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.

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.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

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.

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.

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.

Hepatic copper concentrations in Labrador Retrievers with and without chronic hepatitis: 72 cases (1980-2010).

OBJECTIVE: To evaluate differences in hepatic copper concentrations in Labrador Retrievers with and without chronic hepatitis. DESIGN: etrospective case-control study. SAMPLE: Liver tissue specimens from 36 Labrador Retrievers with chronic hepatitis and 36 age- and sex-matched Labrador Retrievers without chronic hepatitis (control dogs). PROCEDURES: Liver tissue specimens were obtained during 2 study periods (1980 to 1997 and 1998 to 2010). For each tissue specimen, a histologic score was assigned independently by each of 2 interpreters, and the hepatic copper concentration was qualitatively determined via rhodanine staining and quantitatively determined via atomic absorption spectroscopy. RESULTS: Mean hepatic copper concentration was significantly higher in dogs with chronic hepatitis (614 µg/g of dry weight [range, 104 to 4,234 µg/g of dry weight]), compared with that in control dogs (299 µg/g of dry weight [range, 93 to 3,810 µg/g of dry weight]), and increased significantly over time. A higher proportion of liver tissue specimens collected during the 1998-2010 study period had hepatic copper concentrations > 400 µg/g of dry weight (the upper limit of the reference range), compared with the proportion of liver tissue specimens collected during the 1980-1997 study period. The qualitative copper score did not accurately predict quantitative hepatic copper concentration in 33% of study dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that the increase in hepatic copper concentrations in Labrador Retrievers with and without chronic hepatitis over time may be the result of increased exposure of dogs to environmental copper, most likely via the diet.

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.

Olfactory stimulation of Africanized honey bee (Hymenoptera: Apidae) attacks by insect repellents.

Three common insect repellents (N,N-diethyl-m-toluamide [DEET], Pyranha, and Repel X) were tested to determine whether they affected Africanized honey bee attack behavior. Eight Africanized honey bee (Apis mellifera L.) colonies were exposed in an alternating series to the test repellents or blank controls delivered in a stream of air directed toward the colony entrances. The response generated by the repellents and the controls was measured as the number of attacking honey bees recorded with an electronic temper tester. Neither a citronella-based repellent (Pyranha) nor DEET had any effect on colony behavior; however, Repel X consistently caused a greater attack response after exposure.