Keitaro Morishita

Although immune checkpoint inhibitors (ICIs), such as the anti-programmed death-ligand 1 (PD-L1) antibody, have been developed for the treatment of canine malignant melanoma, desirable clinical efficacies have not been achieved. Recent studies in humans have suggested that radiation therapy (RT) combined with ICIs induces robust systemic antitumour immunity in patients with cancer. This study retrospectively examined the therapeutic efficacy of combination therapy (hypofractionated RT and anti-PD-L1 antibody [c4G12]) in dogs with pulmonary metastatic oral malignant melanoma. The intrathoracic clinical benefit rate (CBR)/median overall survival (OS) in the no RT (n = 20, free from the effect of RT), previous RT (n = 9, received RT ≤8 weeks prior to the first c4G12 dose), and concurrent RT (n = 10, c4G12 therapy within ±1 week of the first RT fraction) groups were 10%/185 days, 55.6%/283.5 days (p < 0.05 vs. no RT group), and 20%/129 days (p > 0.05 vs. no RT group), respectively. The adverse events were considered to be tolerable in the combination therapy. Thus, hypofractionated RT before the initiation of c4G12 therapy can be an effective approach for enhancing the therapeutic efficacy of immunotherapy, with acceptable safety profiles. Further prospective clinical studies are required to confirm the findings of this study.

Precursor-targeted immune-mediated anemia (PIMA) in dogs is characterized by persistent non-regenerative anemia and ineffective erythropoiesis, and it is suspected to be an immune-mediated disease. Most affected dogs respond to immunosuppressive therapies; however, some are resistant. In this study, we carried out splenectomy as an alternative therapy for refractory PIMA in dogs, and analyzed gene expression levels in the spleen of dogs with or without PIMA and in serum before and after splenectomy. A total of 1,385 genes were found to express differentially in the spleens from dogs with PIMA compared with healthy dogs by transcriptome analysis, of which 707 genes were up-regulated, including S100A12, S100A8, and S100A9 that are linked directly to the innate immune system and have been characterized as endogenous damage-associated molecular patterns. Furthermore, immunohistochemistry confirmed that S100A8/A9 protein expression levels were significantly higher in dogs with PIMA compared with those in healthy dogs. A total of 22 proteins were found to express differentially between the serum samples collected before and after splenectomy by proteome analysis, of which 12 proteins were up-regulated in the samples before. The lectin pathway of complement activation was identified by pathway analysis in pre-splenectomy samples. We speculated that S100A8/9 expression may be increased in the spleen of dogs with PIMA, resulting in activation of the lectin pathway before splenectomy. These findings further our understanding of the pathology and mechanisms of splenectomy for PIMA.

Objectives: We aimed to determine the response time to immunosuppressive therapy and time required to achieve a 5% increase in haematocrit among dogs with non‐regenerative immune‐mediated anaemia.Materials and Methods: Client‐owned dogs diagnosed with non‐regenerative immune‐mediated anaemia in Hokkaido University Veterinary Teaching Hospital between December 2012 and May 2018 were enrolled. The first treatment regimen included prednisolone (2 mg/kg/day) and ciclosporin (up to 10 mg/kg/day) for 8 weeks. Dogs that did not respond to the first regimen proceeded to the second regimen comprising prednisolone and mycophenolate mofetil (15 mg/kg, twice a day). Reticulocyte count and haematocrit were monitored every 1 to 2 weeks. Treatment response was defined as an absolute reticulocyte count more than 60×10³/μL or increasing haematocrit.Results: During the study period, 23 dogs fulfilled the inclusion criteria for non‐regenerative immune‐mediated anaemia. Twelve dogs were excluded from this study for various reasons and response to therapy was evaluated in the remaining 11 dogs. Treatment responses were observed in 8 of 11 dogs, and the median time to response was 39.5 days (range 8 to 92 days). Two responders were unable to continue the first treatment regimen and were switched to the second regimen owing to anorexia and nausea, possibly induced by ciclosporin; withdrawal of ciclosporin improved their symptoms. The time required to achieve a 5% increase in haematocrit was assessed in the other six dogs, with a median of 55.5 days (range 8 to 135 days).Clinical Significance: Here we report the response to a standardised treatment protocol in dogs with non‐regenerative immune‐mediated anaemia. Knowledge of potential side effects and expected therapeutic outcomes may be of use for veterinary practitioners treating this condition.

Dogs with precursor-targeted immune-mediated anemia (PIMA) are commonly treated with immunosuppressive therapy, but information on predictors of treatment response and response time is limited. Therefore, we retrospectively investigated predictive factors that influenced the treatment response and duration required to observe a response in dogs with PIMA receiving continuous immunosuppressive therapies for more than 105 days. Of 50 client-owned dogs that developed PIMA, 27 were included in this study, of which 18 were responders and 9 were non-responders to immunosuppressive therapies. Sixteen of the 18 responders responded to treatment within 60 days and the remaining 2 responded at 93 and 126 days, respectively. We found that an erythroid-maturation ratio of <0.17 may be a useful predictor for treatment response. In addition, complications of immunosuppressive therapies were investigated further in 50 dogs. Pancreatitis (n=4) and pneumonia (3) occurred over the entire treatment period, and infections such as abscesses (3) tended to be more common in dogs on an extended period of immunosuppressive therapy. These findings may be helpful when planning for the initial treatment and may provide evidence for informed consent about potential comorbidities throughout the treatment course.

Background The interaction between the left ventricle (LV) and systemic arterial systems, known as left ventricular-arterial coupling (VAC), has been evaluated based on the effective arterial elastance (Ea) to LV end-systolic elastance (Ees) ratio (Ea/Ees). The Ea reflects the total arterial load of LV, whereas Ees reflects the LV systolic function. A recent study found that inappropriate VAC based on increased Ea/Ees estimated by echocardiography is associated with advanced disease severity in dogs with myxomatous mitral valve disease (MMVD). Hypothesis Inappropriate VAC assessed by echocardiographic estimation of Ea/Ees is associated with a worse prognosis in dogs with MMVD. Animals Eighty-nine dogs with MMVD. Methods Prospective cohort study. Dogs underwent echocardiographic examinations at enrollment. The Ea was estimated using the formula: mean blood pressure/(forward stroke volume/body weight). The Ees was estimated using the formula: mean blood pressure/(LV end-systolic volume/body weight). The Ea/Ees was calculated. Results By end of study, 22 dogs died of cardiac-related causes with 67 dogs censored. Dogs with increased Ea/Ees (Ea/Ees >0.34; median survival time, 527 days; 95% confidence interval [CI], 322 days-not determinable) had a shorter survival time (P < .0001) than those without increased Ea/Ees (Ea/Ees ≤0.34; median survival time, >1112 days; 95% CI, not determinable). Multivariate Cox proportional hazard analysis showed that Ea/Ees, body weight, peak systolic mitral annular velocity, and the peak early diastolic transmitral velocity-to-peak early diastolic mitral annular velocity ratio were independent predictors of cardiac-related death among echocardiographic indices. Conclusions and Clinical Importance Inappropriate VAC assessed based on echocardiographically-estimated Ea/Ees is associated with a worse prognosis in dogs with MMVD.

Background The effective arterial elastance (Ea) to left ventricular (LV) end‐systolic elastance (Ees) ratio (Ea/Ees) is an index of the interaction between LV and systemic arterial systems, left ventricular‐arterial coupling (VAC). The Ea is an index of total arterial load of the LV, whereas Ees is an index of LV systolic function. In humans, inappropriate VAC based on increased Ea/Ees estimated using echocardiography is associated with more advanced heart disease severity. Hypothesis Left ventricular‐arterial coupling assessed by echocardiographic estimation of Ea/Ees is associated with disease severity in dogs with myxomatous mitral valve disease (MMVD). Animals Ninety MMVD dogs and 61 healthy dogs. Methods Prospective cross‐sectional study. The MMVD dogs were classified into stages B1, B2, or C according to American College of Veterinary Internal Medicine guidelines. Effective arterial elastance was echocardiographically estimated using the formula: mean blood pressure/(forward stroke volume/body weight). End‐systolic elastance was echocardiographically estimated using the formula: mean blood pressure/(LV end‐systolic volume/body weight). The ratio Ea/Ees was calculated. Results The ratio Ea/Ees was higher in stage B2 dogs than in healthy dogs and dogs stage B1 (both P < .0001), and higher in stage C dogs than in healthy dogs and dogs in the other 2 stages (healthy vs C and B1 vs C, P < .0001; B2 vs C, P = .0005). Multivariable logistic regression analysis showed that Ea/Ees and the peak velocity of early diastolic transmitral flow to isovolumic relaxation time ratio were independent predictors of stage C among echocardiographic indices in MMVD dogs. Conclusions and Clinical Importance Inappropriate VAC assessed by echocardiographically estimated Ea/Ees is associated with advanced disease severity in dogs with MMVD.

Background In dogs with protein‐losing enteropathy (PLE), data on the clinical characteristics of food‐responsive PLE (FR‐PLE) remain scarce. Objective To determine the clinical characteristics of FR‐PLE in dogs responsive to ultralow‐fat diet (ULFD) management. Animals Thirty‐three dogs diagnosed with PLE based on standard diagnostic criteria. Methods Retrospective review of medical records. Clinical findings were compared between dogs with FR‐PLE (FR‐PLE group) and those with immunosuppressant‐responsive PLE (IR‐PLE) or nonresponsive PLE (NR‐PLE) (IR/NR‐PLE group). The area under the curve (AUC) of a receiver operating characteristic curve was used to evaluate the ability of factors to differentiate the FR‐PLE and IR/NR‐PLE groups. Survival time was compared between the FR‐PLE and IR/NR‐PLE groups. Results Twenty‐three dogs responded to ULFD management and were diagnosed with FR‐PLE. The canine chronic enteropathy clinical activity index (CCECAI) was significantly lower in the FR‐PLE group than in the IR/NR‐PLE group (P < .001). The AUC of CCECAI for differentiating the FR‐PLE group was 0.935 (95% confidence interval [CI], 0.845‐1.000) with an optimal cutoff value of 8 (sensitivity, 0.826; specificity, 0.889). Survival times were significantly longer in the FR‐PLE group (median, not reached) than in the IR/NR‐PLE group (median, 432 days; P < .001). Conclusions and Clinical Importance Dogs that respond to ULFD management and are diagnosed with FR‐PLE are expected to have a favorable prognosis. Clinical scores, specifically the CCECAI, could be useful for differentiating FR‐PLE from IR‐PLE or NR‐PLE.

Background Two‐dimensional shear wave elastography (2D‐SWE) can noninvasively evaluate hepatic elastic modulus as shear wave velocity (SWV). Additionally, it may predict the presence of clinical relevant hepatic fibrosis (≥F2) in dogs with hepatic disease. Objectives To investigate whether SWV measured by 2D‐SWE can differentiate between dogs with (≥F2) and without (F0‐1) clinically relevant hepatic fibrosis. Animals Twenty‐eight client‐owned dogs with hepatic disease and 8 normal healthy Beagle dogs were enrolled. Methods In this cross‐sectional prospective study, SWVs were measured using 2D‐SWE in all dogs. Hepatic fibrosis stages and necroinflammatory activity grades were histopathologically evaluated using a histological scoring scheme that was adapted from the Ishak schema used in human medicine. Results Median SWVs were significantly higher in dogs with clinically relevant hepatic fibrosis (2.04 m/s; range, 1.81‐2.26 m/s) than in healthy dogs (1.51 m/s; range, 1.44‐1.66 m/s; P = .007), and dogs without clinically relevant hepatic fibrosis (1.56 m/s; range, 1.37‐1.67 m/s; P < .001). However, no significant difference was found in the SWVs between dogs without clinically relevant hepatic fibrosis and healthy dogs (P = .99). Furthermore, median SWVs were not significantly different among dogs with necroinflammatory activity, those without necroinflammatory activity, and healthy dogs (Kruskal‐Wallis test, P = .12). Conclusions and Clinical Importance The 2D‐SWE may be useful for predicting the presence of hepatic fibrosis in dogs with hepatic disease.

Background Lymphocytic‐plasmacytic enteritis is the common form of idiopathic inflammatory bowel disease (IBD) in dogs. In human IBD, disturbances of amino acid metabolism have been demonstrated to be involved in the pathophysiology of IBD. Therefore, plasma amino acid profile might represent a novel marker of human IBD. Objectives To determine the plasma amino acid profiles of dogs with IBD and its usefulness as a novel marker of IBD in dogs. Animals Fasting blood plasma was obtained from 10 dogs with IBD and 12 healthy dogs. Methods All IBD dogs were prospectively included in this study, and heparinized blood samples were collected. The plasma concentrations of 21 amino acids were determined using the ninhydrin method. The relationships among the plasma amino acid concentrations and plasma C‐reactive protein (CRP) concentration, canine chronic enteropathy clinical activity index (CCECAI), and overall World Small Animal Veterinary Association (WSAVA) score were investigated. Results Median concentration (nmol/mL) of methionine [46.2; range, 30.0‐59.3], proline [119.4; range, 76.7‐189.2], serine [115.1; range, 61.4‐155.9], and tryptophan [17.4; range, 11.9‐56.3]) were significantly lower than in control dogs [62.6; range, 51.0‐83.6, 199.1; range, 132.5‐376.7, 164.3; range, 124.7‐222.9, and 68.3; range, 35.7‐94.8, respectively]. A negative correlation was identified between the plasma serine concentration and CCECAI (r _s = −.67, P = .03), but there were no correlations between plasma amino acid concentrations and CRP concentration or overall WSAVA score. Conclusions and Clinical Importance Plasma serine concentration might represent a novel maker of IBD in dogs.