RESUMO
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.
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Vírus da Hepatite E , Hepatite E , Animais , Humanos , Vírus da Hepatite E/genética , Gerbillinae , Tacrolimo , Viremia , GenótipoRESUMO
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.
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Hepatite C Crônica , Hepatite C , Camundongos , Animais , Hepacivirus/genética , Infecção Persistente , Camundongos Endogâmicos C57BL , Linfócitos T CD8-Positivos , FenótipoRESUMO
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.
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Proteínas Cromossômicas não Histona , DNA Polimerase Dirigida por DNA , Vírus da Hepatite A , Hepatite A , Proteínas Intrinsicamente Desordenadas , RNA Nucleotidiltransferases , RNA Viral , Replicação Viral , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Proteínas Cromossômicas não Histona/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Hepatite A/tratamento farmacológico , Hepatite A/metabolismo , Hepatite A/virologia , Vírus da Hepatite A/efeitos dos fármacos , Vírus da Hepatite A/genética , Vírus da Hepatite A/fisiologia , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Camundongos , Camundongos Mutantes , RNA Nucleotidiltransferases/metabolismo , RNA Viral/biossíntese , RNA Viral/genética , Receptor de Interferon alfa e beta/genética , Replicação Viral/efeitos dos fármacosRESUMO
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.
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Ampola Hepatopancreática , Doenças do Gato , Enterite , Neoplasias , Humanos , Gatos , Animais , Ampola Hepatopancreática/patologia , Pâncreas , Inflamação/patologia , Inflamação/veterinária , Enterite/patologia , Enterite/veterinária , Neoplasias/patologia , Neoplasias/veterinária , Doenças do Gato/patologiaRESUMO
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.
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Vírus da Hepatite A , Hepatite A , Animais , Camundongos , Humanos , Vírus da Hepatite A/genética , Peptídeo Hidrolases , Camundongos Endogâmicos C57BL , Imunidade Inata , Proteases ViraisRESUMO
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.
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Hepatite A/metabolismo , Hepatite A/patologia , Fator Regulador 3 de Interferon/metabolismo , Fígado/patologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , TranscriptomaRESUMO
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.
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Carcinoma Hepatocelular , Fluorocarbonos , Neoplasias Hepáticas , Camundongos , Humanos , Animais , Fluorocarbonos/toxicidadeRESUMO
BACKGROUND: COVID-19 outbreaks have disproportionately affected Residential Aged Care Facilities (RACFs) around the world, with devastating impacts for residents and their families. Many factors such as community prevalence, facility layout, and infection control practices have been linked to resident outcomes. At present, there are no scoring systems designed to quantify these factors and assess their level of association with resident attack rates and mortality rates. METHODS: We constructed a novel Infection Prevention and Control (IPC) scoring system to quantify facility layout, ability to cohort residents, and IPC practices in RACFs. We conducted a retrospective observational cohort study of COVID-19 outbreaks, applying our IPC scoring system to all COVID-19 outbreaks occurring in RACFs in Sydney Local Health District during the Delta and Omicron waves of the COVID-19 pandemic in New South Wales, Australia. RESULTS: Twenty-six COVID-19 outbreaks in 23 facilities in the Delta wave, and 84 outbreaks in 53 facilities in the Omicron wave were included in the study. A linear Generalised Estimating Equation model was fitted to the Omicron data. Higher IPC scores were associated with higher attack rates and mortality rates. Facilities with IPC scores greater than 75.0% had attack rates 19.6% higher [95% CI: 6.4%-32.8%] and mortality rates 1.7% higher [95% CI: 0.6%-2.7%] than facilities with an IPC score of less than 60.0%. CONCLUSIONS: The results of this study suggest the utility of the IPC scoring system for identifying facilities at greater risk of adverse outcomes from COVID-19 outbreaks. While further validation and replication of accuracy is required, the IPC scoring system could be used and adapted to improve planning, policy, and resource allocation for future outbreaks.
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COVID-19 , Humanos , Idoso , COVID-19/epidemiologia , COVID-19/prevenção & controle , Pandemias/prevenção & controle , Estudos Retrospectivos , Austrália/epidemiologia , Surtos de Doenças/prevenção & controleRESUMO
Iminosugar compounds are monosaccharide mimetics with broad but generally weak antiviral activities related to inhibition of enzymes involved in glycobiology. Miglustat (N-butyl-1-deoxynojirimycin), which is approved for the treatment of lipid storage diseases in humans, and UV-4 [N-(9-methoxynonyl)-1-deoxynojirimycin] inhibit the replication of hepatitis A virus (HAV) in cell culture (50% inhibitory concentrations [IC50s] of 32.13 µM and 8.05 µM, respectively) by blocking the synthesis of gangliosides essential for HAV cell entry. We used a murine model of hepatitis A and targeted mass spectrometry to assess the capacity of these compounds to deplete hepatic gangliosides and modify the course of HAV infection in vivo. Miglustat, given by gavage to Ifnar1-/- mice (4,800 mg/kg of body weight/day) depleted hepatic gangliosides by 69 to 75% but caused substantial gastrointestinal toxicity and failed to prevent viral infection. UV-4, similarly administered in high doses (400 mg/kg/day), was well tolerated but depleted hepatic gangliosides by only 20% after 14 days. UV-4 depletion of gangliosides varied by class. Several GM2 species were paradoxically increased, likely due to inhibition of ß-glucosidases that degrade gangliosides. Both compounds enhanced, rather than reduced, virus replication. Nonetheless, both iminosugars had surprising anti-inflammatory effects, blocking the accumulation of inflammatory cells within the liver. UV-4 treatment also resulted in a decrease in serum alanine aminotransferase (ALT) elevations associated with acute hepatitis A. These anti-inflammatory effects may result from iminosugar inhibition of cellular α-glucosidases, leading to impaired maturation of glycan moieties of chemokine and cytokine receptors, and point to the potential importance of paracrine signaling in the pathogenesis of acute hepatitis A. IMPORTANCE Hepatitis A virus (HAV) is a common cause of viral hepatitis. Iminosugar compounds block its replication in cultured cells by inhibiting the synthesis of gangliosides required for HAV cell entry but have not been tested for their ability to prevent or treat hepatitis A in vivo. We show that high doses of the iminosugars miglustat and UV-4 fail to deplete gangliosides sufficiently to block HAV infection in mice lacking a key interferon receptor. These compounds nonetheless have striking anti-inflammatory effects on the HAV-infected liver, reducing the severity of hepatitis despite enhancing chemokine and cytokine expression resulting from hepatocyte-intrinsic antiviral responses. We propose that iminosugar inhibition of cellular α-glucosidases impairs the maturation of glycan moieties of chemokine and cytokine receptors required for effective signaling. These data highlight the potential importance of paracrine signaling pathways in the inflammatory response to HAV and add to our understanding of HAV pathogenesis in mice.
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Gangliosídeos , Inibidores de Glicosídeo Hidrolases , Hepatite A , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Antivirais/farmacologia , Gangliosídeos/metabolismo , Hepatite A/tratamento farmacológico , Vírus da Hepatite A , Inflamação/tratamento farmacológico , Camundongos , Camundongos Knockout , Receptor de Interferon alfa e beta/genética , Receptores de Interferon , Internalização do Vírus , alfa-Glucosidases/farmacologiaRESUMO
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.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Strepsirhini , Animais , Biomarcadores Tumorais , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/veterinária , Imuno-Histoquímica , Queratina-19 , Queratina-7 , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/veterinária , Parafina , Estudos RetrospectivosRESUMO
BACKGROUND & AIMS: Hepatitis A virus (HAV) is a common cause of enterically transmitted viral hepatitis. In non-immune individuals, infection results in typically transient but occasionally fulminant and fatal inflammatory liver injury. Virus-specific T cell frequencies peak when liver damage is at its zenith, leading to the prevalent notion that T cells exacerbate liver disease, as suspected for other hepatotropic virus infections. However, the overall contribution of T cells to the control of HAV and the pathogenesis of hepatitis A is unclear and has been impeded by a historic lack of small animal models. METHODS: Ifnar1-/- mice are highly permissive for HAV and develop pathogenesis that recapitulates many features of hepatitis A. Using this model, we identified HAV-specific CD8+ and CD4+ T cells by epitope mapping, and then used tetramers and functional assays to quantify T cells in the liver at multiple times after infection. We assessed the relationships between HAV-specific T cell frequency, viral RNA amounts, and liver pathogenesis. RESULTS: A large population of virus-specific T cells accumulated within the livers of Ifnar1-/- mice during the first 1-2 weeks of infection and persisted over time. HAV replication was enhanced and liver disease exacerbated when mice were depleted of T cells. Conversely, immunization with a peptide vaccine increased virus-specific CD8+ T cell frequencies in the liver, reduced viral RNA abundance, and lessened liver injury. CONCLUSION: These data show that T cells protect against HAV-mediated liver injury and can be targeted to improve liver health. LAY SUMMARY: Hepatitis A virus is a leading cause of acute viral hepatitis worldwide. T cells were thought to contribute to liver injury during acute infection. We now show that virus-specific T cells protect against infection and limit liver injury.
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Hepatite A/prevenção & controle , Hepatopatias/prevenção & controle , Linfócitos T/metabolismo , Análise de Variância , Animais , Modelos Animais de Doenças , Hepatite A/tratamento farmacológico , Hepatite A/epidemiologia , Vírus da Hepatite A/efeitos dos fármacos , Vírus da Hepatite A/patogenicidade , Hepatopatias/tratamento farmacológico , Hepatopatias/epidemiologia , Camundongos , North Carolina , Estatísticas não Paramétricas , Linfócitos T/fisiologiaRESUMO
The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions) Project (www.toxpath.org/inhand.asp) is a joint initiative of the societies of toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying lesions observed in most tissues and organs from the dog used in nonclinical safety studies. Some of the lesions are illustrated by color photomicrographs. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous lesions, lesions induced by exposure to test materials, and relevant infectious and parasitic lesions. A widely accepted and utilized international harmonization of nomenclature for lesions in laboratory animals will provide a common language among regulatory and scientific research organizations in different countries and increase and enrich international exchanges of information among toxicologists and pathologists.
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Animais de Laboratório , Animais , Bases de Dados Factuais , Cães , Europa (Continente) , JapãoRESUMO
Ductal plate malformations are abnormalities in the liver that arise from inappropriate or incomplete remodeling of the embryologic ductal plate. Various types of ductal plate malformations are reported in the human and veterinary literature, most commonly affecting domestic mammalian species but also fish. We investigated the occurrence and described the histopathologic features of ductal plate malformations in captive snakes. Malformations were identified in 18 snakes: 10 colubrids, 6 vipers, and 2 boids. There was no sex predilection, and the mean age was 17 years. The majority of lesions were incidental with most snakes having one or more comorbidities, most commonly neoplasia or systemic inflammation, that resulted in natural death or euthanasia. Ductal plate malformations in all livers were broadly characterized by a well-demarcated nodule of irregular bile ducts embedded within a varying amount of fibrous stroma. Malformations were further categorized based on the amount of fibrous stroma and dilation of the bile ducts as von Meyenburg complexes, cystic liver disease, and/or an intermediate hybrid subtype representative of cysts arising within von Meyenburg complexes. Histochemical and immunohistochemical staining, including Gomori's trichome and pan-cytokeratin, respectively, were applied on select cases to confirm histologic features. Malignant transformation was not identified within this population.
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Cistos , Hepatopatias , Animais , Ductos Biliares , Ductos Biliares Intra-Hepáticos , Cistos/veterinária , Hepatopatias/veterinária , SerpentesRESUMO
Standardization of tumor assessment lays the foundation for validation of grading systems, permits reproducibility of oncologic studies among investigators, and increases confidence in the significance of study results. Currently, there is minimal methodological standardization for assessing tumors in veterinary medicine, with few attempts to validate published protocols and grading schemes. The current article attempts to address these shortcomings by providing standard guidelines for tumor assessment parameters and protocols for evaluating specific tumor types. More detailed information is available in the Supplemental Files, the intention of which is 2-fold: publication as part of this commentary, but more importantly, these will be available as "living documents" on a website (www.vetcancerprotocols.org), which will be updated as new information is presented in the peer-reviewed literature. Our hope is that veterinary pathologists will agree that this initiative is needed, and will contribute to and utilize this information for routine diagnostic work and oncologic studies. Journal editors and reviewers can utilize checklists to ensure publications include sufficient detail and standardized methods of tumor assessment. To maintain the relevance of the guidelines and protocols, it is critical that the information is periodically updated and revised as new studies are published and validated with the intent of providing a repository of this information. Our hope is that this initiative (a continuation of efforts published in this journal in 2011) will facilitate collaboration and reproducibility between pathologists and institutions, increase case numbers, and strengthen clinical research findings, thus ensuring continued progress in veterinary oncologic pathology and improving patient care.
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Neoplasias , Patologia Veterinária , Animais , Neoplasias/diagnóstico , Neoplasias/veterinária , Reprodutibilidade dos TestesRESUMO
Small intestinal (SI) tumors are relatively uncommon outcomes in rodent cancer bioassays, and limited information regarding chemical-induced SI tumorigenesis has been reported in the published literature. Herein, we propose a cytotoxicity-mediated adverse outcome pathway (AOP) for SI tumors by leveraging extensive target species- and site-specific molecular, cellular, and histological mode of action (MOA) research for three reference chemicals, the fungicides captan and folpet and the transition metal hexavalent chromium (Cr(VI)). The gut barrier functions through highly efficient homeostatic regulation of SI epithelial cell sloughing, regenerative proliferation, and repair, which involves the replacement of up to 1011 cells per day. This dynamic turnover in the SI provides a unique local environment for a cytotoxicity mediated AOP/MOA. Upon entering the duodenum, cytotoxicity to the villous epithelium is the molecular initiating event, as indicated by crypt elongation, villous atrophy/blunting, and other morphologic changes. Over time, the regenerative capacity of the gut epithelium to compensate declines as epithelial loss accelerates, especially at higher exposures. The first key event (KE), sustained regenerative crypt proliferation/hyperplasia, requires sufficient durations, likely exceeding 6 or 12 months, due to extensive repair capacity, to create more opportunities for the second KE, spontaneous mutation/transformation, ultimately leading to proximal SI tumors. Per OECD guidance, biological plausibility, essentiality, and empirical support were assessed using modified Bradford Hill considerations. The weight-of-evidence also included a lack of induced mutations in the duodenum after up to 90 days of Cr(VI) or captan exposure. The extensive evidence for this AOP, along with the knowledge that human exposures are orders of magnitude below those associated with KEs in this AOP, supports its use for regulatory applications, including hazard identification and risk assessment.
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Captana/toxicidade , Cromo/toxicidade , Fungicidas Industriais/toxicidade , Hiperplasia , Neoplasias Intestinais/induzido quimicamente , Ftalimidas/toxicidade , Rotas de Resultados Adversos , Animais , Duodeno , Humanos , Camundongos , Medição de RiscoRESUMO
GenX is an alternative to environmentally persistent long-chain perfluoroalkyl and polyfluoroalkyl substances. Mice exposed to GenX exhibit liver hypertrophy, elevated peroxisomal enzyme activity, and other apical endpoints consistent with peroxisome proliferators. To investigate the potential role of peroxisome proliferator-activated receptor alpha (PPARα) activation in mice, and other molecular signals potentially related to observed liver changes, RNA sequencing was conducted on paraffin-embedded liver sections from a 90-day subchronic toxicity study of GenX conducted in mice. Differentially expressed genes were identified for each treatment group, and gene set enrichment analysis was conducted using gene sets that represent biological processes and known canonical pathways. Peroxisome signaling and fatty acid metabolism were among the most significantly enriched gene sets in both sexes at 0.5 and 5 mg/kg GenX; no pathways were enriched at 0.1 mg/kg. Gene sets specific to the PPARα subtype were significantly enriched. These findings were phenotypically anchored to histopathological changes in the same tissue blocks: hypertrophy, mitoses, and apoptosis. In vitro PPARα transactivation assays indicated that GenX activates mouse PPARα. These results indicate that the liver changes observed in GenX-treated mice occur via a mode of action (MOA) involving PPARα, an important finding for human health risk assessment as this MOA has limited relevance to humans.
Assuntos
Hidrocarbonetos Fluorados/toxicidade , Fígado/efeitos dos fármacos , PPAR alfa/efeitos dos fármacos , Propionatos/toxicidade , Animais , Feminino , Humanos , Masculino , Camundongos , Medição de Risco , Transcriptoma/efeitos dos fármacosRESUMO
STUDY OBJECTIVE: Common outcomes of care valued by emergency department (ED) patients who are not hospitalized have been characterized, but no measurement instrument has been developed to date. We developed and validated a patient-reported outcome measure for use with adult ED patients who are discharged home (PROM-ED). METHODS: In previous research, 4 main outcomes of importance to ED patients were defined: symptom relief, understanding, reassurance, and having a plan. We developed a bank of potential questions (phase 1) that were first tested for suitability through cognitive debriefing with patients (phase 2). Revised questions were then tested quantitatively with a large panel of participants who had recently received ED care (phase 3). Informed by these results, a panel of experts used a modified Delphi process to make decisions on item reduction. The resulting instrument (PROM-ED 1.0) was then evaluated for its measurement properties (structural validity, hypothesis testing, and reliability). RESULTS: Sixty-seven questions divided among 4 scales (1 for each outcome domain) were assembled. In accordance with cognitive debriefing with 8 patients (phase 2), 15 questions were modified and 13 removed. Testing of these questions with 444 participants (phase 3) identified problematic floor or ceiling effects (n=10), excessive correlations between items (n=11), and low item-total correlations (n=7). The expert panel (22 participants, phase 4) made decisions using this information on the exclusion of items, resulting in 22 questions across 4 scales that together constitute the PROM-ED 1.0. Testing provided good evidence of validity and test-retest reliability (n=200). CONCLUSION: The PROM-ED enables the measurement of patient-centered outcomes of importance to patients receiving care in the ED who are not hospitalized. These data could have important applications in research and care improvement.
Assuntos
Serviço Hospitalar de Emergência , Conhecimentos, Atitudes e Prática em Saúde , Medidas de Resultados Relatados pelo Paciente , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Fatorial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Alta do Paciente , Reprodutibilidade dos Testes , Inquéritos e Questionários , Resultado do Tratamento , Adulto JovemRESUMO
Limitations in workforce size and access to resources remain perennial challenges to greater progress in academic veterinary medicine and engagement between human and veterinary medicine (One Health). Ongoing resource constraints occur in part due to limited public understanding of the role veterinarians play in improving human health. One Health interactions, particularly through interdisciplinary collaborations in biomedical research, present constructive opportunities to inform resource policies and advance health care. To this end, inter-institutional partnerships between individual veterinary medical education programs (VMEPs) and several National Institutes of Health (NIH) intramural research programs have created synergies beyond those provided by individual programs. In the NIH Comparative Biomedical Scientist Training Program (CBSTP), interdisciplinary cross-training of veterinarians consisting of specialty veterinary medicine coupled with training in human disease research leading to a PhD, occurs collaboratively on both VMEP and NIH campuses. Pre-doctoral veterinary student research opportunities have also been made available. Through the CBSTP, NIH investigators and national biomedical science policy makers gain access to veterinary perspective and expertise, while veterinarians obtain additional opportunities for NIH-funded research training. CBSTP Fellows serve as de facto ambassadors enhancing visibility for the profession while in residence at NIH, and subsequently through a variety of university, industry, and government research appointments, as graduates. Thus, the CBSTP represents an inter-institutional opportunity that not only addresses critical needs for veterinarian-scientists in the biomedical workforce, but also simultaneously exposes national policy makers to veterinarian-scientists' specialized training, leading to more effective realization of One Health goals to benefit human and animal health.
Assuntos
Pesquisa Biomédica , Educação em Veterinária , Saúde Única , Médicos Veterinários , Animais , Objetivos , Humanos , National Institutes of Health (U.S.) , Estados UnidosRESUMO
The lack of a relevant, tractable, and immunocompetent animal model for hepatitis C virus (HCV) has severely impeded investigations of viral persistence, immunity, and pathogenesis. In the absence of immunocompetent models with robust HCV infection, homolog hepaciviruses in their natural host could potentially provide useful surrogate models. We isolated a rodent hepacivirus from wild rats (Rattus norvegicus), RHV-rn1; acquired the complete viral genome sequence; and developed an infectious reverse genetics system. RHV-rn1 resembles HCV in genomic features including the pattern of polyprotein cleavage sites and secondary structures in the viral 5' and 3' untranslated regions. We used site-directed and random mutagenesis to determine that only the first of the two microRNA-122 seed sites in the viral 5' untranslated region is required for viral replication and persistence in rats. Next, we used the clone-derived virus progeny to infect several inbred and outbred rat strains. Our results determined that RHV-rn1 possesses several HCV-defining hallmarks: hepatotropism, propensity to persist, and the ability to induce gradual liver damage. Histological examination of liver samples revealed the presence of lymphoid aggregates, parenchymal inflammation, and macrovesicular and microvesicular steatosis in chronically infected rats. Gene expression analysis demonstrated that the intrahepatic response during RHV-rn1 infection in rats mirrors that of HCV infection, including persistent activation of interferon signaling pathways. Finally, we determined that the backbone drug of HCV direct-acting antiviral therapy, sofosbuvir, effectively suppresses chronic RHV-rn1 infection in rats. CONCLUSION: We developed RHV-rn1-infected rats as a fully immunocompetent and informative surrogate model to delineate the mechanisms of HCV-related viral persistence, immunity, and pathogenesis. (Hepatology 2018).
Assuntos
Hepacivirus/genética , Hepatite C/virologia , Hepatopatias/virologia , Alanina Transaminase , Animais , Antivirais/farmacologia , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Hepacivirus/patogenicidade , Hepatite C/genética , MicroRNAs/genética , RNA Viral/genética , Ratos , Ratos Sprague-Dawley , Análise de Sequência de DNA/métodos , Replicação Viral/genéticaRESUMO
Lemurs are highly endangered mammals inhabiting the forests of Madagascar. In this study, we performed virus discovery on serum samples collected from 84 wild lemurs and identified viral sequence fragments from 4 novel viruses within the family Flaviviridae, including members of the genera Hepacivirus and Pegivirus. The sifaka hepacivirus (SifHV, two genotypes) and pegivirus (SifPgV, two genotypes) were discovered in the diademed sifaka (Propithecus diadema), while other pegiviral fragments were detected in samples from the indri (Indri indri, IndPgV) and the weasel sportive lemur (Lepilemur mustelinus, LepPgV). Although data are preliminary, each viral species appeared host species-specific and frequent infection was detected (18 of 84 individuals were positive for at least one virus). The complete coding sequence and partial 5' and 3' untranslated regions (UTRs) were obtained for SifHV and its genomic organization was consistent with that of other hepaciviruses, with one unique polyprotein and highly structured UTRs. Phylogenetic analyses showed the SifHV belonged to a clade that includes several viral species identified in rodents from Asia and North America, while SifPgV and IndPgV were more closely related to pegiviral species A and C, that include viruses found in humans as well as New- and Old-World monkeys. Our results support the current proposed model of virus-host co-divergence with frequent occurrence of cross-species transmission for these genera and highlight how the discovery of more members of the Flaviviridae can help clarify the ecology and evolutionary history of these viruses. Furthermore, this knowledge is important for conservation and captive management of lemurs.