Intrahepatic mucinous cholangiocarcinoma with recurrent colic in a horse: case report and literature review of cholangiocarcinoma in horses.

A 17-y-old Arabian mare was presented to the Auburn Large Animal Veterinary Teaching Hospital with a long-term history of intermittent mild recurrent colic that responded to medical treatment. CBC revealed mild lymphopenia; serum biochemistry findings were of increased gamma-glutamyl transferase and creatine kinase activities, hyperferremia, hyperglycemia, hypomagnesemia, and hypokalemia. Abdominocentesis was compatible with low-protein transudate. Due to the progression and duration of clinical signs, the owner elected euthanasia. Postmortem examination and histopathology confirmed a cholangiocarcinoma. The neoplastic cells were arranged in large cysts containing lakes of mucin that comprised 90% of the tumor volume; thus, a mucinous variant was determined. The neoplastic cells had strong cytoplasmic immunolabeling for cytokeratin 19 and lacked immunolabeling for hepatocyte paraffin 1, supporting bile duct origin. Cholangiocarcinomas are infrequent tumors in horses with nonspecific and slow progressive clinical signs, including recurrent colic. Mucinous cholangiocarcinomas are seldom reported in veterinary medicine and, to our knowledge, have not been reported previously in horses.

Reply to Piewbang et al. Domestic Cat Hepadnavirus Antigens in Lymphoma Tissues. Comment on "Beatty et al. Domestic Cat Hepadnavirus and Lymphoma. Viruses 2023, 15, 2294".

We are grateful to the authors for providing additional data to demonstrate the presence of domestic cat hepadnavirus in lymphoma tissues [...].

The effects of 6 wk of resistance training on the gut microbiome and cardiometabolic health in young adults with overweight and obesity.

Obesity is a known risk factor for the development of insulin resistance and other cardiometabolic disorders. Recently, the gut microbiome has been associated with obesity and subsequent health complications. Exercise has been regularly utilized as a therapeutic intervention to treat obesity and its associated comorbidities. This study examined the effects of a 6-wk resistance training exercise program (RT) on the diversity, composition, and metabolic pathways of the gut microbiome. Sedentary young adults (age 18-35 yr) with overweight and obesity (BMI 25-45 kg/m2) were recruited to participate in this randomized controlled trial. Participants were randomized to RT (n = 16), a 6-wk resistance training program (3 days/wk), or control (CT) (n = 16), a nonexercising control. Main outcomes of the study included gut microbiome measures (taxa abundances, diversity, and predicted function) and cardiometabolic outcomes [blood pressure (BP) and glucoregulation]. Increased abundances of Roseburia, a short-chain fatty acid (SCFA) producer were observed over 6 wk (W6) with RT compared with CT (group × week, P < 0.05, q < 0.25). RT also induced marginal alterations in predicted microbial metabolic and cell motility pathways compared with CT (group × week, P < 0.05, q < 0.25). However, RT did not significantly impact overall microbial diversity. Furthermore, RT resulted in higher quantitative insulin-sensitivity check index (QUICKI) and lower diastolic BP at W6 compared with CT [baseline (BL)-adjusted P < 0.05]. RT had mixed effects on the gut microbiome. Although RT increased abundances of Roseburia and induced minor changes in microbial pathways, it is important to consider these changes in the context of the overall stability observed in the microbiome composition.NEW & NOTEWORTHY Resistance training induces mixed changes in the gut microbiome, including an increase in the abundances of the Roseburia genus and minor alterations in microbial pathways. However, it is vital to interpret these changes in light of the broader context, where we observe stability in the overall microbiome composition. This stability may be attributed to the microbiome's resilience, demonstrating its capacity to withstand short-term physiological stressors.

Histologic characterization of the major duodenal papilla and association with concurrent biliary, pancreatic, and intestinal pathology in cats.

Conjoining of the major pancreatic duct and common bile duct at the major duodenal papilla (MDP) is suspected to predispose cats to the clinical syndrome of "triaditis." However, microanatomy of the MDP or presence of lesions at the MDP has not been assessed in cats with or without triaditis. The aims of this study were to characterize feline MDP histomorphology and to identify associations between MDP anatomy/disease and the presence of biliary, pancreatic, or intestinal inflammation or neoplasia. Histologic assessment was prospectively performed on the MDP, duodenum, jejunum, ileum, liver, and pancreas from 124 client-owned cats undergoing postmortem examination. The majority of cats (104/124, 84%) had a complex ductular network at the MDP, with no distinction between pancreatic and common bile ducts. Lymphoid aggregates at the MDP were common (63/124, 51%). Inflammation of the MDP (MDPitis) was present in 35 of 124 cats (28%) and was often concurrent with cholangitis, pancreatitis, or enteritis (32/35, 91%), but was only associated with enteritis (19/35, 54%, P < .05). Triaditis was less common (19/124, 15%), but was associated with both conjoined MDP anatomy (19/19, 100%, P < .05) and MDPitis (12/19, 63%, P < .05). Neoplasia was present in 37 of 124 cats (29%), with lymphoma (28/37, 78%) predominating. Enteropathy-associated T-cell lymphoma type 2 (EATL2) was most common (n = 16/37, 43%) and was associated with triaditis and MDPitis (P < .05). These findings suggest that anatomy, immune activation, and/or inflammation of the MDP may play a role in the pathogenesis of triaditis. Further studies are needed to elucidate the relationships between triaditis, MDPitis, and EATL2.

Severe bronchiectasis resulting from chronic bacterial bronchitis and bronchopneumonia in a jungle cat.

Bronchiectasis is irreversible bronchial dilation that can be congenital or acquired secondary to chronic airway obstruction. Feline bronchiectasis is rare and, to our knowledge, has not been reported previously in a non-domestic felid. An ~10-y-old female jungle cat (Felis chaus) was presented for evaluation of an abdominal mass and suspected pulmonary metastasis. The animal died during exploratory laparotomy and was submitted for postmortem examination. Gross examination revealed consolidation of the left caudal lung lobe and hila of the cranial lung lobes. Elsewhere in the lungs were several pale-yellow pleural foci of endogenous lipid pneumonia. On cut section, there was severe distension of bronchi with abundant white mucoid fluid. The remaining lung lobes were multifocally expanded by marginal emphysema. Histologically, ectatic bronchi, bronchioles, and fewer alveoli contained degenerate neutrophils, fibrin, and mucin (suppurative bronchopneumonia) with rare gram-negative bacteria. Aerobic culture yielded low growth of Proteus mirabilis and Escherichia coli. There was chronic bronchitis, marked by moderate bronchial gland hyperplasia, lymphoplasmacytic inflammation, and lymphoid hyperplasia. The palpated abdominal mass was a uterine endometrial polyp, which was considered an incidental, but novel, finding. Chronic bronchitis and bronchopneumonia should be considered as a cause of bronchiectasis and a differential diagnosis for respiratory disease in non-domestic felids.

Eosinophilic pericardial effusion and pericarditis in a cat.

Case summary: A 10-year-old domestic shorthair cat presented for lethargy, anorexia and labored breathing. Significant pleural and pericardial effusions prompted thoracocentesis and pericardiocentesis. Cytologic evaluation of the pericardial effusion revealed a highly cellular hemorrhagic, eosinophilic (12%) effusion, with many markedly atypical suspected mesothelial cells, interpreted as concerning for neoplasia. Thoracoscopic subtotal pericardiectomy and histology of the pericardium revealed predominantly eosinophilic inflammation with multifocal mesothelial hypertrophy and ulceration. A peripheral eosinophilia was not present on serial complete blood counts. Initial infectious disease testing was mostly negative. Toxoplasma gondii titers were most consistent with prior exposure, although reactivation could not be excluded. The owner's medical history included a prior diagnosis of bartonellosis. Owing to the challenges of definitive Bartonella species exclusion, the cat was treated empirically with pradofloxacin and doxycycline, and a subtotal pericardectomy. There was improvement at first but pleural effusion recurred approximately 3 months after discharge. The cat was euthanized and a necropsy was not performed. Subsequent pericardial effusion Piroplasma/Bartonella/Borrelia droplet digital PCR detected DNA of Bartonella vinsonii subspecies berkhoffii, and peripheral blood culture and sequencing revealed a rare apicomplexan organism (90% homology with Colpodella species) of unknown clinical significance. Testing for filamentous bacteria and fungal pathogens was not performed. Relevance and novel information: This case offers several unique entities - eosinophilic pericardial effusion and eosinophilic pericarditis of unknown etiology - and illustrates the well-known marked atypia that may occur in reactive and hyperplastic mesothelial cells, particularly of infrequently sampled and cytologically described feline pericardial effusion, supporting a cautious interpretation of this cytology finding.

Domestic Cat Hepadnavirus and Lymphoma.

We write to comment on Piewbang C et al [...].

Phenotype and fate of liver-resident CD8 T cells during acute and chronic hepacivirus infection.

Immune correlates of hepatitis C virus (HCV) clearance and control remain poorly defined due to the lack of an informative animal model. We recently described acute and chronic rodent HCV-like virus (RHV) infections in lab mice. Here, we developed MHC class I and class II tetramers to characterize the serial changes in RHV-specific CD8 and CD4 T cells during acute and chronic infection in C57BL/6J mice. RHV infection induced rapid expansion of T cells targeting viral structural and nonstructural proteins. After virus clearance, the virus-specific T cells transitioned from effectors to long-lived liver-resident memory T cells (TRM). The effector and memory CD8 and CD4 T cells primarily produced Th1 cytokines, IFN-γ, TNF-α, and IL-2, upon ex vivo antigen stimulation, and their phenotype and transcriptome differed significantly between the liver and spleen. Rapid clearance of RHV reinfection coincided with the proliferation of virus-specific CD8 TRM cells in the liver. Chronic RHV infection was associated with the exhaustion of CD8 T cells (Tex) and the development of severe liver diseases. Interestingly, the virus-specific CD8 Tex cells continued proliferation in the liver despite the persistent high-titer viremia and retained partial antiviral functions, as evident from their ability to degranulate and produce IFN-γ upon ex vivo antigen stimulation. Thus, RHV infection in mice provides a unique model to study the function and fate of liver-resident T cells during acute and chronic hepatotropic infection.

Distinct disease features of acute and persistent genotype 3 hepatitis E virus infection in immunocompetent and immunosuppressed Mongolian gerbils.

Hepatitis E virus (HEV) causes self-limited acute hepatitis in immunocompetent individuals and can establish chronic infection in solid organ transplant recipients taking immunosuppressive drugs. A well characterized small animal model is needed to understand HEV pathogenesis. In this study, we established a robust model to study acute and persistent HEV infection using Mongolian gerbils (Meriones unguiculatus) with or without immunosuppression. Gerbils were implanted subcutaneously with continuous release tacrolimus pellet to induce immunosuppression. Gerbils with or without tacrolimus treatment were inoculated with HEV intraperitoneally. Viremia, fecal virus shedding, serum antibody and ALT levels, liver histopathological lesions, hepatocyte apoptosis, and liver macrophage distribution were assessed. Mild to moderate self-limited hepatitis and IgM and IgG antibody responses against HEV ORF2 were observed in immunocompetent gerbils. Levels of HEV-specific IgM responses were higher and lasted longer in immunocompetent gerbils with higher peak viremia. Persistent viremia and fecal virus shedding with either weak, or absent HEV antibody levels were seen in immunosuppressed gerbils. Following HEV infection, serum ALT levels were increased, with lower and delayed peaks observed in immunosuppressed compared to immunocompetent gerbils. In immunocompetent gerbils, foci of apoptotic hepatocytes were detected that were distributed with inflammatory infiltrates containing CD68+ macrophages. However, these foci were absent in immunosuppressed gerbils. The immunosuppressed gerbils showed no inflammation with no increase in CD68+ macrophages despite high virus replication in liver. Our findings suggest adaptive immune responses are necessary for inducing hepatocyte apoptosis, CD68+ macrophage recruitment, and inflammatory cell infiltration in response to HEV infection. Our studies show that Mongolian gerbils provide a promising model to study pathogenesis during acute and persistent HEV infection.

Immunohistochemical characterization of clear cell variant of hepatocellular carcinoma in a sheep.

The cadaver and viscera of a mature female sheep (Ovis aries) underwent routine abattoir meat inspection. The liver was expanded by an infiltrative neoplastic mass comprising multifocal to coalescing, well-demarcated, pink to white-yellow nodules, up to 25 mm in diameter. An unencapsulated, moderately densely cellular, infiltrative neoplasm was present within the hepatic parenchyma. The neoplastic cells were arranged in solid sheets and acini supported by a moderately fine collagenous vascularized stroma. The neoplastic cells were moderately sized and polygonal, with clearly delineated cell borders and a moderate amount of cytoplasm that was clear or exhibited either globular eosinophilic deposits or fine fibrillar eosinophilic strands. The neoplastic cell nuclei were round and centrally located. The chromatin was lightly stippled and there was frequently a single, prominent, basophilic nucleolus. There were eight mitoses in 10 high-power fields (2.37 mm2). Most of the neoplastic cells had intense cytoplasmic immunolabelling for arginase 1, with frequent concurrent nuclear positivity, and mild to moderately intense punctate cytoplasmic labelling for hepatocyte specific antigen (Hep Par-1). The neoplastic cells did not label with anti-cytokeratin 19 antibody. Based on the histological appearance and the immunolabelling pattern, the neoplasm was diagnosed as the clear cell variant of a hepatocellular carcinoma.

Assessment of Mouse Liver Histopathology Following Exposure to HFPO-DA With Emphasis on Understanding Mechanisms of Hepatocellular Death.

Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate (HFPO-DA) is a short chain member of per- and polyfluoroalkyl substances (PFAS). To better understand the relevance of histopathological effects seen in livers of mice exposed to HFPO-DA for human health risk assessment, histopathological effects were summarized from hematoxylin and eosin (H&E)-stained sections in several repeat-dose toxicity studies in mice. Findings across studies revealed histopathological changes consistent with peroxisomal proliferation, whereas two reports of steatosis could not be confirmed in the published figures. In addition, mechanisms of hepatocellular death were assessed in H&E sections as well as with the apoptotic marker cleaved caspase-3 (CCasp3) in newly cut sections from archived liver blocks from select studies. A comparison of serially CCasp3 immunolabeled and H&E-stained sections revealed that mechanisms of hepatocellular death cannot be clearly discerned in H&E-stained liver sections alone as several examples of putatively necrotic cells were positive for CCasp3. Published whole genome transcriptomic data were also reevaluated for enrichment of various forms of hepatocellular death in response to HFPO-DA, which revealed enrichment of apoptosis and autophagy, but not ferroptosis, pyroptosis, or necroptosis. These morphological and molecular findings are consistent with transcriptomic evidence for peroxisome proliferator-activated receptor alpha (PPARα) signaling in HFPO-DA exposed mice.

Gallbladder microbiota in healthy dogs and dogs with mucocele formation.

To date studies have not investigated the culture-independent microbiome of bile from dogs, a species where aseptic collection of bile under ultrasound guidance is somewhat routine. Despite frequent collection of bile for culture-based diagnosis of bacterial cholecystitis, it is unknown whether bile from healthy dogs harbors uncultivable bacteria or a core microbiota. The answer to this question is critical to understanding the pathogenesis of biliary infection and as a baseline to exploration of other biliary diseases in dogs where uncultivable bacteria could play a pathogenic role. A pressing example of such a disease would be gallbladder mucocele formation in dogs. This prevalent and deadly condition is characterized by excessive secretion of abnormal mucus by the gallbladder epithelium that can eventually lead to rupture of the gallbladder or obstruction of bile flow. The cause of mucocele formation is unknown as is whether uncultivable, and therefore unrecognized, bacteria play any systematic role in pathogenesis. In this study we applied next-generation 16S rRNA gene sequencing to identify the culture-negative bacterial community of gallbladder bile from healthy dogs and gallbladder mucus from dogs with mucocele formation. Integral to our study was the use of 2 separate DNA isolations on each sample using different extraction methods and sequencing of negative control samples enabling recognition and curation of contaminating sequences. Microbiota findings were validated by simultaneous culture-based identification, cytological examination of bile, and fluorescence in-situ hybridization (FISH) performed on gallbladder mucosa. Using culture-dependent, cytological, FISH, and 16S rRNA sequencing approaches, results of our study do not support existence of a core microbiome in the bile of healthy dogs or gallbladder mucus from dogs with mucocele formation. Our findings further document how contaminating sequences can significantly contribute to the results of sequencing analysis when performed on samples with low bacterial biomass.

Patent hepatic ciliated foregut remnant resulting in an umbilicobiliary sinus tract, with gallbladder agenesis, in an 8-wk-old male French Bulldog.

Hepatic ciliated foregut remnants or cysts are congenital abnormalities resulting from retention of embryonic ciliated foregut within the liver. These structures are rarely reported in the human medical literature and have not been reported in the veterinary literature previously, to our knowledge. We describe here a case of an 8-wk-old male French Bulldog with a congenital patent hepatic ciliated foregut remnant resulting in an umbilicobiliary sinus tract. The dog also had concurrent gallbladder agenesis. The patient had yellow fluid discharging from the umbilicus, mimicking a patent urachus. Surgical exploration, removal, and histology provided a conclusive diagnosis of a hepatic foregut remnant and therapeutic resolution of the clinical signs. The histologic appearance of a hepatic foregut remnant is classical, namely a duct composed of 4 layers: an inner ciliated epithelial lining, loose connective tissue, smooth muscle, and a fibrous capsule.

Assessment of the mode of action underlying development of liver lesions in mice following oral exposure to HFPO-DA and relevance to humans.

HFPO-DA (ammonium, 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoate) is a short-chain polyfluorinated alkyl substance (PFAS) used in the manufacture of some types of fluorinated polymers. Like many PFAS, toxicity studies with HFPO-DA indicate the liver is the primary target of toxicity in rodents following oral exposure. Due to the structural diversity of PFAS, the mode of action (MOA) can differ between PFAS for the same target tissue. There is significant evidence for involvement of peroxisome proliferator-activated receptor alpha (PPARα) activation based on molecular and histopathological responses in the liver following HFPO-DA exposure, but other MOAs have also been hypothesized based on limited evidence. The MOA underlying the liver effects in mice exposed to HFPO-DA was assessed in the context of the Key Events (KEs) outlined in the MOA framework for PPARα activator-induced rodent hepatocarcinogenesis. The first 3 KEs (ie, PPARα activation, alteration of cell growth pathways, and perturbation of cell growth/survival) are supported by several lines of evidence from both in vitro and in vivo data available for HFPO-DA. In contrast, alternate MOAs, including cytotoxicity, PPARγ and mitochondrial dysfunction are generally not supported by the scientific literature. HFPO-DA-mediated liver effects in mice are not expected in humans as only KE 1, PPARα activation, is shared across species. PPARα-mediated gene expression in humans produces only a subset (ie, lipid modulating effects) of the responses observed in rodents. As such, the adverse effects observed in rodent livers should not be used as the basis of toxicity values for HFPO-DA for purposes of human health risk assessment.

Viral protease cleavage of MAVS in genetically modified mice with hepatitis A virus infection.

BACKGROUND & AIMS: Consistent with its relatively narrow host species range, hepatitis A virus (HAV) cannot infect C57BL/6 mice. However, in Mavs-/- mice with genetic deficiency of the innate immune signaling adaptor MAVS, HAV replicates robustly in the absence of disease. The HAV 3ABC protease cleaves MAVS in human cells, thereby disrupting virus-induced IFN responses, but it cannot cleave murine MAVS (mMAVS) due to sequence differences at the site of scission. Here, we sought to elucidate the role of 3ABC MAVS cleavage in determining HAV pathogenesis and host species range. METHODS: Using CRISPR/Cas9 gene editing, we established two independent lineages of C57BL/6 mice with knock-in mutations altering two amino acids in mMAVS ('mMAVS-VS'), rendering it susceptible to 3ABC cleavage without loss of signaling function. We challenged homozygous Mavsvs/vs mice with HAV, and compared infection outcomes with C57BL/6 and genetically deficient Mavs-/- mice. RESULTS: The humanized murine mMAVS-VS protein was cleaved as efficiently as human MAVS when co-expressed with 3ABC in Huh-7 cells. In embyronic fibroblasts from Mavsvs/vs mice, mMAVS-VS was cleaved by ectopically expressed 3ABC, significantly disrupting Sendai virus-induced IFN responses. However, in contrast to Mavs-/- mice with genetic MAVS deficiency, HAV failed to establish infection in Mavsvs/vs mice, even with additional genetic knockout of Trif or Irf1. Nonetheless, when crossed with permissive Ifnar1-/- mice lacking type I IFN receptors, Mavsvs/vsIfnar1-/- mice demonstrated enhanced viral replication coupled with significant reductions in serum alanine aminotransferase, hepatocellular apoptosis, and intrahepatic inflammatory cell infiltrates compared with Ifnar1-/- mice. CONCLUSIONS: MAVS cleavage by 3ABC boosts viral replication and disrupts disease pathogenesis, but it is not by itself sufficient to break the host-species barrier to HAV infection in mice. IMPACT AND IMPLICATIONS: The limited host range of human hepatitis viruses could be explained by species-specific viral strategies that disrupt innate immune responses. Both hepatitis A virus (HAV) and hepatitis C virus express viral proteases that cleave the innate immune adaptor protein MAVS, in human but not mouse cells. However, the impact of this immune evasion strategy has never been assessed in vivo. Here we show that HAV 3ABC protease cleavage of MAVS enhances viral replication and lessens liver inflammation in mice lacking interferon receptors, but that it is insufficient by itself to overcome the cross-species barrier to infection in mice. These results enhance our understanding of how hepatitis viruses interact with the host and their impact on innate immune responses.

A systematic review on the effects of exercise on gut microbial diversity, taxonomic composition, and microbial metabolites: identifying research gaps and future directions.

The gut microbiome, hosting a diverse microbial community, plays a pivotal role in metabolism, immunity, and digestion. While the potential of exercise to influence this microbiome has been increasingly recognized, findings remain incongruous. This systematic review examined the effects of exercise on the gut microbiome of human and animal models. Databases (i.e., PubMed, Cochrane Library, Scopus, and Web of Science) were searched up to June 2022. Thirty-two exercise studies, i.e., 19 human studies, and 13 animal studies with a minimum of two groups that discussed microbiome outcomes, such as diversity, taxonomic composition, or microbial metabolites, over the intervention period, were included in the systematic review (PROSPERO registration numbers for human review: CRD42023394223). Results indicated that over 50% of studies found no significant exercise effect on human microbial diversity. When evident, exercise often augmented the Shannon index, reflecting enhanced microbial richness and evenness, irrespective of disease status. Changes in beta-diversity metrics were also documented with exercise but without clear directionality. A larger percentage of animal studies demonstrated shifts in diversity compared to human studies, but without any distinct patterns, mainly due to the varied effects of predominantly aerobic exercise on diversity metrics. In terms of taxonomic composition, in humans, exercise usually led to a decrease in the Firmicutes/Bacteroidetes ratio, and consistent increases with Bacteroides and Roseburia genera. In animal models, Coprococcus, another short chain fatty acid (SCFA) producer, consistently rose with exercise. Generally, SCFA producers were found to increase with exercise in animal models. With regard to metabolites, SCFAs emerged as the most frequently measured metabolite. However, due to limited human and animal studies examining exercise effects on microbial-produced metabolites, including SCFAs, clear patterns did not emerge. The overall risk of bias was deemed neutral. In conclusion, this comprehensive systematic review underscores that exercise can potentially impact the gut microbiome with indications of changes in taxonomic composition. The significant variability in study designs and intervention protocols demands more standardized methodologies and robust statistical models. A nuanced understanding of the exercise-microbiome relationship could guide individualized exercise programs to optimize health. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=394223, identifier CRD42023394223.

Hepatocellular carcinomas in captive prosimians.

We performed a retrospective examination of spontaneous hepatocellular carcinomas (HCCs) (primary and metastatic tumors) in 14 captive prosimians brought to the Veterinary Medical Diagnostic Laboratory in North Carolina State University over a period of 11 years (2003 to 2014) to characterize the tumors. These animals are endangered primates; a better understanding of the main fatal neoplasms is crucial. In addition to the histologic evaluation, an immunohistochemical study was also performed, using a hepatocyte marker (hepatocyte paraffin 1 [HepPar-1]) and 2 cholangiocyte markers (keratin 7 [K7] and keratin 19 [K19]), in an attempt to identify a specific profile for HCCs with metastatic behavior. Six of the 14 HCCs had pulmonary metastases. The most frequent histopathological findings were a trabecular pattern (14/14, 100%), presence of multinucleated cells (12/14, 85.7%), and foci of extramedullary hematopoiesis (9/14, 64.3%). The mitotic count was significantly higher in the metastatic HCCs (P < .05). HepPar-1 was detected in all primary and metastatic HCCs, with a strong intensity of staining. Labeling for K7 and K19 was positive in 12 HCCs (85.7%) and 1 HCC (7.1%), respectively. Contrary to the less aggressive HCCs, most of the metastatic HCCs (5/6) expressed K7 in more than 15% of cells. The percentage of K7-positive neoplastic hepatocytes was significantly higher in metastatic HCCs. This study suggests that K7 might be a prognostically relevant marker in HCCs of captive prosimians.

The ZCCHC14/TENT4 complex is required for hepatitis A virus RNA synthesis.

Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5' untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3' poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3' poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent in vitro antiviral activity against HAV (IC50 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1-/- mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.

IRF3-mediated pathogenicity in a murine model of human hepatitis A.

HAV-infected Ifnar1-/- mice recapitulate many of the cardinal features of hepatitis A in humans, including serum alanine aminotransferase (ALT) elevation, hepatocellular apoptosis, and liver inflammation. Previous studies implicate MAVS-IRF3 signaling in pathogenesis, but leave unresolved the role of IRF3-mediated transcription versus the non-transcriptional, pro-apoptotic activity of ubiquitylated IRF3. Here, we compare the intrahepatic transcriptomes of infected versus naïve Mavs-/- and Ifnar1-/- mice using high-throughput sequencing, and identify IRF3-mediated transcriptional responses associated with hepatocyte apoptosis and liver inflammation. Infection was transcriptionally silent in Mavs-/- mice, in which HAV replicates robustly within the liver without inducing inflammation or hepatocellular apoptosis. By contrast, infection resulted in the upregulation of hundreds of genes in Ifnar1-/- mice that develop acute hepatitis closely modeling human disease. Upregulated genes included pattern recognition receptors, interferons, chemokines, cytokines and other interferon-stimulated genes. Compared with Ifnar1-/- mice, HAV-induced inflammation was markedly attenuated and there were few apoptotic hepatocytes in livers of infected Irf3S1/S1Ifnar1-/- mice in which IRF3 is transcriptionally-inactive due to alanine substitutions at Ser-388 and Ser-390. Although transcriptome profiling revealed remarkably similar sets of genes induced in Irf3S1/S1Ifnar1-/- and Ifnar1-/- mice, a subset of genes was differentially expressed in relation to the severity of the liver injury. Prominent among these were both type 1 and type III interferons and interferon-responsive genes associated previously with apoptosis, including multiple members of the ISG12 and 2'-5' oligoadenylate synthetase families. Ifnl3 and Ifnl2 transcript abundance correlated strongly with disease severity, but mice with dual type 1 and type III interferon receptor deficiency remained fully susceptible to liver injury. Collectively, our data show that IRF3-mediated transcription is required for HAV-induced liver injury in mice and identify key IRF3-responsive genes associated with pathogenicity, providing a clear distinction from the transcription-independent role of IRF3 in liver injury following binge exposure to alcohol.

Using rheology to quantify the effects of localized collagenase treatments on uterine fibroid digestion.

Uterine fibroids are stiff, benign tumors containing excessive, disordered collagens that occur in 70-80% of women before age 50 and cause bleeding and pain. Collagenase Clostridium histolyticum (CCH) is a bacterial enzyme capable of digesting the collagens present in fibroids. By combining CCH with injectable drug delivery systems to enhance effectiveness, a new class of treatments could be developed to reduce the stiffness of fibroids, preventing the need for surgical removal and preserving fertility. In this work, we achieved localization of CCH via physical entrapment by co-injecting a thermoresponsive pNIPAM-based polymeric delivery system called LiquoGel (LQG), which undergoes a sol-gel transition upon heating. Toxicity study results for LQG injected subcutaneously into mice demonstrate that LQG does not induce lesions or other adverse effects. We then used rheology to quantify the effects of localized CCH injections on the modulus and viscoelasticity of uterine fibroids, which exhibit gel-like behavior, through ex vivo and in vivo digestion studies. Ex vivo CCH injections reduce the tissue modulus by over two orders of magnitude and co-injection of LQG enhances this effect. Rheological results from an in vivo digestion study in mice show a significant reduction in tissue modulus and increase in tissue viscoelasticity 7 days after a single injection of LQG+CCH. Parallel histological staining validates that the observed rheological changes correspond to an increase in collagen lysis after treatment by LQG+CCH. These results show promise for development of injectable and localized enzymatic therapies for uterine fibroids and other dense tumors. STATEMENT OF SIGNIFICANCE: Uterine fibroids are stiff, benign tumors containing high collagen levels that cause bleeding and pain in women. Fertility-preserving and minimally-invasive treatments to soften fibroids are needed as an alternative to surgical removal via hysterectomy. We demonstrate through ex vivo and in vivo studies that co-injecting a thermoresponsive polymer delivery system (LQG) alongside a bacterial collagenase (CCH) enzyme significantly increases treatment effectiveness at softening fibroids through CCH localization. We use rheology to measure the modulus and viscoelasticity of fibroids and histology to show that fibroid softening corresponds to a decrease in collagen after treatment with LQG+CCH. These results highlight the utility of rheology at quantifying tissue properties and present a promising injectable therapy for fibroids and other dense tumors.