Cathepsin W, T-cell receptor-associated transmembrane adapter 1, lymphotactin and killer cell lectin like receptor K1 are sensitive and specific RNA biomarkers of canine epitheliotropic lymphoma.

Cutaneous T-cell lymphoma (CTCL) is an uncommon type of lymphoma involving malignant skin-resident or skin-homing T cells. Canine epitheliotropic lymphoma (EL) is the most common form of CTCL in dogs, and it also spontaneously arises from T lymphocytes in the mucosa and skin. Clinically, it can be difficult to distinguish early-stage CTCLs apart from other forms of benign interface dermatitis (ID) in both dogs and people. Our objective was to identify novel biomarkers that can distinguish EL from other forms of ID, and perform comparative transcriptomics of human CTCL and canine EL. Here, we present a retrospective gene expression study that employed archival tissue from biorepositories. We analyzed a discovery cohort of 6 canines and a validation cohort of 8 canines with EL which occurred spontaneously in client-owned companion dogs. We performed comparative targeted transcriptomics studies using NanoString to assess 160 genes from lesional skin biopsies from the discovery cohort and 800 genes from the validation cohort to identify any significant differences that may reflect oncogenesis and immunopathogenesis. We further sought to determine if gene expression in EL and CTCL are conserved across humans and canines by comparing our data to previously published human datasets. Similar chemokine profiles were observed in dog EL and human CTCL, and analyses were performed to validate potential biomarkers and drivers of disease. In dogs, we found enrichment of T cell gene signatures, with upregulation of IFNG, TNF, PRF1, IL15, CD244, CXCL10, and CCL5 in EL in dogs compared to healthy controls. Importantly, CTSW, TRAT1 and KLRK1 distinguished EL from all other forms of interface dermatitis we studied, providing much-needed biomarkers for the veterinary field. XCL1/XCL2 were also highly specific of EL in our validation cohort. Future studies exploring the oncogenesis of spontaneous lymphomas in companion animals will expand our understanding of these disorders. Biomarkers may be useful for predicting disease prognosis and treatment responses. We plan to use our data to inform future development of targeted therapies, as well as for repurposing drugs for both veterinary and human medicine.

Using cultured canine cardiac slices to model the autophagic flux with doxorubicin.

Chemotherapy-induced impairment of autophagy is implicated in cardiac toxicity induced by anti-cancer drugs. Imperfect translation from rodent models and lack of in vitro models of toxicity has limited investigation of autophagic flux dysregulation, preventing design of novel cardioprotective strategies based on autophagy control. Development of an adult heart tissue culture technique from a translational model will improve investigation of cardiac toxicity. We aimed to optimize a canine cardiac slice culture system for exploration of cancer therapy impact on intact cardiac tissue, creating a translatable model that maintains autophagy in culture and is amenable to autophagy modulation. Canine cardiac tissue slices (350 µm) were generated from left ventricular free wall collected from euthanized client-owned dogs (n = 7) free of cardiovascular disease at the Foster Hospital for Small Animals at Tufts University. Cell viability and apoptosis were quantified with MTT assay and TUNEL staining. Cardiac slices were challenged with doxorubicin and an autophagy activator (rapamycin) or inhibitor (chloroquine). Autophagic flux components (LC3, p62) were quantified by western blot. Cardiac slices retained high cell viability for >7 days in culture and basal levels of autophagic markers remained unchanged. Doxorubicin treatment resulted in perturbation of the autophagic flux and cell death, while rapamycin co-treatment restored normal autophagic flux and maintained cell survival. We developed an adult canine cardiac slice culture system appropriate for studying the effects of autophagic flux that may be applicable to drug toxicity evaluations.

Retrospective evaluation of canine primary, multicentric, and metastatic intraocular neoplasia.

OBJECTIVE: To provide an updated characterization of the prevalence of primary, multicentric, and metastatic intraocular tumors in the canine patient. PROCEDURES: Medical records databases from 4 veterinary referral hospitals were reviewed from 1999 to present to identify dogs with a diagnosis of intraocular neoplasia histopathologically confirmed following enucleation or necropsy. RESULTS: One hundred seventy-two dogs with 173 intraocular neoplasms met the inclusion criteria. Primary intraocular neoplasms were the most common tumors in the study (128); the two most common types were melanocytic neoplasia (90), followed by iridociliary neoplasia (33). There were 28 cases of intraocular involvement secondary to round cell neoplasia, with 18 cases of lymphoma, seven histiocytic sarcomas, and three undifferentiated round cell neoplasms. There were 17 cases of metastatic intraocular neoplasia, with hemangiosarcoma being the most common (9). CONCLUSIONS: The majority of intraocular tumors in dogs arise from the ocular tissues. However, the eye may also be involved in patients with multicentric neoplasia, and, less commonly, as a site for metastatic disease. Ocular screening for patients with multicentric neoplasia should be considered during staging, and ocular signs should be viewed with suspicion in dogs with neoplasia in other sites.

Development and validation of a multivariable model and online decision-support calculator to aid in preoperative discrimination of benign from malignant splenic masses in dogs.

OBJECTIVE: To develop a multivariable model and online decision-support calculator to aid in preoperative discrimination of benign from malignant splenic masses in dogs. ANIMALS: 522 dogs that underwent splenectomy because of splenic masses. PROCEDURES: A multivariable model was developed with preoperative clinical data obtained retrospectively from the records of 422 dogs that underwent splenectomy. Inclusion criteria were the availability of complete abdominal ultrasonographic examination images and splenic histologic slides or histology reports for review. Variables considered potentially predictive of splenic malignancy were analyzed. A receiver operating characteristic curve was created for the final multivariable model, and area under the curve was calculated. The model was externally validated with data from 100 dogs that underwent splenectomy subsequent to model development and was used to create an online calculator to estimate probability of splenic malignancy in individual dogs. RESULTS: The final multivariable model contained 8 clinical variables used to estimate splenic malignancy probability: serum total protein concentration, presence (vs absence) of ≥ 2 nRBCs/100 WBCs, ultrasonographically assessed splenic mass diameter, number of liver nodules (0, 1, or ≥ 2), presence (vs absence) of multiple splenic masses or nodules, moderate to marked splenic mass inhomogeneity, moderate to marked abdominal effusion, and mesenteric, omental, or peritoneal nodules. Areas under the receiver operating characteristic curves for the development and validation populations were 0.80 and 0.78, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The online calculator (T-STAT.net or T-STAT.org) developed in this study can be used as an aid to estimate the probability of malignancy in dogs with splenic masses and has potential to facilitate owners' decisions regarding splenectomy.

LC-ESI-QTOF-MS/MS Characterization of Seaweed Phenolics and Their Antioxidant Potential.

Seaweed is an important food widely consumed in Asian countries. Seaweed has a diverse array of bioactive compounds, including dietary fiber, carbohydrate, protein, fatty acid, minerals and polyphenols, which contribute to the health benefits and commercial value of seaweed. Nevertheless, detailed information on polyphenol content in seaweeds is still limited. Therefore, the present work aimed to investigate the phenolic compounds present in eight seaweeds [Chlorophyta (green), Ulva sp., Caulerpa sp. and Codium sp.; Rhodophyta (red), Dasya sp., Grateloupia sp. and Centroceras sp.; Ochrophyta (brown), Ecklonia sp., Sargassum sp.], using liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS). The total phenolic content (TPC), total flavonoid content (TFC) and total tannin content (TTC) were determined. The antioxidant potential of seaweed was assessed using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, a 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) free radical scavenging assay and a ferric reducing antioxidant power (FRAP) assay. Brown seaweed species showed the highest total polyphenol content, which correlated with the highest antioxidant potential. The LC-ESI-QTOF-MS/MS tentatively identified a total of 54 phenolic compounds present in the eight seaweeds. The largest number of phenolic compounds were present in Centroceras sp. followed by Ecklonia sp. and Caulerpa sp. Using high-performance liquid chromatography-photodiode array (HPLC-PDA) quantification, the most abundant phenolic compound was p-hydroxybenzoic acid, present in Ulva sp. at 846.083 ± 0.02 µg/g fresh weight. The results obtained indicate the importance of seaweed as a promising source of polyphenols with antioxidant properties, consistent with the health potential of seaweed in food, pharmaceutical and nutraceutical applications.

Evaluation of cytology and histopathology for the diagnosis of feline orbital neoplasia: 81 cases (2004-2019) and review of the literature.

OBJECTIVE: To provide an updated overview of feline orbital neoplasia, to compare diagnostic utility of cytology and histopathology, and to evaluate minimally invasive sampling modalities. PROCEDURES: A medical records search was performed to identify cats with orbital neoplasia. Data were collected regarding signalment, diagnosis, vision status, imaging modalities, and sample collection methods. A reference population with orbital neoplasia was also identified via literature search for comparison with regard to final diagnosis. RESULTS: Eighty-one cats met selection criteria and 140 cases were identified in the literature. In the study and reference populations, respectively, diagnoses were grouped as follows: round cell tumors 47% and 24%, epithelial tumors 38% and 40%, mesenchymal tumors 14% and 34%, and neurologic origin tumors 1% and 2%. The most common diagnoses in both groups were lymphoma and squamous cell carcinoma (SCC). Feline restrictive orbital myofibroblastic sarcoma (FROMS) was common in the reference population but not diagnosed in the study population. Cytology results were available for 41 cats; histopathology results were available for 65 cats. Both cytology and histopathology results were available for 25 cats, in 44% of which cytologic results were overturned. No significant complications were associated with any sampling method. Lack of cats with multiple samples available for histopathology limited comparison between tissue sampling methods. CONCLUSIONS: Orbital neoplasia is common in cats, with round cell and epithelial tumors diagnosed most commonly in the study population. Histopathology is superior to cytology in providing a definitive diagnosis. Minimally invasive tissue biopsy techniques appear to be safe and effective.

Evaluation of cytology and histopathology for the diagnosis of canine orbital neoplasia: 112 cases (2004-2019) and review of the literature.

OBJECTIVE: To provide an updated overview of canine orbital neoplasia, to compare diagnostic utility of cytology and histopathology, and to evaluate alternative sampling modalities, particularly image-guided core needle biopsy. PROCEDURES: A medical records search was performed to identify dogs with orbital neoplasia. Data were collected regarding signalment, diagnosis, vision status, imaging modalities, and sample collection methods. A reference population with orbital neoplasia was also identified via literature search for comparison with regard to final diagnosis. RESULTS: One hundred and twelve dogs met selection criteria. In the study and reference populations, respectively, diagnoses were grouped as follows: mesenchymal tumors 40% and 35%, epithelial tumors 35% and 18%, tumors of neural origin 8% and 37%, and round cell 17% and 10%. The most common diagnoses in the study group were nasal adenocarcinoma, osteosarcoma, lymphoma, and meningioma. Cytology results were available for 47 dogs and histopathology results were available for 95 dogs. Both cytology and histopathology results were available for 30 dogs, in 53% of which results were discordant. Cytology samples were nondiagnostic or provided a diagnosis that was later overturned in 32% of cases in which they were obtained. Results from core needle biopsy samples were nondiagnostic or overturned by surgical biopsy results in only 13% of cases. No significant complications were associated with any sampling method. CONCLUSIONS: Orbital neoplasia is common in dogs. Histopathology is superior to cytology in providing a definitive diagnosis. Image-guided core needle biopsy appears to be a safe and effective means of obtaining samples.

Dysregulation of valvular interstitial cell let-7c, miR-17, miR-20a, and miR-30d in naturally occurring canine myxomatous mitral valve disease.

Canine myxomatous mitral valve disease (MMVD) resembles the early stages of myxomatous pathology seen in human non-syndromic mitral valve prolapse, a common valvular heart disease in the adult human population. Canine MMVD is seen in older subjects, suggesting age-related epigenetic dysregulation leading to derangements in valvular cell populations and matrix synthesis or degradation. We hypothesized that valvular interstitial cells (VICs) undergo disease-relevant changes in miRNA expression. In primary VIC lines from diseased and control valves, miRNA expression was profiled using RT-qPCR and next generation sequencing. VICs from diseased valves showed phenotypic changes consistent with myofibroblastic differentiation (vimentinlow+, α-SMAhigh+), increases in senescence markers (p21, SA-ß-gαl), and decreased cell viability and proliferation potential. RT-qPCR and miRNA sequencing analyses both showed significant (p<0.05) downregulation of let-7c, miR-17, miR-20a, and miR-30d in VICs from diseased valves compared to controls. Decreased let-7c, miR-17, and miR-20a may contribute to myofibroblastic differentiation in addition to cell senescence, and decreased miR-30d may disinhibit cell apoptosis. These data support the hypothesis that epigenetic dysregulation plays an important role in age-related canine MMVD.

Impact of a refined advanced design for left atrial appendage exclusion.

OBJECTIVES: Exclusion of the left atrial appendage has been proposed to reduce the risk of stroke in patients with atrial fibrillation. The aim of this study was to evaluate the feasibility and efficacy of the AtriClip PRO·V device (AOD2), now in development, for left atrial appendage exclusion in a canine model. METHODS: The newest AtriClip design comprises a dual-spring mechanism that allows the clip to open into a 'V' shape while still providing equivalent force along the length of the beam. The AOD2's hallmark is a distal tip closure to help retain the appendage during clip closure. Six dogs were implanted via thoracotomy with the clinically available AtriClip device (AOD1) on the right atrial appendage and the AOD2 on the left. At 90 days after implantation, all devices were evaluated by epicardial echocardiography, computed tomography, gross pathology and histology. System performance at the initial surgery was evaluated as well. RESULTS: The ease of use of the clinically available AtriClip device (AOD1) and AOD2 was deemed comparable in all cases. All animals survived for the planned 90-day duration without complications. The atrial appendages were fully occluded in all cases without device migration. On histology, all AtriClip devices demonstrated an acceptable biocompatibility response. CONCLUSIONS: The AOD2 achieved easy, reliable and safe exclusion of the left atrial appendage, with favourable histologic findings. Once approved for clinical application, the AOD2 could provide a new therapeutic option to lower the risks of stroke in patients with atrial fibrillation.

Epipodial Tentacle Gene Expression and Predetermined Resilience to Summer Mortality in the Commercially Important Greenlip Abalone, Haliotis laevigata.

"Summer mortality" is a phenomenon that occurs during warm water temperature spikes that results in the mass mortality of many ecologically and economically important mollusks such as abalone. This study aimed to determine whether the baseline gene expression of abalone before a laboratory-induced summer mortality event was associated with resilience to summer mortality. Tentacle transcriptomes of 35 greenlip abalone (Haliotis laevigata) were sequenced prior to the animals being exposed to an increase in water temperature-simulating conditions which have previously resulted in summer mortality. Abalone derived from three source locations with different environmental conditions were categorized as susceptible or resistant to summer mortality depending on whether they died or survived after the water temperature was increased. We detected two genes showing significantly higher expression in resilient abalone relative to susceptible abalone prior to the laboratory-induced summer mortality event. One of these genes was annotated through the NCBI non-redundant protein database using BLASTX to an anemone (Exaiptasia pallida) Transposon Ty3-G Gag Pol polyprotein. Distinct gene expression signatures were also found between resilient and susceptible abalone depending on the population origin, which may suggest divergence in local adaptation mechanisms for resilience. Many of these genes have been suggested to be involved in antioxidant and immune-related functions. The identification of these genes and their functional roles have enhanced our understanding of processes that may contribute to summer mortality in abalone. Our study supports the hypothesis that prestress gene expression signatures are indicative of the likelihood of summer mortality.

Assessment of eosinophil peroxidase as a potential diagnostic and prognostic marker in dogs with inflammatory bowel disease.

OBJECTIVE To evaluate a method for identifying intact and degranulated eosinophils in the small intestine of dogs with inflammatory bowel disease (IBD) by use of a monoclonal antibody (mAb) against eosinophil peroxidase (EPX). ANIMALS 11 untreated dogs with IBD, 5 dogs with IBD treated with prednisolone, and 8 control dogs with no clinical evidence of gastrointestinal tract disease and no immunosuppressive treatment. PROCEDURES 4-µm-thick sections of paraffin-embedded tissues from necropsy specimens were immunostained with EPX mAb. Stained intact and degranulated eosinophils in consecutive microscopic fields (400X magnification) of the upper (villus tips) and lower (between the muscularis mucosae and crypts) regions of the lamina propria of the jejunum were manually counted. RESULTS Compared with control and treated IBD dogs, untreated IBD dogs had a significantly higher number of degranulated eosinophils in the lower region of the lamina propria. However, no significant differences were detected in the number of intact eosinophils in this region among groups. In the upper region of the lamina propria, untreated IBD dogs had a significantly higher number of degranulated and intact eosinophils, compared with control and treated IBD dogs. Number of degranulated and intact eosinophils did not differ significantly between control and treated IBD dogs. CONCLUSIONS AND CLINICAL RELEVANCE Immunohistologic analysis with EPX mAb yielded prominent granule staining that allowed reliable morphological identification of degranulated and intact eosinophils, which may provide a strategy for quantitative and selective evaluation of eosinophils in gastrointestinal biopsy specimens and a potential method to diagnose IBD and evaluate treatment outcome.

Ovarian mixed germ cell tumor with yolk sac and teratomatous components in a dog.

Mixed germ cell tumors of the ovary have rarely been reported in veterinary species. A 3-year-old intact female Labrador Retriever dog was presented for lethargy, abdominal distention, and a midabdominal mass. An exploratory laparotomy revealed a large (23 cm in diameter) left ovarian tumor and multiple small (2-3 cm in diameter) pale tan masses on the peritoneum and abdominal surface of the diaphragm. Histological examination of the left ovary revealed a mixed germ cell tumor with a yolk sac component with rare Schiller-Duval bodies and a teratomatous component comprised primarily of neural differentiation. The abdominal metastases were solely comprised of the yolk sac component. The yolk sac component was diffusely immunopositive for cytokeratin with scattered cells reactive for α-fetoprotein and placental alkaline phosphatase. Within the teratomatous component, the neuropil was diffusely immunopositive for S100, neuron-specific enolase, and neurofilaments with a few glial fibrillary acidic protein immunopositive cells. Ovarian germ cell tumors may be pure and consist of only 1 germ cell element or may be mixed and include more than 1 germ cell element, such as teratoma and yolk sac tumor.

Management of adverse side-effects after chemotherapy in macaques as exemplified by streptozotocin: case studies and recommendations.

The chemotherapeutic streptozotocin is used for induction of diabetes in animal models including non-human primates. Being a cytotoxic nitrosourea compound, it can be associated with adverse events (AEs), mainly nausea and emesis, nephrotoxicity, elevated liver transaminase levels, pulmonary oedema and, most prominently, metabolic acidosis: these can be severe in some cases. The incidence and gravity are to some extent related to the characteristics of the individual animal, diagnostic tools, prompt recognition of symptoms and supportive measures. Careful animal selection, dose adaptation and supportive actions such as renal protective hydration are the main tools in managing AEs, but do not fully eliminate unavoidable and sometimes life-threatening conditions. In our centre we have built experience in a cohort of 78 cynomolgus and rhesus macaques in which six cases manifested severe AEs (8%). This experience has prompted implementation of strategies for early detection and management of adverse effects, together with an animal refinement programme. We present here specific pretreatment regimens, post-infusion laboratory evaluations, and flow charts to assess/treat metabolic acidosis and precipitating factors. Case reports of the six animals with severe AEs are presented to illustrate management of AEs, especially metabolic acidosis, and criteria for early euthanasia where appropriate. We conclude that improved monitoring and validated tools allow for optimal management of adverse effects in an early stage of their manifestation. Reduced morbidity and mortality not only improve individual animal wellbeing but also avoid model-induced confounding that diminishes the translational value of the experimental protocol.

Tubular hypoplasia of the aorta and right atrioventricular valve dysplasia in a Bulldog.

A Bulldog puppy that died at 1 day of age was presented for postmortem evaluation. Macroscopically, there was marked hypoplasia of the ascending, transverse, and proximal segments of the descending thoracic aorta and almost complete secondary thrombosis of the left ventricle causing a functional stenosis of the left atrioventricular valve. Separately, there was right atrioventricular valve dysplasia with secondary dilation of the right atrium. Microscopically, the left ventricular outflow tract was occluded by chondroid metaplasia, fibrosing recanalization of a left-ventricular thrombus, and isolated Purkinje fiber degeneration and necrosis.