Neurosurgical Management of Spinal Epithelioid Hemangioendothelioma: Systematic Review & Illustrative Case Presentation.

BACKGROUND: Epithelioid hemangioendothelioma (EHE) is a rare vascular tumor predominantly arising in soft tissue. We report a rare case of thoracic spinal EHE with pulmonary metastasis. METHODS: Case report and systematic review of spinal EHE. RESULTS: A 36-year-old man presented with bilateral lower extremity weakness, progressive paresthesia, and urinary incontinence. He underwent open surgical resection of the tumor and decompression of the spinal cord, with subsequent improvement in neurological function. Systematic review identified 84 cases of spinal EHE, 73 of which were primary, and 14 of which developed extra-spinal metastases. CONCLUSION: EHE is an exceedingly rare tumor that may present with a wide swath of clinical symptoms. At present, no guidelines or formal treatment recommendations have been established. Surgical debulking has demonstrated efficacy as a front-line treatment, particularly in the setting of compressive neurological dysfunction; data regarding adjuvant chemoradiation are less consistently reported, mandating further study.

RNAscope: a novel method for the detection of Heterobilharzia americana ova in canine liver.

Canine schistosomiasis caused by Heterobilharzia americana can lead to severe morbidity and eventual mortality, in part due to the deposition of fluke ova in the liver and gastrointestinal tract, which promotes an influx of peri-ova inflammatory cells. Although fluke eggs can be identified in H&E-stained histologic sections, cases exist in which only fragments of the ova persist, or the egg is obscured by inflammatory infiltrates, which can confound definitive histologic diagnosis. Unfortunately, antibodies specific to Heterobilharzia are not commercially available for immunohistochemical labeling. Therefore, we aimed to use an RNA in situ hybridization strategy to fluorescently label Heterobilharzia ova. Using the H. americana 18S rRNA sequence, we developed an RNA probe and validated its performance on archival formalin-fixed, paraffin-embedded canine tissue. A positive signal was observed for all identifiable ova, fragmented and whole. Use of this methodology could aid understanding of the pathogenesis of H. americana infection in dogs. This technique augments standard diagnostic methodology, enabling spatial colocalization of fluke ova and inflammatory infiltrates when using fluorescent techniques.

Deletion of PGAM5 Downregulates FABP1 and Attenuates Long-Chain Fatty Acid Uptake in Hepatocellular Carcinoma.

Phosphoglycerate mutase 5 (PGAM5) is a Ser/His/Thr phosphatase responsible for regulating mitochondrial homeostasis. Overexpression of PGAM5 is correlated with a poor prognosis in hepatocellular carcinoma, colon cancer, and melanoma. In hepatocellular carcinoma, silencing of PGAM5 reduces growth, which has been attributed to decreased mitophagy and enhanced apoptosis. Yet in colon cancer, PGAM5's pro-tumor survival effect is correlated to lipid metabolism. We sought to identify whether deletion of PGAM5 modulated lipid droplet accrual in hepatocellular carcinoma. HepG2 and Huh7 PGAM5 knockout cell lines generated using CRISPR/Cas9 technology were used to measure cell growth, cellular ATP, and long-chain fatty acid uptake. Expression of hepatocellular fatty acid transporters, cluster of differentiation 36 (CD36), solute carrier family 27 member 2 (SLC27A2), solute carrier family 27 member 5 (SLC27A5), and fatty acid binding protein 1 (FABP1) was measured by quantitative PCR and Western blot. We found that deletion of PGAM5 attenuates hepatocellular carcinoma cell growth and ATP production. Further, PGAM5 knockout ameliorates palmitate-induced steatosis and reduces expression of FABP1 in HepG2 and Huh7 cell lines. PGAM5's role in hepatocellular carcinoma includes regulation of fatty acid metabolism, which may be related to expression of the fatty acid transporter, FABP1.

Prevalence of antibiotic use for dogs and cats in United States veterinary teaching hospitals, August 2020.

BACKGROUND: Awareness of prescribing practices helps identify opportunities to improve antibiotic use (AU). OBJECTIVES: To estimate AU prevalence in dogs and cats in U.S. veterinary teaching hospitals (VTHs) and identify antibiotic drugs commonly prescribed, indications for use, and evidence of bacterial infection. ANIMALS: Medical record data were collected from dogs and cats examined at 14 VTHs. METHODS: Data were collected from VTH medical records of dogs and cats examined by primary care, urgent care, emergency and critical care, internal medicine, and surgery services on a single day during August 13-September 3, 2020. Data included signalment; clinical service; inpatient or outpatient status; clinical conditions; diagnostic tests; evidence of bacterial infection; intended reason for AU; name and route of antibiotics prescribed. RESULTS: Of 883 dogs and cats, 322 (36.5%) were prescribed at least 1 antibiotic. Among 285 antibiotics administered systemically intended for treatment of infection, 10.9% were prescribed without evidence of infection. The most common class of antibiotics presribed for systemic administration was potentiated penicillin for dogs (115/346, 33.3%) and cats (27/80, 33.8%). For dogs and cats, first-generation cephalosporins (93/346, 26.9% and 11/80, 13.8%, respectively) and fluoroquinolones (51/346, 14.7% and 19/80, 23.8%, respectively) was second or third most-prescribed. Common AU indications included skin, respiratory, and urinary conditions, and perioperative use. CONCLUSIONS AND CLINICAL IMPORTANCE: Collaborative data collection provides a sustainable methodology to generate national AU prevalence estimates and bring attention to areas requiring additional research and detailed data collection. These efforts can also identify practice improvement opportunities in settings where future veterinarians are trained.

Shear wave velocity values measured by 2D-shear wave elastography are not different between awake and anesthetized cats without clinically significant hepatic fibrosis.

Two-dimensional shear wave elastography (2D-SWE) is widely used as a noninvasive method to quantify liver stiffness. In humans, liver stiffness approximates histologic hepatic fibrosis. While histology is the gold standard for diagnosing liver disease, 2D-SWE may be a minimally invasive alternative to biopsy in feline patients. The objectives of this prospective, observational, crossover study were trifold: (1) to assess the feasibility of performing 2D-SWE in awake cats, (2) to determine whether anesthesia altered shear wave velocity (SWV) measurements, and (3) to correlate hepatic stiffness with histologically quantified hepatic fibrosis. Eleven healthy, purpose-bred cats underwent 2D-SWE in awake and anesthetized states. SWV measurements were compared with histologic fibrosis measurements obtained from liver biopsies during the anesthetic period. The mean velocities were not significantly different between awake (1.47 ± 0.18 m/s) and anesthetized (1.47 ± 0.24 m/s) cats. Premedication and anesthetic drugs did not impact mean SWV. There was a higher variability in the SWV values in the awake group. The data points were reliably replicated, with an interquartile range of 0.24 and 0.32 in anesthetized and awake groups, respectively. There was moderate agreement between observers (intraclass correlation coefficient = 0.66). All cats had clinically insignificant fibrosis. There was no correlation between the SWV measurements and the histological fibrosis values. This study demonstrates that 2D-SWE is feasible in awake cats and that the anesthetic protocol employed did not significantly alter mean SWV. This work is the first to histologically validate normal SWV values in cats and show that 2D-SWE cannot differentiate minimal differences in feline hepatic fibrosis.

Lymphopenia predicts reduced survival in canine hepatocellular carcinoma.

Platelet to lymphocyte ratio (PLR) is a prognostic marker in human hepatocellular carcinoma (HCC) however, its utility in canine HCC has not been explored. The aim of the study was to determine if PLR could predict survival outcomes in 42 dogs with HCC. PLR was not a significant predictive factor (p = 0.15) but lymphopenia alone was significantly correlated with a reduced probability of survival (p = 0.024). Further studies are needed to evaluate if peripheral lymphocyte count mirrors that of the tumor microenvironment in canine HCC.

Neoplastic signatures: Comparative proteomics of canine hepatobiliary neuroendocrine tumors to normal niche tissue.

Hepatobiliary neuroendocrine neoplasms are rare cancers in humans and dogs. To date, no large-scale primary hepatobiliary neoplasm omics analyses exist in any species. This limits the development of diagnostic biomarkers and targeted therapeutics. Neuroendocrine cancers are a heterogenous group of neoplasms categorized by their tissue-of-origin. Because the anatomic niche of neuroendocrine neoplasms shapes tumor phenotype, we sought to compare the proteomes of 3 canine hepatobiliary neoplasms to normal hepatobiliary tissue and adrenal glands with the objective of identifying unique protein signatures. Protein was extracted from formalin-fixed paraffin-embedded samples and submitted for tandem mass spectroscopy. Thirty-two upregulated and 126 downregulated differentially expressed proteins were identified. Remarkably, 6 (19%) of the upregulated proteins are correlated to non-hepatobiliary neuroendocrine neoplasia and 16 (50%) are functionally annotated within the exosome cellular compartment key to neuroendocrine signaling. Twenty-six (21%) downregulated proteins are enriched in metabolic pathways consistent with alterations in cancer. These results suggests that characteristic neoplastic protein signatures can be gleaned from small data sets using a comparative proteomics approach.

Pathologic complete response following low-dose radiation for advanced oral cavity cancer in a patient with human immunodeficiency virus.

BACKGROUND: Advanced squamous cell carcinoma (SCCa) of the oral cavity is often not amenable to curative-intent therapy due to tumor location, tumor size, or comorbidities. CASE PRESENTATION: A 51-year-old male patient with human immunodeficiency virus and on highly active antiretroviral therapy (HAART) presented with a cT4aN2c SCCa of the tongue. He received a preoperative single course of Quad-Shot radiation therapy to 14 Gy in 4 fractions followed by surgical resection. Patient had no residual carcinoma on surgical pathology and no evidence of disease on subsequent clinical and radiological exams. CONCLUSIONS: To our knowledge, this is the first case of pathologic complete response for a patient on HAART following a single cycle of the Quad-Shot regimen for advanced oral cavity SCCa. Protease inhibitors in HAART can induce spontaneous tumor regression via inhibition of proteasome function and activation of apoptosis, and thus act as a cancer therapeutic.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Root system architecture and environmental flux analysis in mature crops using 3D root mesocosms.

Current methods of root sampling typically only obtain small or incomplete sections of root systems and do not capture their true complexity. To facilitate the visualization and analysis of full-sized plant root systems in 3-dimensions, we developed customized mesocosm growth containers. While highly scalable, the design presented here uses an internal volume of 45 ft3 (1.27 m3), suitable for large crop and bioenergy grass root systems to grow largely unconstrained. Furthermore, they allow for the excavation and preservation of 3-dimensional root system architecture (RSA), and facilitate the collection of time-resolved subterranean environmental data. Sensor arrays monitoring matric potential, temperature and CO2 levels are buried in a grid formation at various depths to assess environmental fluxes at regular intervals. Methods of 3D data visualization of fluxes were developed to allow for comparison with root system architectural traits. Following harvest, the recovered root system can be digitally reconstructed in 3D through photogrammetry, which is an inexpensive method requiring only an appropriate studio space and a digital camera. We developed a pipeline to extract features from the 3D point clouds, or from derived skeletons that include point cloud voxel number as a proxy for biomass, total root system length, volume, depth, convex hull volume and solidity as a function of depth. Ground-truthing these features with biomass measurements from manually dissected root systems showed a high correlation. We evaluated switchgrass, maize, and sorghum root systems to highlight the capability for species wide comparisons. We focused on two switchgrass ecotypes, upland (VS16) and lowland (WBC3), in identical environments to demonstrate widely different root system architectures that may be indicative of core differences in their rhizoeconomic foraging strategies. Finally, we imposed a strong physiological water stress and manipulated the growth medium to demonstrate whole root system plasticity in response to environmental stimuli. Hence, these new "3D Root Mesocosms" and accompanying computational analysis provides a new paradigm for study of mature crop systems and the environmental fluxes that shape them.

The BPH/5 Mouse Model of Superimposed Preeclampsia Is Not a Model of HELLP Syndrome.

Preeclampsia (PE) is a multisystemic disease of pregnancy affecting 2-8% of women worldwide. PE-induced liver disease is a rare but important complication of pregnancy. The pathogenesis of liver dysfunction in PE is poorly understood, but is correlated with dysregulated angiogenic, inflammatory, and hypoxic events in the early phase of placental development. Because BPH/5 mice develop the maternal and fetal hallmarks of PE during pregnancy, we hypothesized that they may also share the clinicopathologic findings of the human PE-associated hemolysis elevated liver transaminases low platelets (HELLP) syndrome. Using this model, we determined that microangiopathic hemolysis, thrombocytopenia, and elevated liver enzymes do not occur in mid to late gestation. Pregnant BPH/5 mice do not develop histologic evidence of hepatic inflammation, but they do have increased microsteatosis scores at preconception and in mid to late gestation that progress to macrosteatosis in a subset of mice in late gestation. The transcriptional upregulation of TNF-α, CXCL-10, and TLR-2 occurs in mid gestation prior to the onset of macrosteatosis. The BPH/5 female mouse is not a model of HELLP syndrome, but may be a model of fatty liver disease associated with pregnancy.

Quantification of serum fibroblast growth factor-19 concentration in healthy dogs before and after feeding.

OBJECTIVE: To measure serum fibroblast growth factor-19 (FGF-19) concentration and gallbladder volume in healthy dogs before and after feeding to determine whether serum FGF-19 concentration increases following gallbladder contraction and to assess FGF-19 stability in blood samples kept under different storage conditions after collection in tubes containing no anticoagulant or in serum separator tubes. ANIMALS: 10 healthy dogs of various ages and breeds (30 blood samples and 30 gallbladder volume measurements). PROCEDURES: Serum FGF-19 concentration was measured with a commercially available ELISA. Gallbladder volume was determined ultrasonographically. Blood samples and gallbladder measurements were obtained from the dogs after food had been withheld for 12 hours (baseline) and at 1 and 3 hours after feeding. The stability of serum FGF-19 was assessed in samples collected in tubes containing no anticoagulant or in serum separator tubes and stored at -80°C for variable intervals or 4°C for 1 or 5 days. RESULTS: Serum FGF-19 concentration was significantly increased from baseline at 1 and 3 hours after feeding. There was a significant decrease in gallbladder volume 1 hour after feeding, compared with baseline findings. Regardless of collection tube used, concentrations of FGF-19 in serum obtained from blood samples that were collected and immediately stored at -80°C differed significantly from concentrations in serum obtained from blood samples that had been collected and stored at 4°C for 5 days. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that postprandial gallbladder contraction results in increases of serum FGF-19 concentration in healthy dogs. Assessment of circulating FGF-19 concentration could be used to detect disruptions in the enterohepatic-biliary axis in dogs.

Hepatocyte ploidy in cats with and without hepatocellular carcinoma.

BACKGROUND: Domestic cats rarely develop hepatocellular carcinoma. The reason for the low prevalence is unknown. Reductions in hepatocellular ploidy have been associated with hepatic carcinogenesis. Recent work in mice has shown that livers with more polyploid hepatocytes are protected against the development of hepatocellular carcinoma. Hepatocyte ploidy in the domestic cat has not been evaluated. We hypothesized that ploidy would be reduced in peri-tumoral and neoplastic hepatocytes compared to normal feline hepatocytes. Using integrated fluorescence microscopy, we quantified the spectra of ploidy in hepatocellular carcinoma and healthy control tissue from paraffin embedded tissue sections. RESULTS: Feline hepatocytes are predominantly mononuclear and the number of nuclei per hepatocyte did not differ significantly between groups. Normal cats have a greater number of tetraploid hepatocytes than cats with hepatocellular carcinoma. CONCLUSIONS: Total hepatocellular polyploidy in normal cat liver is consistent with values reported in humans, yet cellular ploidy (nuclei per cell) is greater in humans than in cats. Tetraploid cat hepatocytes are predominantly mononuclear.

HSP70 promotes MLKL polymerization and necroptosis.

Mixed lineage kinase domain-like protein (MLKL) is the proposed executioner of necroptosis. Our recent findings identify a novel inhibitor necroptosis-blocking compound 1 (NBC1) which specifically conjugates to two cysteines of heat shock protein 70 (HSP70) to block its function. Importantly, HSP70 promotes MLKL polymerization to activate necroptosis.

Necroptosis-blocking compound NBC1 targets heat shock protein 70 to inhibit MLKL polymerization and necroptosis.

Necroptosis is a regulated necrotic cell death pathway involved in development and disease. Its signaling cascade results in the formation of disulfide bond-dependent amyloid-like polymers of mixed lineage kinase domain-like protein (MLKL), which mediate proinflammatory cell membrane disruption. We screened compound libraries provided by the National Cancer Institute and identified a small-molecule inhibitor of necroptosis named necroptosis-blocking compound 1 (NBC1). Biotin-labeled NBC1 specifically conjugates to heat shock protein Hsp70. NBC1 and PES-Cl, a known Hsp70 substrate-binding inhibitor, block the formation of MLKL polymers, but not MLKL tetramers in necroptosis-induced cells. In vitro, recombinant Hsp70 interacts with the N-terminal domain (NTD) of MLKL and promotes NTD polymerization, which has been shown to mediate the cell killing activity. Furthermore, the substrate-binding domain (SBD) of Hsp70 is sufficient to promote MLKL polymerization. NBC1 covalently conjugates cysteine 574 and cysteine 603 of the SBD to block its function. In addition, an SBD mutant with both cysteines mutated to serines loses its ability to promote MLKL polymerization. Interestingly, knockdown of Hsp70 in cells leads to MLKL destabilization, suggesting that MLKL might also be a client protein of Hsp70. In summary, using NBC1, an inhibitor of necroptosis, we identified Hsp70 as a molecular chaperone performing dual functions in necroptosis. It stabilizes MLKL protein under normal condition and promotes MLKL polymerization through its substrate-binding domain during necroptosis.

Necroptosis: MLKL Polymerization.

Necroptosis is a subtype of regulated necrosis that occurs when caspases are inhibited or fail to activate. Stimulus of cell death receptors results in a signaling cascade that triggers caspase independent, immunogenic cell death. The core pathway relies on receptor interacting protein kinase (RIPK) 1 and 3, which interact through their receptor homotypic interacting motif (RHIM) domains, and form amyloid-like structures termed the necrosome. RIPK3 recruits and phosphorylates mixed lineage kinase domain-like pseudokinase (MLKL), the terminal mediator in the necroptotic pathway. MLKL polymerizes to form a second amyloid-like structure that causes cell membrane disruption resulting in cell death. Although the core necroptosis pathway has been elucidated, the details of MLKL membrane translocation and membrane disruption remain an open area of research.

MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis.

Mixed-lineage kinase domain-like protein (MLKL) is essential for TNF-α-induced necroptosis. How MLKL promotes cell death is still under debate. Here we report that MLKL forms SDS-resistant, disulfide bond-dependent polymers during necroptosis in both human and mouse cells. MLKL polymers are independent of receptor-interacting protein kinase 1 and 3 (RIPK1/RIPK3) fibers. Large MLKL polymers are more than 2 million Da and are resistant to proteinase K digestion. MLKL polymers are fibers 5 nm in diameter under electron microscopy. Furthermore, the recombinant N-terminal domain of MLKL forms amyloid-like fibers and binds Congo red dye. MLKL mutants that cannot form polymers also fail to induce necroptosis efficiently. Finally, the compound necrosulfonamide conjugates cysteine 86 of human MLKL and blocks MLKL polymer formation and subsequent cell death. These results demonstrate that disulfide bond-dependent, amyloid-like MLKL polymers are necessary and sufficient to induce necroptosis.

Combined Chemoinformatics Approach to Solvent Library Design Using clusterSim and Multidimensional Scaling.

Reported here is a rational approach for the selection of solvents intended for use in physical form screening based on a novel chemoinformatics analysis of solvent properties. A comprehensive assessment of eight clustering methods was carried out on a series of 94 solvents described by calculated molecular descriptors using the clusterSim package in R. The effectiveness of clustering methods was evaluated using a range of statistical measures as well as increasing efficiency of solid form discovery using a cluster-based solvent selection approach. Multidimensional scaling was used to illustrate cluster analysis on a two-dimensional solvent map. The map presented here is a valuable tool to aid efficient solvent selection in physical form screens. This tool is equally applicable to any scientific area which requires a solubility dependent decision on solvent choice.

Clinical findings, diagnostics and outcome in 33 cats with adrenal neoplasia (2002-2013).

OBJECTIVES: The objective of this retrospective study was to describe the clinical signs and diagnostic findings in cats with histopathologically confirmed adrenal neoplasms, and to assess correlations with survival data. METHODS: Study data were acquired by reviewing medical records for all cats diagnosed with adrenal neoplasms at seven referral institutions between 2002 and 2013. Inclusion criteria required a histopathologic diagnosis of an adrenal neoplasm (ante-mortem or on necropsy). RESULTS: Thirty-three cats met the inclusion criteria for the study. The most common presenting complaints included weakness (n = 12), respiratory signs (n = 4), blindness (n = 4) or gastrointestinal signs (n = 3). Laboratory abnormalities included hypokalemia (n = 18), alkalemia (n = 12), elevated creatine kinase (>3000, n = 5) and azotemia (n = 4). In addition, hypertension was noted in 13 cats. Thirty cats were diagnosed with cortical tumors (17 carcinomas, 13 adenomas) and three cats were diagnosed with pheochromocytomas. Twenty-five cats underwent tests to evaluate the function of the adrenal tumors; 19/25 cats had functional tumors (hyperaldosteronism [n = 16], hypercortisolemia [n = 1], high estradiol [n = 1], and hypersecretion of aldosterone, estradiol and progesterone [n = 1]). Twenty-six cats underwent adrenalectomy, one cat was medically managed and six were euthanized without treatment. Long-term survival postoperatively ranged from 4-540 weeks, with 20 (77%) cats surviving the perioperative period of 2 weeks. The only variable that was found to be negatively associated with survival was female sex. The most common complications noted during the perioperative period were hemorrhage and progressive lethargy and anorexia. CONCLUSIONS AND RELEVANCE: Surgical treatment for feline adrenal tumors (regardless of tumor type) resulted in good long-term survival. Given that pre- and postoperative hypocortisolemia was identified in this study, and, in addition, hypersecretion of more than one adrenal hormone occurred in one cat, adrenal panels prior to surgery may be beneficial as part of the preoperative work-up.