Remdesivir increases mtDNA copy number causing mild alterations to oxidative phosphorylation.

SARS-CoV-2 causes the severe respiratory disease COVID-19. Remdesivir (RDV) was the first fast-tracked FDA approved treatment drug for COVID-19. RDV acts as an antiviral ribonucleoside (adenosine) analogue that becomes active once it accumulates intracellularly. It then diffuses into the host cell and terminates viral RNA transcription. Previous studies have shown that certain nucleoside analogues unintentionally inhibit mitochondrial RNA or DNA polymerases or cause mutational changes to mitochondrial DNA (mtDNA). These past findings on the mitochondrial toxicity of ribonucleoside analogues motivated us to investigate what effects RDV may have on mitochondrial function. Using in vitro and in vivo rodent models treated with RDV, we observed increases in mtDNA copy number in Mv1Lu cells (35.26% increase ± 11.33%) and liver (100.27% increase ± 32.73%) upon treatment. However, these increases only resulted in mild changes to mitochondrial function. Surprisingly, skeletal muscle and heart were extremely resistant to RDV treatment, tissues that have preferentially been affected by other nucleoside analogues. Although our data suggest that RDV does not greatly impact mitochondrial function, these data are insightful for the treatment of RDV for individuals with mitochondrial disease.

Inhibitory checkpoint molecule mRNA expression in canine soft tissue sarcoma.

Canine soft tissue sarcomas (STS) are common neoplasms and considered immune deserts. Tumour infiltrating lymphocytes are sparse in STS and, when present, tend to organize around blood vessels or at the periphery of the neoplasm. This pattern is associated with an immunosuppressive tumour microenvironment linked to overexpression of molecules of the PD-axis. PD-1, PD-L1 and PD-L2 expression correlates with malignancy and poor prognosis in other neoplasms in humans and dogs, but little is known about their role in canine STS, their relationship to tumour grade, and how different therapies affect expression. The objective of this study was to evaluate the expression of checkpoint molecules across STS tumour grades and after tumour ablation treatment. Gene expression analysis was performed by reverse-transcriptase real-time quantitative PCR in soft tissue sarcomas that underwent histotripsy and from histologic specimens of STS from the Virginia Tech Animal Laboratory Services archives. The expression of PD-1, PD-L1 and PD-L2 was detected in untreated STS tissue representing grades 1, 2, and 3. Numerically decreased expression of all markers was observed in tissue sampled from the treatment interface relative to untreated areas of the tumour. The relatively lower expression of these checkpoint molecules at the periphery of the treated area may be related to liquefactive necrosis induced by the histotripsy treatment, and would potentially allow TILs to infiltrate the tumour. Relative increases of these checkpoint molecules in tumours of a higher grade and alongside immune cell infiltration are consistent with previous reports that associate their expression with malignancy.

Characterization of the Expression of Angiogenic Factors in Cutaneous Squamous Cell Carcinoma of Domestic Cats.

Cutaneous squamous cell carcinoma (CSCC) is a common malignant skin cancer with a significant impact on health, and it is important to determine the degree of reliance of CSCC on angiogenesis for growth and metastasis. Major regulators of angiogenesis are the vascular endothelial growth factor (VEGF) family and their associated receptors. Alternative pre-mRNA splicing produces multiple isoforms of VEGF-A and PLGF with distinct biological properties. Several studies highlight the function of VEGF-A in CSCC, but there are no studies of the different isoforms of VEGF-A and PLGF for this neoplasm. We characterized the expression of three isoforms of VEGF-A, two isoforms of PLGF, and their receptors in cat CSCC biopsies compared to normal haired skin (NHS). Although our results revealed no significant changes in transcript levels of panVEGF-A or their isoforms, the mRNA levels of PLGF I and the receptors Flt-1 and KDR were downregulated in CSCC compared to NHS. Differences were observed in ligand:receptor mRNA expression ratio, with the expression of VEGF-A relative to its receptor KDR higher in CSCC, which is consistent with our hypothesis and prior human SCC studies. Immunolocalization in tissue showed increased expression of all measured factors and receptors in tumor cells compared to NHS and surrounding vasculature. We conclude that the factors measured may play a pivotal role in CSCC growth, although further studies are needed to clarify the role of angiogenic factors in feline CSCC.

Interstitial pneumonia and diffuse alveolar damage in domestic animals.

Classification of pneumonia in animals has been controversial, and the most problematic pattern is interstitial pneumonia. This is true from the gross and histologic perspectives, and also from a mechanistic point of view. Multiple infectious and noninfectious diseases are associated with interstitial pneumonia, all of them converging in the release of inflammatory mediators that generate local damage and attract inflammatory cells that inevitably trigger a second wave of damage. Diffuse alveolar damage is one of the more frequently identified histologic types of interstitial pneumonia and involves injury to alveolar epithelial and/or endothelial cells, with 3 distinct stages. The first is the "exudative" stage, with alveolar edema and hyaline membranes. The second is the "proliferative" stage, with hyperplasia and reactive atypia of type II pneumocytes, infiltration of lymphocytes, plasma cells, and macrophages in the interstitium and early proliferation of fibroblasts. These stages are reversible and often nonfatal. If damage persists, there is a third "fibrosing" stage, characterized by fibrosis of the interstitium due to proliferation of fibroblasts/myofibroblasts, persistence of type II pneumocytes, segments of squamous metaplasia of alveolar epithelium, plus inflammation. Understanding the lesion patterns associated with interstitial pneumonias, their causes, and the underlying mechanisms aid in accurate diagnosis that involves an interdisciplinary collaborative approach involving pathologists, clinicians, and radiologists.

Canine melanoma: A review of diagnostics and comparative mechanisms of disease and immunotolerance in the era of the immunotherapies.

Melanomas in humans and dogs are highly malignant and resistant to therapy. Since the first development of immunotherapies, interest in how the immune system interacts within the tumor microenvironment and plays a role in tumor development, progression, or remission has increased. Of major importance are tumor-infiltrating lymphocytes (TILs) where distribution and cell frequencies correlate with survival and therapeutic outcomes. Additionally, efforts have been made to identify subsets of TILs populations that can contribute to a tumor-promoting or tumor-inhibiting environment, such as the case with T regulatory cells versus CD8 T cells. Furthermore, cancerous cells have the capacity to express certain inhibitory checkpoint molecules, including CTLA-4, PD-L1, PD-L2, that can suppress the immune system, a property associated with poor prognosis, a high rate of recurrence, and metastasis. Comparative oncology brings insights to comprehend the mechanisms of tumorigenesis and immunotolerance in humans and dogs, contributing to the development of new therapeutic agents that can modulate the immune response against the tumor. Therapies that target signaling pathways such as mTOR and MEK/ERK that are upregulated in cancer, or immunotherapies with different approaches such as CAR-T cells engineered for specific tumor-associated antigens, DNA vaccines using human tyrosinase or CGSP-4 antigen, anti-PD-1 or -PD-L1 monoclonal antibodies that intercept their binding inhibiting the suppression of the T cells, and lymphokine-activated killer cells are already in development for treating canine tumors. This review provides concise and recent information about diagnosis, comparative mechanisms of tumor development and progression, and the current status of immunotherapies directed toward canine melanoma.

PD-1, PD-L1, and PD-L2 Gene Expression and Tumor Infiltrating Lymphocytes in Canine Melanoma.

Melanoma in humans and dogs is considered highly immunogenic; however, the function of tumor-infiltrating lymphocytes (TILs) is often suppressed in the tumor microenvironment. In humans, current immunotherapies target checkpoint molecules (such as PD-L1, expressed by tumor cells), inhibiting their suppressive effect over TILs. The role of PD-L2, an alternative PD-1 ligand also overexpressed in malignant tumors and in patients with anti-PD-L1 resistance, remains poorly understood. In the current study, we evaluated the expression of checkpoint molecule mRNAs in canine melanoma and TILs. Analysis of checkpoint molecule gene expression was performed by RT-qPCR (real-time quantitative polymerase chain reaction) using total RNA isolated from formalin-fixed and paraffin-embedded melanomas (n = 22) and melanocytomas (n = 9) from the Virginia Tech Animal Laboratory Services archives. Analysis of checkpoint molecule expression revealed significantly higher levels of PDCD1 (PD-1) and CD274 (PD-L1) mRNAs and an upward trend in PDCD1LG2 (PD-L2) mRNA in melanomas relative to melanocytomas. Immunohistochemistry revealed markedly increased numbers of CD3+ T cells in the highest PD-1-expressing subgroup of melanomas compared to the lowest PD-1 expressors, whereas densities of IBA1+ cells (macrophages) were similar in both groups. CD79a+ cell numbers were low for both groups. As in human melanoma, overexpression of the PD-1/PD-L1/PD-L2 axis is a common feature of canine melanoma. High expression of PD-1 and PD-L1 correlates with increased numbers of CD3+ cells. Additionally, the high level of IBA1+ cells in melanomas with low PD-1 expression and low CD3+ cells levels suggest that the expression of checkpoint molecules is modulated by interactions between T cells and cancer cells rather than histiocytes.

Fibrous osteodystrophy in a dromedary camel.

A 6-y-old female dromedary camel (Camelus dromedarius L.) was presented for assessment of firm, bilateral swellings rostral and ventral to the eyes. Serum biochemistry revealed hyperglycemia (28.5 mmol/L), hypocalcemia (1.27 mmol/L), hyperphosphatemia (3.39 mmol/L), hypoproteinemia (total protein 50 g/L), and hypoalbuminemia (20 g/L). Based on the poor prognosis associated with the presumptive diagnosis of fibrous osteodystrophy, the camel was euthanized. Gross postmortem findings revealed expanded fibrous tissue replacing the maxilla and mandible, and bilaterally prominent parathyroid glands. Histology of the maxilla revealed proliferative loose fibrous tissue with widely scattered, regularly spaced, small spicules of mineralized bone. The parathyroid glands were prominent bilaterally; the internal and external parathyroid glands were composed of plump cells with abundant pale basophilic cytoplasm and open nuclei. The pathologic findings were consistent with the antemortem diagnosis of fibrous osteodystrophy. The camel's diet, which was not specifically balanced for a camel, included grass hay, sweet feed, and alfalfa pellets. The camel's caregivers reported feeding her treats of cookies. A feed analysis was not available. The biochemistry abnormalities and clinical and postmortem findings, along with a diet that was not balanced for a camel, are consistent with a diagnosis of nutritional secondary hyperparathyroidism.

Blood mineral concentrations in the endangered huemul deer (Hippocamelus bisulcus) from Chilean Patagonia.

Concentrations of calcium, magnesium, copper, zinc, and selenium were measured in plasma from 11 huemul (Hippocamelus bisulcus) from Chilean Patagonia. Except for zinc and copper, concentrations of these minerals were similar to those of other deer species.