Comparison of fibroblast growth factor 19 concentrations between dogs with and without gallbladder mucoceles.

BACKGROUND: Fibroblast growth factor 19 (FGF19) is an enterohepatic hormone the synthesis of which is stimulated by bile acid activation of the nuclear farnesoid X receptor (FXR) in ileal enterocytes. Increased production of FGF19 downregulates hepatocyte bile acid synthesis and gluconeogenesis, while concurrently upregulating hepatocyte glycogenesis and gallbladder (GB) filling. The physiologic impact of this regulatory cycle is illustrated in cholecystectomized humans, in whom the disturbed meal-related flux of GB bile decreases serum FGF19 concentrations. OBJECTIVE: Determine if serum FGF19 concentrations are lower in dogs with clinical GB mucoceles (GBMs) than in control dogs. ANIMALS: Seven dogs with GBM diagnosed using abdominal ultrasonography, biochemical markers, and GB histopathology. Forty-two control dogs without gastrointestinal or hepatobiliary disorders also were evaluated. Health status of controls was assessed by physical examination and diagnostic hematologic and biochemical test results. METHODS: Prospective cross-sectional study to compare fasting plasma or serum FGF19 concentrations between groups. Concentrations of FGF19 were quantified by a commercially available FGF19 ELISA. RESULTS: Concentrations of FGF19 were significantly lower in dogs with clinical GBM (median, 14.0 pg/mL; range, 12.8-67.2) than in control dogs (median, 145.3 pg/mL; range, 36.5-285.1). CONCLUSIONS AND CLINICAL IMPORTANCE: In dogs, GBM is associated with significantly decreased serum FGF19 concentrations. We speculate that this finding reflects compromised GB contraction and decreased enterohepatic circulation of bile flow. Subnormal FGF19 concentrations may influence bile acid synthesis and hepatic metabolism.

Gallbladder Neuroendocrine Neoplasms in Dogs and Humans.

Gallbladder neuroendocrine neoplasms (GB NENs) are among the rarest cancers reported in humans and dogs. This review provides a detailed review of the canine GB NEN literature and an interspecies comparison of demographics, clinical pathophysiology, pathobiology, and therapeutic response of GB NENs. The aim of this work is to explore the relevance of dogs as a spontaneous model for human GB NENs.

Neurosurgical Management of Spinal Epithelioid Hemangioendothelioma: Systematic Review and 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 neurologic 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 neurologic dysfunction; data regarding adjuvant chemoradiation are less consistently reported, mandating further study.

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.

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.

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.

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.

Protein kinase Cδ protects against bile acid apoptosis by suppressing proapoptotic JNK and BIM pathways in human and rat hepatocytes.

Retained bile acids, which are capable of inducing cell death, activate protein kinase Cδ (PKC-δ) in hepatocytes. In nonhepatic cells, both pro- and antiapoptotic effects of PKC-δ are described. The aim of this study was to determine the role of PKC-δ in glycochenodeoxycholate (GCDC)-induced apoptosis in rat hepatocytes and human HUH7-Na-taurocholate-cotransporting polypeptide (Ntcp) cells. Apoptosis was monitored morphologically by Hoechst staining and biochemically by immunoblotting for caspase 3 cleavage. The role of PKC-δ was evaluated with a PKC activator (phorbol myristate acetate, PMA) and PKC inhibitors (chelerythrine, H-7, or calphostin), PKC-δ knockdown, and wild-type (WT) or constitutively active (CA) PKC-δ. PKC-δ activation was monitored by immunoblotting for PKC-δ Thr505 and Tyr311 phosphorylation or by membrane translocation. JNK and Akt phosphorylation and the amount of total bisindolylmaleimide (BIM) were determined by immunoblotting. GCDC induced the translocation of PKC-δ to the mitochondria and/or plasma membrane in rat hepatocytes and HUH7-Ntcp cells and increased PKC-δ phosphorylation on Thr505, but not on Tyr311, in HUH7-Ntcp cells. GCDC-induced apoptosis was attenuated by PMA and augmented by PKC inhibition in rat hepatocytes. In HUH-Ntcp cells, transfection with CA or WT PKC-δ attenuated GCDC-induced apoptosis, whereas knockdown of PKC-δ increased GCDC-induced apoptosis. PKC-δ silencing increased GCDC-induced JNK phosphorylation, decreased GCDC-induced Akt phosphorylation, and increased expression of BIM. GCDC translocated BIM to the mitochondria in rat hepatocytes, and knockdown of BIM in HUH7-Ntcp cells decreased GCDC-induced apoptosis. Collectively, these results suggest that PKC-δ does not mediate GCDC-induced apoptosis in hepatocytes. Instead PKC-δ activation by GCDC stimulates a cytoprotective pathway that involves JNK inhibition, Akt activation, and downregulation of BIM.

Influence of biopsy specimen size, tissue fixation, and assay variation on copper, iron, and zinc concentrations in canine livers.

OBJECTIVE-To determine whether metal concentrations in canine liver specimens were influenced by specimen size, assay variability, tissue processing (formalin fixation and deparaffinization), or storage in paraffin blocks. SAMPLE POPULATION-Liver specimens (fresh frozen and deparaffinized) from 2 dogs with chronic hepatitis (high copper but unremarkable iron concentration [liver 1] and unremarkable copper but high iron concentration [liver 2]) as well as fresh and deparaffinized-archived liver specimens from 20 dogs with various hepatopathies. PROCEDURES-Fresh frozen liver specimens (obtained via simulated needle-core and wedge biopsy), fresh hepatic tissue, and deparaffinized-archived specimens (0.5 to 14 years old) were analyzed for concentrations of copper, iron, and zinc by atomic absorption flame spectrometry. Clinical severity scores were assigned on the basis of tissue metal concentrations. RESULTS-Interassay variation of metal standards was < 4%. Measurements of liver tissues on 8 consecutive days yielded high coefficients of variation (3.6% to 50%) reflecting heterogenous histologic metal distribution; variation was highest in liver 1 and deparaffinized-archived tissues. Heterogenous metal distribution was confirmed by histologic evaluation. The largest range of metal concentrations was detected in wedge biopsy specimens. In tissues with high metal concentrations, copper and iron concentrations were significantly lower in needle-core versus wedge biopsy specimens. A higher zinc concentration in deparaffinized-archived specimens masked a low zinc concentration in fresh liver tissue of 10 of 20 (50%) dogs. CONCLUSIONS AND CLINICAL RELEVANCE-Retrospective measurement of copper and iron concentrations but not zinc concentrations in deparaffinized-archived liver specimens provided relevant information. The value of needle-core biopsy specimens for measurement of metal concentrations is questionable.