and spleen by a commercial laboratory test. Consequently, treatment with toceranib was started. After complete remission, because of recurrence on day 117, the spleen and skin tumors were removed, but the cat eventually died on day 191. The analysis of ten cDNA clones of the c-KIT gene cloned from the surgically removed spleen revealed that seven different cDNA patterns were included, indicating the heterogeneity of this gene in the splenic MCT. The seven cDNA nucleotide patterns can be classified into four protein sequence patterns. In addition to the previously known mutations in exon 8, we identified novel mutations in exons 9, 10, and 18; four amino acids deletion in exon 9, and a point mutation in exons 10 and 18. Mouse IL-3-dependent cell line, Ba/F3, was transduced with these mutant clones, and c-KIT phosphorylation and proliferation assays were performed. We found that certain mutations affected the c-KIT phosphorylation status and cell proliferation. This suggests that heterogeneity among the population of tumor cells exists in MCTs, and that the dominant clones of this heterogeneity may contribute to the subsequent tumor cell growth.

Prekallikrein deficiency in a dog.

Prekallikrein (PK) deficiency is an uncommon disorder in dogs. In this report, we describe a case of a dog that was referred for neurological defects and had a prolonged activated partial thromboplastin time (aPTT) and normal prothrombin time (PT) with no hemostatic defects. By using human PK-deficient plasma, the dog was diagnosed to have PK deficiency. The nucleotide sequence of normal canine PK cDNA was determined and compared with the genomic sequences of PK in the affected dog. The comparison revealed that the dog had a point mutation in exon 8 that leads to an amino acid substitution in the fourth apple domain of PK. This is the first report showing a point mutation of PK in a dog with PK deficiency.

Effect of simvastatin on cell proliferation and Ras activation in canine tumour cells.

Statins are inhibitors of the mevalonate cascade that is responsible for cholesterol biosynthesis and the formation of intermediate metabolites, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) used in the prenylation of proteins. Although statins are widely used in the treatment of hypercholesterolemia, recent studies suggest that they also inhibit proliferation of tumour cells by reducing prenylation of small GTP-binding proteins, such as, Ras. This study aimed to evaluate the effect of simvastatin on cell proliferation and Ras activation in various canine tumour cell lines, including hemangiosarcoma (HSA), melanoma, and lymphoma cell lines. Simvastatin inhibited cell proliferation of all cell lines tested in a concentration- and time-dependent manner, but the susceptibilities were different amongst the cell lines. Simvastatin induced apoptotic cell death via activation of caspase-3 and cell cycle arrest. The cytotoxic effects of simvastatin were attenuated by GGPP and FPP. Simvastatin decreased the amount of prenylated Ras and GTP-bound Ras in HSA and melanoma cell lines, but not in lymphoma cell lines. These results indicate that simvastatin induces cytotoxic effects through the depletion of GGPP and FPP in a variety of canine tumour cells, whereas multiple mechanisms are involved in the effects. Further study is required to elucidate the underlying mechanisms of simvastatin-induced cytotoxic effects in a variety of canine tumour cells.

Expression of DEP Domain-Containing 1B in Canine Lymphoma and Other Types of Canine Tumours.

Canine lymphoma is the most common haematological malignancy in dogs and is typically treated with multidrug chemotherapy. Most cases are at risk of relapse after several courses of chemotherapy and the oncogenic mechanism remains unknown. This study was aimed at identifying genes expressed in canine lymphoma by cDNA microarray. We found elevated expression of Dishevelled, EGL-10 and pleckstrin (DEP) domain-containing 1B (DEPDC1B) in canine lymphoma cells compared with cells and tissues from healthy dogs. Canine DEPDC1B protein was detected in 13 of 41 lymphoma specimens by immunohistochemistry, but was not detected in lymph nodes from normal dogs. Immunoreactive DEPDC1B protein was also detected in several other types of canine tumour. This is the first report documenting the association of DEPDC1B with canine cancer and the results suggest that DEPDC1B might serve as a potential marker or therapeutic target for canine malignancies.