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Supplementary Figure from CCR4 Blockade Depletes Regulatory T Cells and Prolongs Survival in a Canine Model of Bladder Cancer

Supplementary Data from CCR4 Blockade Depletes Regulatory T Cells and Prolongs Survival in a Canine Model of Bladder Cancer

Regulatory T-cell (Treg) infiltration can be targeted as a cancer immunotherapy. Here, we describe therapeutic efficacy of this strategy in a canine model of bladder cancer. We used dogs with naturally occurring bladder cancer to study the molecular mechanism of Treg infiltration into bladder cancer tissues and the effect of anti-Treg treatment. Tumor-infiltrating Tregs were evaluated by immunohistochemistry, and their association with prognosis was examined in dogs with bladder cancer. The molecular mechanism of Treg infiltration was explored by RNA sequencing and protein analyses. Murine xenograft experiments and canine studies were used to explore the therapeutic potential of anti-Treg treatment for bladder cancer. We found that tumor-infiltrating Tregs were associated with poor prognosis in dogs bearing spontaneous bladder cancer. Treg infiltration was caused by interaction between the tumor-producing chemokine CCL17 and the receptor CCR4 expressed on Tregs. CCR4 blockade inhibited tumor growth and Treg infiltration into the tissues in a xenograft mouse model. Dogs with spontaneous bladder cancer responded to anti-CCR4 treatment with improved survival and low incidence of clinically relevant toxicities. In human patients with bladder cancer, immunohistochemistry showed that tumor-infiltrating Tregs expressed CCR4. Thus, anti-CCR4 treatment may be a rational approach to test in clinical trials for human patients with bladder cancer.

Supplementary Table from CCR4 Blockade Depletes Regulatory T Cells and Prolongs Survival in a Canine Model of Bladder Cancer

Protein carbonylation is an irreversible and degenerative modification that can be used to evaluate oxidative stress caused by glucocorticoids. In this study, we focused on protein carbonylation in dogs with hypercortisolism (HC). Sera samples were collected from 14 dogs diagnosed with HC and treated with trilostane, 12 dogs with inflammatory diseases (disease control group), and eight clinically healthy dogs. When the carbonylated protein levels were detected by the immunoblot analysis, one band of approximately 40 kDa was predominantly increased in the dogs with HC. The band was identified as haptoglobin using the liquid chromatography tandem mass spectrometry method. Furthermore, haptoglobin immune reactivity was higher in the dogs with HC. Although the average protein carbonylation level of the HC group was not significantly different from that of the other groups, the carbonylation level was significantly higher for the poorly controlled HC cases than for the well-controlled HC group. Additionally, the primary culture of canine hepatocytes was used to clarify the direct effect of glucocorticoids on protein carbonylation in dog livers. Both the carbonylated protein and haptoglobin clearly increased after 72 h. These findings suggest that haptoglobin and its carbonylated form are increased with canine HC, and that the protein carbonylation ratio and/or haptoglobin level could be related to disease management. These factors could be useful as biomarkers for an oxidative stress reaction, at least in the liver, and for treatment monitoring of HC.

Urethral sphincter mechanism incompetence (USMI) is a common cause of urinary incontinence in dogs. Although estrogen is often prescribed for the medical therapy of USMI for spayed female dogs, they are known to have limited effectiveness and potential adverse effects. In castrated male dogs with USMI, testosterone reagents have been attempted besides estrogen. In this study, the effect of testosterone drugs, mainly methyltestosterone, on spayed female dogs with USMI was retrospectively evaluated. Ten spayed female dogs with USMI were included. Diagnosis of USMI was based on the results of the dogs’ medical history, clinical signs, and no abnormalities in physical examinations, urinalysis, ultrasonography, X-ray imaging, and neurological examinations. Methyltestosterone was administered at doses of 0.32–1.27 mg/kg BW p.o. semel in die (sid.) to twice a week. Nine of the ten dogs had good or excellent responses 2 to 4 weeks after the start of treatment. The minimum effective dose was 0.32 mg/kg/day. Although no severe adverse symptoms occurred in any dog, a mild increase in alanine aminotransferase was temporally observed at doses of 1.0 and 1.1 mg/kg/day in the two dogs. After dose reduction or withdrawal, two of eight dogs had recurrence of urinary incontinence. Resumption of testosterone treatment clearly improved the symptoms in the two dogs. These results indicate that testosterone reagents might be an option for treating USMI in spayed female dogs as well.

Urothelial carcinoma (UC) is the most common tumor affecting the urinary bladder of dogs. Protein overexpression of ErbB2 (the canine homolog of HER2) has been observed in dogs with UC. However, no study regarding ErbB2 copy number aberration (CNA) is reported in dogs with UC. In this study, a digital PCR assay for detecting CNA of canine ErbB2 was developed. DNA samples were isolated from 83 formalin-fixed, paraffin-embedded urinary bladder tissues (36 UC, 8 polypoid cystitis, and 39 normal) and 94 urinary sediments (54 UC, 30 nonneoplastic, and 10 normal). The copy number of canine chromosome 8 ( CFA8) was used as a control. In the urinary bladder tissues, ErbB2 CNA was detected in 12 of 36 (33%) UC, 2 of 8 (25%) polypoid cystitis, and 0 of 39 (0%) normal controls. In the urinary sediments, ErbB2 CNA was also detected in 19 of 54 (35%) UC; however, no ErbB2 CNA was detected in nonneoplastic diseases or normal controls. The sensitivity and specificity of ErbB2 CNA in urinary sediment for the detection of UC were 35% and 100%, respectively. There was a positive correlation between the copy number ratios of ErbB2 to CFA8 in the urinary bladder tissues and urinary sediments. Our findings indicate that the digital PCR assay of urinary sediments may be a useful, noninvasive method for detecting ErbB2 CNA in dogs with UC.

In humans, plasma amino acids (AAs) levels are used as dynamic nutritional markers. Moreover, some AAs are associated with chronic inflammation. In this study, we analyzed plasma AA profiles in cats with chronic gastrointestinal (GI) diseases. Eight healthy controls (HCs) and 12 client-owned cats with chronic GI diseases including chronic enteritis (n=8) and neoplasms (n=4) were recruited. Plasma albumin, total protein, and 22 AAs (11 essential and 11 non-essential AAs) levels were estimated. There was no significant difference in plasma albumin and total protein concentrations between the cats with chronic GI diseases and HCs. The plasma concentrations of 7 essential AAs (arginine, histidine, lysine, methionine, phenylalanine, taurine, and tryptophan) and 7 non-essential AAs (asparagine, aspartic acid, glutamic acid, glycine, hydroxyproline, proline, and serine) were significantly decreased in the cats with chronic GI diseases (P

Short chain fatty acids (SCFAs) play an important role in the maintenance of colonic homeostasis, and their depletion has been reported in various gastrointestinal disorders. Inflammatory colorectal polyps (ICRPs) are a recently recognized disease specific to miniature dachshunds (MDs), and fecal dysbiosis with a reduction of SCFA-producing bacteria has been reported with this disease. Therefore, this study was performed based on the hypothesis that a reduced SCFA concentration associates with the development of ICRPs. We recruited 11 ICRP-affected MDs and 25 control MDs. Their fecal SCFA concentrations and bacterial proportions were quantified using high performance liquid chromatography and quantitative real-time PCR, respectively. The feces of ICRP-affected MDs contained lower amounts of propionic acid and lower proportions of Bifidobacterium than the feces of control MDs. Furthermore, fecal proportions of Bifidobacterium, Firmicutes and Lactobacillus exhibited significant positive correlations with fecal concentrations of total SCFAs and/or propionic acid; fecal Escherichia coli proportions correlated negatively with fecal concentrations of total SCFAs, as well as acetic, propionic and butyric acid. This result indicates an association between fecal dysbiosis and fecal SCFA concentrations; these phenomena may contribute to ICRP pathogenesis in MDs. Potential therapeutic targeting of the reduced propionic acid concentration using probiotics, prebiotics or SCFA enemas merits further study.

Although alteration of commensal microbiota is associated with chronic gastrointestinal (GI) diseases such as inflammatory bowel disease (IBD) in dogs, the microbiota composition in intestinal lymphoma, an important differential diagnosis of canine IBD, has not been investigated. The objective of this study was to compare the fecal microbiota in dogs with IBD, dogs with intestinal lymphoma, and healthy dogs. Eight dogs with IBD, eight dogs with intestinal lymphoma, and fifteen healthy dogs were included in the study. Fecal samples were analyzed by 16S rRNA gene next-generation sequencing. Rarefaction analysis failed to reveal any difference in bacterial diversity among healthy dogs and diseased dogs. Based on PCoA plots of unweighted UniFrac distances, the bacterial composition in dogs with intestinal lymphoma was different from those observed in dogs with IBD and healthy dogs. When compared with healthy dogs, intestinal lymphoma subjects showed significant increases in organisms belonging to the Eubacteriaceae family. The proportion of the family Paraprevotellaceae and the genus Porphyromonas was significantly higher in dogs with IBD compared to healthy dogs. These observations suggest that dysbiosis is associated with intestinal lymphoma as well as IBD in dogs.

A 4-y-old cat exhibited neurologic signs such as wobbling, right head tilt, and intention tremor, and MRI revealed a mass in the cerebellum. The cat died 5 mo after initial presentation, and no neoplastic lesions, other than the cerebellar mass, were observed at autopsy. Histologically, large atypical cells resembling Hodgkin cells, with single large inclusion-like nucleoli, and those resembling Reed–Sternberg cells, with symmetrically arranged nuclei, had infiltrated the left side of the cerebellum and were admixed with small lymphocytes. These atypical cells were positive for feline leukemia virus (FeLV), CD20, BLA36, vimentin, p16, p53, and Pax5, and negative for CD3, CD79a, and Iba1 by immunohistochemistry. Multiplex PCR for immunoglobulin heavy-chain gene rearrangement revealed monoclonal proliferation of B-lymphocytes. We describe this feline primary cerebellar B-cell lymphoma that displayed Hodgkin lymphoma–like tumor cells with FeLV protein expression.

Inflammatory bowel disease (IBD) is a common gastrointestinal disease in dogs. Decreased production of intestinal immunoglobulin A (IgA) has been suggested as a possible pathogenesis in a subset of canine IBD; however, the underlying cause remains unclear. Sphingosine-1-phosphate (S1P) is a lipid mediator that regulates intestinal IgA production by controlling lymphocyte trafficking in mice. The objectives of this study were to clarify the role of S1P in IgA production in dogs and to evaluate the expression of S1P-related molecules in dogs with IBD. First, an S1P receptor antagonist was administrated to five healthy dogs. The S1P receptor antagonist significantly decreased the IgA concentration in sera and feces but did not affect the IgG concentration. Moreover, the immunoreactivity of intestinal IgA was significantly decreased by S1P signal blockade. These results indicate that S1P signaling specifically regulates the intestinal IgA production in dogs. Subsequently, the intestinal S1P concentration and the expression of S1P-related molecules were measured in dogs with IBD and healthy dogs. The intestinal concentration of S1P was significantly lower in dogs with IBD than in healthy dogs. In addition, the gene expression levels of S1P receptor (S1P1) and S1P synthase (SK1) were significantly lower in dogs with IBD than in healthy dogs. Taken together, these observations suggest that decreased S1P production, likely caused by a lower expression of S1P synthetase, leads to attenuation of S1P/S1P1 signaling pathway and the production of intestinal IgA in dogs with IBD.

Contagious yawning is triggered by others yawning, and it has previously been reported in humans, primates and several experimental and companion mammals. Whereas it might be a response to an innate releasing mechanism, contagious yawning is also considered to involve emotional contagion. Here, we demonstrate that sheep, the animal model of livestock animals, also experience contagious yawning. Twelve adult castrated Corriedale sheep were used in this study. Pairs of sheep were adjacently restrained with or without a wooden divider panel to shield them from viewing the other. Their behaviors were video-recorded for 3 days in each condition. Sheep yawned 2.0 ± 1.1 and 1.2 ± 1.1 times/day/head in the unshielded and shielded conditions, respectively. Unshielded restrained sheep yawned within 1 min after the other one 11.1% of the time, while shielded pairs did not exhibit contagious yawning. Rumination was also highly synchronized under the unshielded condition. These data reveal that contagious yawning and behavioral synchronicity occur in ruminants like sheep, making them a suitable animal model to investigate contagious yawning and the underlying mechanism.

Canine urothelial carcinoma (UC) has a poor prognosis and high metastatic rate. Human epidermal growth factor receptor 2 (HER2), a receptor tyrosine kinase involved in cell proliferation and differentiation regulation, has been attracting interest as a therapeutic target molecule for human breast cancer. This study investigated expression of the canine homolog of HER2 (ERBB2) in canine UC, and its association with clinical factors. Since it has been controversial whether commercial anti-human HER2 antibody (Dako A0485) correctly recognizes the canine homolog of HER2, an application of the antibody using a canine UC cell line was validated first. By Western blot, a single band at the appropriate size for canine HER2 (185 kDa) was recognized. Immunohistochemistry for HER2 was performed on 23 samples of UC, 8 samples of polypoid cystitis, and 8 samples of normal urinary bladder, and the results were scored as either 0, 1+, 2+, or 3+ with reference to the evaluation method for human UC. Intense membranous HER2 immunoreactivity was frequently observed in neoplastic cells, especially in grade 2 UC. Minor HER2 expression was found in the epithelial cells of polypoid cystitis and normal bladder. The incidence of HER2 positivity (scores of 2+ or 3+) was 14 of 23 (60.9%) in UC, 3 of 8 (37.5%) in polypoid cystitis, and 0 of 8 (0%) in normal bladder. There was no significant correlation between HER2 positivity and clinical factors. While increased HER2 expression was observed in a subset of urothelial carcinomas, further mechanistic studies are needed to determine its role in the pathogenesis and targeted therapy of this cancer.

Combined_supplemental_materials-Sakai_et_al for ErbB2 Copy Number Aberration in Canine Urothelial Carcinoma Detected by a Digital Polymerase Chain Reaction Assay by Kosei Sakai, Shingo Maeda, Kohei Saeki, Ryohei Yoshitake, Yuko Goto-Koshino, Takayuki Nakagawa, Ryohei Nishimura, Tomohiro Yonezawa and Naoaki Matsuki in Veterinary Pathology

Supplemental Material, DS1_VET_10.1177_0300985818817024 for Assessment of Human Epidermal Growth Factor Receptor 2 Expression in Canine Urothelial Carcinoma of the Urinary Bladder by Masaya Tsuboi, Kosei Sakai, Shingo Maeda, James K. Chambers, Tomohiro Yonezawa, Naoaki Matsuki, Kazuyuki Uchida, and Hiroyuki Nakayama in Veterinary Pathology

Although antimicrobial peptides (AMPs) play an integral role in the regulation of intestinal microbiota and homeostasis, their expression in canine gastrointestinal diseases, including idiopathic inflammatory bowel disease (IBD) and intestinal lymphoma, remains unknown. The objective of this study was to investigate the intestinal expression of AMPs in dogs with IBD or intestinal lymphoma. IBD was diagnosed in 44 dogs, small cell intestinal lymphoma in 25 dogs, and large cell intestinal lymphoma in 19 dogs. Twenty healthy beagles were used as normal controls. Duodenal mRNA expression of six representative AMPs - lactoferrin, lysozyme, cathelicidin, secretory leukocyte peptidase inhibitor (SLPI), bactericidal/permeability increasing protein (BPI), and canine beta defensin (CBD103) - was quantified by real-time reverse transcription polymerase chain reaction. The relative expression of BPI, lactoferrin, and SLPI was significantly higher in dogs with IBD and intestinal lymphomas than in healthy controls. Interestingly, the expression patterns of AMPs differed between dogs with IBD and those with intestinal lymphomas, especially small cell lymphoma. Increased expression of BPI differentiated IBD from dogs with small cell intestinal lymphoma, with a sensitivity of 93.2%, a specificity of 100%, and an area under the curve of 0.955. These results suggest that the expression patterns of AMP aid in the diagnosis of canine IBD and intestinal lymphoma, although it remains uncertain whether the altered AMP expression is the cause or effect of mucosal inflammation.

Regulatory T-cell (Treg) infiltration can be targeted as a cancer immunotherapy. Here, we describe therapeutic efficacy of this strategy in a canine model of bladder cancer. We used dogs with naturally occurring bladder cancer to study the molecular mechanism of Treg infiltration into bladder cancer tissues and the effect of anti-Treg treatment. Tumor-infiltrating Tregs were evaluated by immunohistochemistry, and their association with prognosis was examined in dogs with bladder cancer. The molecular mechanism of Treg infiltration was explored by RNA sequencing and protein analyses. Murine xenograft experiments and canine studies were used to explore the therapeutic potential of anti-Treg treatment for bladder cancer. We found that tumor-infiltrating Tregs were associated with poor prognosis in dogs bearing spontaneous bladder cancer. Treg infiltration was caused by interaction between the tumor-producing chemokine CCL17 and the receptor CCR4 expressed on Tregs. CCR4 blockade inhibited tumor growth and Treg infiltration into the tissues in a xenograft mouse model. Dogs with spontaneous bladder cancer responded to anti-CCR4 treatment with improved survival and low incidence of clinically relevant toxicities. In human patients with bladder cancer, immunohistochemistry showed that tumor-infiltrating Tregs expressed CCR4. Thus, anti-CCR4 treatment may be a rational approach to test in clinical trials for human patients with bladder cancer.

A 6-year 5-month-old spayed female Scottish Fold cat presented with a one-month history of gait abnormalities, increased salivation, and decreased activity. A blood test showed hyperammonemia and increased serum bile acids. Imaging tests revealed multiple shunt vessels indicating acquired portosystemic shunt. Histopathologic analysis of liver biopsy showed features consistent with liver hypoperfusion, such as a barely recognizable portal vein, increased numbers of small arterioles, and diffuse vacuolar degeneration of hepatocytes. These findings supported the diagnosis of primary hypoplasia of the portal vein/microvascular dysplasia, (PHPV/MVD). To our knowledge, this is the first case of feline PHPV/MVD that developed multiple acquired portosystemic shunts and presented with hepatic encephalopathy.

Background Invasive urothelial carcinoma (iUC) is a major cause of death in humans, and approximately 165,000 individuals succumb to this cancer annually worldwide. Comparative oncology using relevant animal models is necessary to improve our understanding of progression, diagnosis, and treatment of iUC. Companion canines are a preferred animal model of iUC due to spontaneous tumor development and similarity to human disease in terms of histopathology, metastatic behavior, and treatment response. However, the comprehensive molecular characterization of canine iUC is not well documented. In this study, we performed transcriptome analysis of tissue samples from canine iUC and normal bladders using an RNA sequencing (RNA-Seq) approach to identify key molecular pathways in canine iUC. Methods Total RNA was extracted from bladder tissues of 11 dogs with iUC and five healthy dogs, and RNA-Seq was conducted. Ingenuity Pathway Analysis (IPA) was used to assign differentially expressed genes to known upstream regulators and functional networks. Results Differential gene expression analysis of the RNA-Seq data revealed 2531 differentially expressed genes, comprising 1007 upregulated and 1524 downregulated genes, in canine iUC. IPA revealed that the most activated upstream regulator was PTGER2 (encoding the prostaglandin E2 receptor EP2), which is consistent with the therapeutic efficiency of cyclooxygenase inhibitors in canine iUC. Similar to human iUC, canine iUC exhibited upregulated ERBB2 and downregulated TP53 pathways. Biological functions associated with cancer, cell proliferation, and leukocyte migration were predicted to be activated, while muscle functions were predicted to be inhibited, indicating muscle-invasive tumor property. Conclusions Our data confirmed similarities in gene expression patterns between canine and human iUC and identified potential therapeutic targets (PTGER2, ERBB2, CCND1, Vegf, and EGFR), suggesting the value of naturally occurring canine iUC as a relevant animal model for human iUC. Electronic supplementary material The online version of this article (10.1186/s12885-018-4409-3) contains supplementary material, which is available to authorized users.

Apoptosis inhibitor of macrophage (AIM) is initially reported to protect macrophages from apoptosis. In this study, we determined the effect of AIM on the macrophage-derived tumor, histiocytic sarcoma cell lines (HS) of dogs. Five HS and five other tumor cell lines were used. When recombinant canine AIM was applied to non-serum culture media, cell numbers of all the HS and two of other tumor cell lines decreased dose-dependently. The DNA fragmentation, TUNEL staining and flow cytometry tests revealed that AIM induced both of apoptosis and cell cycle arrest in the HS. Although AIM is known as an apoptosis inhibitor, these results suggest that a high dose of AIM could have an opposite function in HS and some tumor cell lines.

The activity of matrix metalloproteinase (MMP)-2 and MMP-9 in synovial fluids (SF) sampled from dogs with joint disorders was investigated by gelatin zymography and densitometry. Pro-MMP-2 showed similar activity levels in dogs with idiopathic polyarthritis (IPA; n=17) or canine rheumatoid arthritis (cRA; n=4), and healthy controls (n=10). However, dogs with cranial cruciate ligament rupture (CCLR; n=5) presented significantly higher pro-MMP-2 activity than IPA and healthy dogs. Meanwhile, dogs with IPA exhibited significantly higher activity of pro- and active MMP-9 than other groups. Activity levels in pro- and active MMP-9 in cRA and CCLR dogs were not significantly different from those in healthy controls. Different patterns of MMP-2 and MMP-9 activity may reflect the differences in the underlying pathological processes.

Regulatory T cells (Tregs) infiltrate into a variety of tumour tissues and associate with poor prognosis in humans. However, data on association of Treg infiltration with prognosis is limited in canine tumours. The purpose of this study was to examine the number of tumour-infiltrating Tregs and its association with overall survival (OS) in dogs with malignant tumours. The following 168 canine tumours were included: 37 oral malignant melanomas (OMMs); 14 oral squamous cell carcinomas (OSCCs); 16 pulmonary adenocarcinomas (PAs); 37 mammary carcinomas (MCs); 36 mast cell tumours (MCTs) and 28 hepatocellular carcinomas (HCCs). Normal tissues were obtained from 8 healthy dogs as controls. The number of forkhead box P3 (Foxp3)-positive Tregs in intratumoral and peritumoral areas was investigated by immunohistochemistry. OS was compared between high and low Treg groups. The number of intratumoral and peritumoral Foxp3-positive Tregs was significantly higher in OMM, OSCC, PA and MC compared with each normal tissue. There were few Foxp3-positive Tregs in MCT and HCC. With intratumoral Tregs, the OS in the high Treg group was significantly shorter than that in the low Treg group in OMM, OSCC and PA. With peritumoral Tregs, there was no significant difference for OS between the 2 groups in each tumour type. These results suggest that Tregs infiltrate into a variety of canine tumours and the abundance of Tregs are associated with poor prognosis in some solid tumour types.

Objective To evaluate the utility of the chemiluminescent enzyme immunoassay (CLEIA) method for point-of-care (POC) measurement of canine plasma thrombin-antithrombin complex (TAT) concentration. Assessment and main results Plasma TAT concentration was measured in 54 healthy dogs and in 72 dogs with various diseases. A significant correlation was found between TAT concentration measured by CLEIA and that measured by an ELISA that was previously used in dogs. The upper limit of the reference value of TAT concentrations measured by CLEIA was determined to be 0.2 ng/mL based on the TAT concentration in 54 healthy dogs. TAT concentrations exceeded the reference interval in a portion of dogs when a hypercoagulable state may be present. Conclusions Canine plasma TAT concentrations measured using CLEIA were correlated with that measured using ELISA. Hence, a POC testing instrument may be used for early detection of activation of thrombin generation in emergency and critical care settings.

Although cytology is a rapid diagnostic procedure in dogs, the cytologic criteria of endoscopic biopsies for chronic enteritis and intestinal lymphoma are not well defined. An immediate diagnosis using cytology would benefit patients by enabling prompt initiation of therapy. The objective of this study was to investigate the correlation between the results of endoscopic cytology and histopathology. In this study, 167 dogs with clinical signs of chronic gastrointestinal disease were included. On the basis of histopathology, the following diagnoses were determined: lymphocytic-plasmacytic enteritis in 93 dogs; eosinophilic enteritis in 5 dogs; small cell intestinal lymphoma in 45 dogs; and large cell intestinal lymphoma in 24 dogs. Two clinical pathologists retrospectively evaluated the endoscopic cytology of squash-smear preparations. The cytologic diagnoses of inflammation, small cell lymphoma, and large cell lymphoma were based on the severity of lymphocyte infiltration, the size of infiltrated lymphocytes, and eosinophil/mast cell infiltration. The clinical severity score was significantly increased along with the degree of lymphocyte infiltration evaluated by cytology. The cytologic diagnosis was in complete agreement with the histopathologic diagnosis in 136 of 167 (81.4%) cases. For the differentiation between enteritis and lymphoma, endoscopic cytology had a sensitivity of 98.6%, a specificity of 73.5%, a positive predictive value of 72.3%, and a negative predictive value of 98.6%. The log-rank test and Cox regression analysis showed that the results of cytology predicted the prognosis. These results suggest that endoscopic cytology is a useful technique to aid diagnosis of intestinal inflammation and lymphoma in dogs.

Although tumor-infiltrating lymphocytes (TILs) play a key role in anti-tumor immunity, their involvement in canine transitional cell carcinoma (TCC) is not well-documented. The objective of this study was to investigate the association between TIL number and prognosis in dogs with urinary bladder TCC. Immunohistochemical analysis of CD3 and granzyme B was performed using canine TCC (n=32) and normal bladder (n=10) tissues. The numbers of CD3+ and granzyme B+ cells located in peritumoral stroma of canine TCC were significantly higher than those in normal controls. In TCC cases, the number of CD3+ TILs was not significantly related to prognosis, whereas the abundant granzyme B+ TILs were associated with favorable outcome. Since granzyme B+ TILs were not associated with the tumor stage, the presence of granzyme B+ TILs may be an independent prognostic factor. These results suggest that granzyme B+ TILs play a role in anti-tumor immunity and inhibit tumor progression in canine TCC.

Summary A strain of familial spontaneous epileptic cats (FSECs) with typical limbic seizures was identified in 2010. The electroencephalographic features suggested that an epileptogenic zone is present in the mesial temporal structures (i.e., amygdala and/or hippocampus). In this study, visual evaluations and quantitative analyses were performed by using 3D MR hippocampal volumetry in comparing FSECs with age-matched controls. Visual hippocampal asymmetries were seen in 8 of 14 (57.1%) FSECs. The FSEC group showed a significantly higher asymmetric ratio (4.15%) than the control group (0.99%). The smaller side of hippocampal volume (HV) (0.206cm 3 ) in FSECs was significantly smaller than the mean HV in controls (0.227cm 3 ). However, the means of left and right HVs and total HVs in FSECs showed no differences because the laterality of hippocampal atrophy was different in each individual. Therefore, since FSECs represent a true model of spontaneous epilepsy, hippocampal volumetry should be evaluated in each individual as well as in human patients. The significant asymmetry of HV suggests the potential for hippocampal atrophy in FSECs.

Idiopathic polyarthritis (IPA) is a very common inflammatory arthropathy in the dog. Canine IPA is diagnosed mainly by detecting increased number of leukocytes in the synovial fluid (SF), which is easily influenced by glucocorticoid therapy. We obtained 31 SF samples from 24 IPA dogs prior to (n=19) and/or after (n=12) 1 to 10 weeks of glucocorticoid therapy. The SF total protein concentrations of IPA dogs were significantly higher than those of dogs with non-arthritis diseases (n=34) and healthy controls (n=10). Our data revealed that the SF total protein concentrations are not influenced by several weeks of glucocorticoid therapy. Hence, the SF total protein concentration is applicable as a diagnostic marker of canine IPA even when the patients are receiving glucocorticoid therapy.

Apoptosis inhibitor of macrophage (AIM) plays roles in survival of macrophages. In this study, we cloned canine AIM cDNA and observed its transcriptional expression levels in various tissues. The coding sequence of canine AIM was 1,023 bp encoding 340 amino acid residues, which had around 65% homology with those of the human, mouse and rat. Transcriptional expression of AIM was observed in the spleen, lung, liver and lymph node, which confirmed the expression of canine AIM in tissue macrophages. Moreover, AIM was highly expressed in one of the canine histiocytic sarcoma cell lines. CD36, the receptor of AIM, was also expressed in various tissues and these cell lines. These findings are useful to reveal the actual functions of canine AIM.

Contagious yawning is triggered by others yawning, and it has previously been reported in humans, primates and several experimental and companion mammals. Whereas it might be a response to an innate releasing mechanism, contagious yawning is also considered to involve emotional contagion. Here, we demonstrate that sheep, the animal model of livestock animals, also experience contagious yawning. Twelve adult castrated Corriedale sheep were used in this study. Pairs of sheep were adjacently restrained with or without a wooden divider panel to shield them from viewing the other. Their behaviors were video-recorded for 3 days in each condition. Sheep yawned 2.0 ± 1.1 and 1.2 ± 1.1 times/day/head in the unshielded and shielded conditions, respectively. Unshielded restrained sheep yawned within 1 min after the other one 11.1% of the time, while shielded pairs did not exhibit contagious yawning. Rumination was also highly synchronized under the unshielded condition. These data reveal that contagious yawning and behavioral synchronicity occur in ruminants like sheep, making them a suitable animal model to investigate contagious yawning and the underlying mechanism.

Canine idiopathic polyarthritis (IPA) is characterized by increased numbers of polymorphonuclear leukocytes (PMNs) in the synovial fluid (SF). In humans, CC chemokine ligand 2 (CCL2) and CXC chemokine ligand 8 (CXCL8) recruit monocytes and neutrophils, respectively, and are involved in various inflammatory disorders. The aim of this study was to assess the roles of these chemokines in driving PMNs infiltration into the joints of dogs with IPA. SF samples were collected from dogs with IPA (n=19) and healthy controls (n=8), and the concentrations of SF CCL2 and CXCL8 were determined by ELISA. Dogs with IPA had significantly higher concentrations of CCL2 (3316±2452pg/ml, mean±SD) and CXCL8 (3668±3879pg/ml) compared with the healthy controls (235±45pg/ml and

A disease characterized by tongue and facial muscle atrophy has been recognized sporadically among Pembroke Welsh Corgi (PWC) dogs in Japan. The present study describes the pathologic findings of this canine syndrome. Histopathologic examinations were performed in 2 dogs, including a case of muscular biopsy. Identification and characterization of autoantibodies were attempted by fluorescent antibody test (FAT) and Western blot (WB) by using sera from 7 PWC dogs with typical clinical features, 6 PWC dogs with other clinical signs, and 2 from other breeds with polymyositis. Clinically, the 7 affected PWC dogs exhibited dysphagia with severe tongue atrophy, facial muscular atrophy, and occasional walking difficulty. Histopathologic examinations of the 2 dogs with clinical symptoms revealed moderate to severe inflammatory lesions characterized by lymphohistiocytic infiltration and muscular atrophy in the tongue and/or femoral muscles. The tongue lesions were very severe and accompanied by diffuse fatty infiltration. There were no major lesions in the nervous tissues examined. By FAT, an autoantibody against the cross striation of skeletal muscle was detected in sera from 5 affected PWC dogs. By using WB analysis, the autoantibodies recognized a 42-kDa molecule in striated muscle but not in the nervous tissues. All of the findings indicated that the unique disease of PWC dogs might be generalized inflammatory myopathy, whereas the detailed etiology concerning the dominant involvement of tongue muscles and the role of the autoantibody in the canine disease remain to be clarified.

Some of the pharmacological properties of phytosteryl ferulates may be linked to their antioxidant potential. In this study, 2,2-diphenyl-1-picrylhydrazyl (DPPH), electron spin resonance (ESR), and thiobarbituric acid–reactive substances (TBARS) assays demonstrated that phytosteryl ferulates such as cycloartenyl ferulate (CAF), 24-methylenecycloartanyl ferulate (24-mCAF), and β-sitosteryl ferulate (β-SF) and ferulic acid (FA) each exerted strong free radical scavenging and antioxidation of lipid membrane, which were comparable to α-tocopherol. However, the sterol moiety alone, such as cycloartenol (CA), had neither activity. Since, the reactive oxygen species (ROS) production in the cell complex defense mechanism cannot be ruled out with the cell free system, we measured ROS production in NIH 3T3 fibroblast cells induced by H2O2. CAF and ethyl ferulate (eFA) greatly decreased the ROS level in this system. CA also significantly inhibited the ROS level, suggesting that CA could inhibit ROS production in living cells. Besides these, CAF, 24-mCAF, β-SF, as well as eFA and CA, all these chemicals significantly inhibited the NF-κB activity as analyzed by measuring translocation of NF-κB p65 in LPS-stimulated RAW 264.7 macrophages. These observations revealed that phytosteryl ferulates are responsible for the antioxidant and anti-inflammatory activity via ROS scavenging and inhibition of ROS production. Keywords:: phytosteryl ferulate, cycloartenyl ferulate, NF-κB, antioxidant, reactive oxygen species (ROS)

To clarify the involvement of excitatory and inhibitory amino acids in canine necrotizing meningoencephalitis (NME), glutamate, aspartate, taurine and gamma-aminobutylic acid (GABA) were determined in the cerebrospinal fluids (CSF) from eight NME cases and ten healthy controls. NME dogs exhibited significantly higher concentrations of glutamate and aspartate than those in controls (p

To investigate in vitro differentiation of canine adipose tissue-derived stromal cells (ATSCs) into neuronal cells, ATSCs from celiac adipose tissue in clinically healthy beagle dogs were treated with 100 muM dibutyryl cyclic adenosine monophosphate (dbcAMP) and 125 muM isobuthylmethylxanthine (IBMX). ATSCs were morphologically changed into differentiated ATSCs from spindle-shaped cells to neuron-like cells with numerous processes after the treatment. Expression of neuron-specific enolase (NSE) as an early neuron specific marker protein was detected in both ATSCs and differentiated ATSCs, however diachronic increase of NSE expression was observed in differentiated ATSCs after the treatment with dbcAMP/IBMX. In addition, neurofilament-68 (NF-68) as an early to mature neuron specific marker protein was weakly expressed in differentiated ATSCs. Neuron specific glutamate and glucose transporter (EAAC1 and GLUT-3, respectively) mRNAs were strongly expressed in differentiated ATSCs compared with those in ATSCs, although glia specific glutamate transporter mRNA (GLT-1) was also detected in differentiated ATSCs. ATSCs can differentiate into early to mature neuronal cells and are candidate cells for autologous nerve regeneration therapy, although additional research is needed to examine functional characteristics of differentiated ATSCs.

UTokyo Research掲載「月の満ち欠けがウシの出産に影響を与える」 URI: http://www.u-tokyo.ac.jp/ja/utokyo-research/research-news/lunar-cycle-affects-timing-of-birth-in-cows.html, UTokyo Research "Lunar cycle affects timing of birth in cows" URI: http://www.u-tokyo.ac.jp/en/utokyo-research/research-news/lunar-cycle-affects-timing-of-birth-in-cows.html

To establish clinical markers for canine necrotising meningoencephalitis (NME) and to elucidate its pathogenesis, glial fibrillary acidic protein (GFAP) and anti-GFAP autoantibodies were measured in the cerebrospinal fluid (CSF) of 32 dogs with NME, 23 dogs with other inflammatory central nervous system (CNS) diseases, 27 dogs with miscellaneous CNS diseases and 25 healthy dogs, including five pugs. The dogs with NME had the highest levels of anti-GFAP autoantibodies. The diagnostic sensitivity and specificity of anti-GFAP autoantibodies for NME were 91 per cent and 73 per cent, respectively. Some of the dogs with NME and the healthy pugs, had high CSF concentrations of GFAP, suggesting a breed-specific fragility of astrocytes. The leakage of GFAP and the development of autoimmunity may be key to understanding the pathogenesis of NME.

Little information is available regarding triple-phase computed tomography (CT) of canine pancreatic insulinoma. A few case reports with small numbers of cases have indicated that hyper-attenuation in the arterial phase was a common finding on multi-phasic CT in dogs with insulinoma. Our purpose was to clarify the characteristic findings of dogs with insulinoma on triple-phase CT. Nine dogs with insulinoma that underwent triple-phase CT were included in the present study. Attenuation patterns in the arterial phase indicated hypo-attenuation in 4 cases and hyper-attenuation in 2 cases. In the remaining 3 cases, 1 case showed hypo-attenuation and 1 case showed hyper-attenuation in the pancreatic phase, and 1 case presented hyper-attenuation in the later phase. Altogether, 5 cases showed hypo and 4 cases showed hyper-attenuation in at least one phase. The enhancement pattern was homogenous in 7 cases and heterogeneous in 2 cases. Tumor margins were well-defined in 5 cases and ill-defined in 4 cases. Capsule formation was present in 5 cases and absent in 4 cases. In conclusion, it is important to note that hypo-attenuation was as common as hyper-attenuation in dogs with insulinoma in triple-phase CT in at least one phase. Additionally, mass lesions were most conspicuous not only in the arterial phase but in the pancreatic and later phases in some cases. Therefore, it is important to perform triple-phase CT and notice about variable findings for the detection of canine pancreatic insulinoma.

Seven novel cell lines from canine histiocytic sarcoma (HS), three of which were disseminated cutaneous HS and four of which were synovial HS, were established. All of the established cell lines had the same morphological (by light and electron microscopic findings), cytochemical (alpha-naphthyl butyrate esterase-positive), and immunohistochemical (vimentin- and lysozyme-positive, and cyto-keratin-negative) characteristics as the original HS tumor cells. All of the established cell lines injected into nude mice subcutaneously produced solid tumors. Because the established cell lines also showed phagocytic and processing activities, the HS tumor cells appear to originate from the mononuclear phagocytic system cells, despite their differences in locations or organs.

Age-associated changes of visual evoked potentials by flash stimulation (flash VEP) were evaluated in 53 beagle dogs aged from 1- to 15-year-old. Among the components of flash VEP consisted of 3 positive (P1, P2 and P3) and 2 negative (N1 and N2) peaks by 150 msec, the latency of P2 and the later peaks (N2 and P3) were significantly delayed with aging. Both amplitudes of the P2-N2 and N2-P3 also showed a significant correlation with aging. The flash VEP is considered to be an available and useful technique to evaluate not only for visual pathway, but also some disturbance of neurological functions, like as those reported in demented human.

The expression of parathyroid hormone-related protein (PTHrP) mRNA was examined in mammary gland with or without lactation, and during periparturient period in a Holstein cow and a Jersey cow. In the lactating mammary gland, PTHrP was detected in alveolar epithelial cells and the lumen by immunohistochemical analysis. The relative expression levels of PTHrP mRNA in mammary gland from lactating cows were significantly higher than those from non-lactating cows (P

Visual evoked potentials by flash stimulation (flash VEP) were analyzed in dogs using a topographic method. The flash VEP consisted of 3 positive (P1, P2 and P3) and 2 negative (N1 and N2) components by 150 msec after the flash stimuli. On the topographic mappings, a negative response area was observed in the frontal region of the scalp in the stimulated site followed by the shifting of the area to the contralateral frontal region and occipital region, during the first 100 msec. The negative response area in the frontal region in the stimulated site, contralateral frontal and temporal region, and occipital region were corresponded to N1, P2, and N2 on the flash VEP, respectively, according to their latencies. In the dogs with experimentally impaired the right lateral geniculate body, the latency of P2 was prolonged, and N2 and P3 were disappeared after the left eye stimulation. On the topographic mapping, only the early negative response area was detected on the stimulated site of the frontal region of the brain. Therefore, it is concluded that P1 and N1, P2, and N2 are referred to the retinal potentials, the potentials from the retina to the brainstem included the lateral geniculate body, and those from the brainstem to the visual cortex, respectively.