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2.
Emerg Microbes Infect ; 10(1): 1881-1889, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34490832

RESUMO

SARS-CoV-2 has been the causative pathogen of the pandemic of COVID-19, resulting in catastrophic health issues globally. It is important to develop human-like animal models for investigating the mechanisms that SARS-CoV-2 uses to infect humans and cause COVID-19. Several studies demonstrated that the non-human primate (NHP) is permissive for SARS-CoV-2 infection to cause typical clinical symptoms including fever, cough, breathing difficulty, and other diagnostic abnormalities such as immunopathogenesis and hyperplastic lesions in the lung. These NHP models have been used for investigating the potential infection route and host immune response to SARS-CoV-2, as well as testing vaccines and drugs. This review aims to summarize the benefits and caveats of NHP models currently available for SARS-CoV-2, and to discuss key topics including model optimization, extended application, and clinical translation.


Assuntos
COVID-19/virologia , Modelos Animais de Doenças , Primatas/virologia , SARS-CoV-2/fisiologia , Animais , Antivirais/administração & dosagem , COVID-19/tratamento farmacológico , COVID-19/imunologia , COVID-19/patologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/imunologia , Humanos , Primatas/imunologia , SARS-CoV-2/genética
4.
Sci Transl Med ; 13(606)2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34285130

RESUMO

Multiple safe and effective vaccines that elicit immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are necessary to respond to the ongoing coronavirus disease 2019 (COVID-19) pandemic. Here, we developed a protein subunit vaccine composed of spike ectodomain protein (StriFK) plus a nitrogen bisphosphonate-modified zinc-aluminum hybrid adjuvant (FH002C). StriFK-FH002C generated substantially higher neutralizing antibody titers in mice, hamsters, and cynomolgus monkeys than those observed in plasma isolated from COVID-19 convalescent individuals. StriFK-FH002C also induced both TH1- and TH2-polarized helper T cell responses in mice. In hamsters, StriFK-FH002C immunization protected animals against SARS-CoV-2 challenge, as shown by the absence of virus-induced weight loss, fewer symptoms of disease, and reduced lung pathology. Vaccination of hamsters with StriFK-FH002C also reduced within-cage virus transmission to unvaccinated, cohoused hamsters. In summary, StriFK-FH002C represents an effective, protein subunit-based SARS-CoV-2 vaccine candidate.


Assuntos
COVID-19 , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra COVID-19 , Cricetinae , Humanos , Camundongos , Subunidades Proteicas , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética
5.
Theranostics ; 11(13): 6607-6615, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995679

RESUMO

SARS-CoV-2 infection, which is responsible for the current COVID-19 pandemic, can cause life-threatening pneumonia, respiratory failure and even death. Characterizing SARS-CoV-2 pathogenesis in primary human target cells and tissues is crucial for developing vaccines and therapeutics. However, given the limited access to clinical samples from COVID-19 patients, there is a pressing need for in vitro/in vivo models to investigate authentic SARS-CoV-2 infection in primary human lung cells or tissues with mature structures. The present study was designed to evaluate a humanized mouse model carrying human lung xenografts for SARS-CoV-2 infection in vivo. Methods: Human fetal lung tissue surgically grafted under the dorsal skin of SCID mice were assessed for growth and development after 8 weeks. Following SARS-CoV-2 inoculation into the differentiated lung xenografts, viral replication, cell-type tropism and histopathology of SARS-CoV-2 infection, and local cytokine/chemokine expression were determined over a 6-day period. The effect of IFN-α treatment against SARS-CoV-2 infection was tested in the lung xenografts. Results: Human lung xenografts expanded and developed mature structures closely resembling normal human lung. SARS-CoV-2 replicated and spread efficiently in the lung xenografts with the epithelial cells as the main target, caused severe lung damage, and induced a robust pro-inflammatory response. IFN-α treatment effectively inhibited SARS-CoV-2 replication in the lung xenografts. Conclusions: These data support the human lung xenograft mouse model as a useful and biological relevant tool that should facilitate studies on the pathogenesis of SARS-CoV-2 lung infection and the evaluation of potential antiviral therapies.


Assuntos
COVID-19/imunologia , Modelos Animais de Doenças , Pulmão/patologia , Mucosa Respiratória/citologia , SARS-CoV-2/imunologia , Feto Abortado , Animais , COVID-19/patologia , COVID-19/virologia , Células Cultivadas , Células Epiteliais/virologia , Xenoenxertos , Humanos , Pulmão/imunologia , Pulmão/virologia , Transplante de Pulmão , Masculino , Camundongos , Camundongos SCID , Cultura Primária de Células , SARS-CoV-2/patogenicidade , Replicação Viral
6.
Signal Transduct Target Ther ; 6(1): 136, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790236

RESUMO

Epidemiological studies of the COVID-19 patients have suggested the male bias in outcomes of lung illness. To experimentally demonstrate the epidemiological results, we performed animal studies to infect male and female Syrian hamsters with SARS-CoV-2. Remarkably, high viral titer in nasal washings was detectable in male hamsters who presented symptoms of weight loss, weakness, piloerection, hunched back and abdominal respiration, as well as severe pneumonia, pulmonary edema, consolidation, and fibrosis. In contrast with the males, the female hamsters showed much lower shedding viral titers, moderate symptoms, and relatively mild lung pathogenesis. The obvious differences in the susceptibility to SARS-CoV-2 and severity of lung pathogenesis between male and female hamsters provided experimental evidence that SARS-CoV-2 infection and the severity of COVID-19 are associated with gender.


Assuntos
COVID-19 , SARS-CoV-2/metabolismo , Caracteres Sexuais , Animais , COVID-19/metabolismo , COVID-19/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Masculino , Mesocricetus
7.
Small Methods ; 5(2): 2001031, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33614907

RESUMO

The ongoing corona virus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system, a genetically engineered sensor of fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process is developed. In ACE2-expressing cells, it is found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.

8.
Stem Cell Res ; 47: 101900, 2020 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-32622343

RESUMO

BACKGROUND & AIMS: Biliary injury is one of the main pathological mechanisms of fulminant hepatic failure (FHF). Delta-like ligand 4 (DLL4)-mediated Notch activation contributes to reversing biliary injury; however, the specific role of DLL4 in biliary restoration is still unclear. This study aimed to determine whether human bone marrow mesenchymal stem cells (hBMSCs) can differentiate into biliary epithelial cells (cholangiocytes) in vitro and in vivo and to clarify the role of DLL4 in restoring damaged liver by enhancing cholangiocyte differentiation. METHODS: hBMSCs were transplanted into immunodeficient mice (FRGS) with FHF induced by the hamster-anti-mouse CD95 antibody JO2. The appearance of human cholangiocytes was evaluated in the generated hBMSC-FRGS mice by q-PCR expression, flow cytometry and immunohistochemistry. The potency of DLL4 in inducing cholangiocyte differentiation from hBMSCs was assessed by observing the cell morphology and measuring the expression of cholangiocyte-specific genes and proteins. RESULTS: Human KRT19- and KRT7-double-positive cholangiocyte-like cells appeared in hBMSC-FRGS mice at 12 weeks after transplantation. After these cells were separated and collected by fluorescent-activated cell sorting (FACS), there were high levels of expression of eight typical human cholangiocyte-specific genes and proteins (e.g., KRT19 and KRT7). Furthermore, hBMSC-derived cholangiocytes induced by DLL4 had a better shape with higher nucleus/cytoplasm ratios and showed a specific increase in the expression of cholangiocyte-specific genes and proteins (e.g., KRT19, KRT7, SOX9 and CFTR). CONCLUSIONS: Cholangiocytes can be efficiently differentiated from hBMSCs in vivo and in vitro. DLL4 restores damaged liver by enhancing cholangiocyte differentiation from hBMSCs and has the potential to be used in future clinical therapeutic applications.

9.
Emerg Microbes Infect ; 9(1): 949-961, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32378471

RESUMO

The emergences of coronaviruses have caused a serious global public health problem because their infection in humans caused the severe acute respiratory disease and deaths. The outbreaks of lethal coronaviruses have taken place for three times within recent two decades (SARS-CoV in 2002, MERS-CoV in 2012 and SARS-CoV-2 in 2019). Much more serious than SARS-CoV in 2002, the current SARS-CoV-2 infection has been spreading to more than 213 countries, areas or territories and causing more than two million cases up to date (17 April 2020). Unfortunately, no vaccine and specific anti-coronavirus drugs are available at present time. Current clinical treatment at hand is inadequate to suppress viral replication and inflammation, and reverse organ failure. Intensive research efforts have focused on increasing our understanding of viral biology of SARS-CoV-2, improving antiviral therapy and vaccination strategies. The animal models are important for both the fundamental research and drug discovery of coronavirus. This review aims to summarize the animal models currently available for SARS-CoV and MERS-CoV, and their potential use for the study of SARS-CoV-2. We will discuss the benefits and caveats of these animal models and present critical findings that might guide the fundamental studies and urgent treatment of SARS-CoV-2-caused diseases.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronaviridae/patologia , Infecções por Coronaviridae/prevenção & controle , Infecções por Coronavirus/patologia , Infecções por Coronavirus/prevenção & controle , Modelos Animais de Doenças , Pandemias/prevenção & controle , Pneumonia Viral/patologia , Pneumonia Viral/prevenção & controle , Pesquisa/tendências , Animais , COVID-19 , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Vírus da SARS/fisiologia , SARS-CoV-2
10.
Theranostics ; 9(7): 2115-2128, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31037160

RESUMO

Rationale: Hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (hiPSCs) have been developed to address the shortage of primary human hepatocytes (PHHs) for therapeutic applications. However, the in vivo repopulation capacity of HLCs remains limited. This study investigated the roles of agonist antibody activating the c-Met receptor in promoting the in vivo proliferation and repopulation of engrafted PHHs and/or HLCs in mice with liver injuries due to different causes. Methods: An agonist c-Met receptor antibody (5D5) was used to treat PHHs and hiPSC-HLCs in both cell culture and hepatocyte-engrafted immunodeficient mice mimicking various inherited and acquired liver diseases. The promoting roles and potential influence on the hepatic phenotype of the 5D5 regimen in cell transplantation-based therapeutic applications were systematically evaluated. Results: In hiPSC-HLC cell cultures, 5D5 treatment significantly stimulated c-Met receptor downstream signalling pathways and accelerated cell proliferation in dose-dependent and reversible manners. In contrast, only slight but nonsignificant promotion was observed in 5D5-treated PHHs. In vivo administration of 5D5 greatly promoted the expansion of implanted hiPSC-HLCs in fumarylacetoacetate hydrolase (Fah) deficient mice, resulting in significantly increased human albumin levels and high human liver chimerism (over 40%) in the transplanted mice at week 8 after transplantation. More importantly, transplantation of hiPSC-HLCs in combination with 5D5 significantly prolonged animal survival and ameliorated liver pathological changes in mice with acute and/or chronic liver injuries caused by Fas agonistic antibody treatment, carbon tetrachloride treatment and/or tyrosinemic stress. Conclusion: Our results demonstrated that the proliferation of hiPSC-HLCs can be enhanced by antibody-mediated modulation of c-Met signalling and facilitate hiPSC-HLC-based therapeutic applications for life-threatening liver diseases.


Assuntos
Anticorpos Monoclonais/farmacologia , Proliferação de Células/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Falência Hepática/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Transplante de Células/métodos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Modelos Animais de Doenças , Hepatócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Falência Hepática/metabolismo , Camundongos
11.
Gut ; 68(11): 2044-2056, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-30700543

RESUMO

OBJECTIVE: Developing a small animal model that accurately delineates the natural history of hepatitis B virus (HBV) infection and immunopathophysiology is necessary to clarify the mechanisms of host-virus interactions and to identify intervention strategies for HBV-related liver diseases. This study aimed to develop an HBV-induced chronic hepatitis and cirrhosis mouse model through transplantation of human bone marrow mesenchymal stem cells (hBMSCs). DESIGN: Transplantation of hBMSCs into Fah-/-Rag2-/-IL-2Rγc-/- SCID (FRGS) mice with fulminant hepatic failure (FHF) induced by hamster-anti-mouse CD95 antibody JO2 generated a liver and immune cell dual-humanised (hBMSC-FRGS) mouse. The generated hBMSC-FRGS mice were subjected to assessments of sustained viremia, specific immune and inflammatory responses and liver pathophysiological injury to characterise the progression of chronic hepatitis and cirrhosis after HBV infection. RESULTS: The implantation of hBMSCs rescued FHF mice, as demonstrated by robust proliferation and transdifferentiation of functional human hepatocytes and multiple immune cell lineages, including B cells, T cells, natural killer cells, dendritic cells and macrophages. After HBV infection, the hBMSC-FRGS mice developed sustained viremia and specific immune and inflammatory responses and showed progression to chronic hepatitis and liver cirrhosis at a frequency of 55% after 54 weeks. CONCLUSION: This new humanised mouse model recapitulates the liver cirrhosis induced by human HBV infection, thus providing research opportunities for understanding viral immune pathophysiology and testing antiviral therapies in vivo.


Assuntos
Modelos Animais de Doenças , Hepatite B Crônica/etiologia , Cirrose Hepática/etiologia , Transplante de Células-Tronco Mesenquimais , Animais , Hepatite B Crônica/patologia , Humanos , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID
12.
Angew Chem Int Ed Engl ; 57(38): 12499-12503, 2018 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-30088325

RESUMO

A facile route is presented for fabricating a new class of nanomimics that overexpress hepatitis B virus (HBV) receptor by a natural biosynthetic procedure against HBV infection. A nine-transmembrane HBV-specific receptor, human sodium taurocholate co-transporting polypeptide (hNTCP), was engineered to naturally immobilize it onto the cellular surface and subsequently trigger the budding of hNTCP-anchoring membrane vesicles (hNTCP-MVs) that favor the HBV virion. hNTCP-MVs could rapidly block HBV infection in cell models. Furthermore, hNTCP-MVs treatment could effectively prevent viral infection, spreading, and replication in a human-liver-chimeric mouse model of HBV infection. Our findings demonstrate the receptor-mediated antiviral effect of hNTCP-MVs to trick HBV and offer novel opportunities for further development of antiviral strategies in nanomedicine.


Assuntos
Membrana Celular/metabolismo , Vírus da Hepatite B/fisiologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Simportadores/química , Animais , Biomimética , Membrana Celular/química , Células Hep G2 , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Camundongos , Microscopia Confocal , Modelos Biológicos , Nanomedicina , Nanoestruturas/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Simportadores/genética , Simportadores/metabolismo , Internalização do Vírus , Replicação Viral
13.
Emerg Microbes Infect ; 7(1): 144, 2018 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-30097574

RESUMO

The human liver chimeric mouse with primary human hepatocytes (PHHs) engraftment has been demonstrated to be a useful animal model to study hepatitis B virus (HBV) pathogenesis and evaluate anti-HBV drugs. However, the disadvantages of using PHHs include the inability for cellular expansion in vitro, limited donor availability, individual differences, and ethical issues, necessitating the development of alternatives. To obtain in vitro expandable hepatocytes, we optimized the hepatic differentiation procedure of the human liver progenitor cell line, HepaRG, using four functional small molecules (4SM) and enriched the precursor hepatocyte-like cells (HLCs). HepaRG cells of different hepatic differentiation states were engrafted to immunodeficient mice (FRGS) with weekly 4SM treatment. The HepaRG-engrafted mice were challenged with HBV and/or treated with several antivirals to evaluate their effects. We demonstrated that the 4SM treatment enhanced hepatic differentiation and promoted cell proliferation capacity both in vitro and in vivo. Mice engrafted with enriched HepaRG of prehepatic differentiation and treated with 4SM displayed approximately 10% liver chimerism at week 8 after engraftment and were maintained at this level for another 16 weeks. Therefore, we developed a HepaRG-based human liver chimeric mouse model: HepaRG-FRGS. Our experimental results showed that the liver chimerism of the mice was adequate to support chronic HBV infection for 24 weeks and to evaluate antivirals. We also demonstrated that HBV infection in HepaRG cells was dependent on their hepatic differentiation state and liver chimerism in vivo. Overall, HepaRG-FRGS mice provide a novel human liver chimeric mouse model to study chronic HBV infection and evaluate anti-HBV drugs.


Assuntos
Quimera/virologia , Modelos Animais de Doenças , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/virologia , Hepatócitos/virologia , Fígado/virologia , Animais , Diferenciação Celular , Linhagem Celular , Quimera/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/fisiopatologia , Hepatócitos/citologia , Humanos , Fígado/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Replicação Viral
14.
Cell Death Dis ; 9(7): 762, 2018 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-29988038

RESUMO

Hepatitis B virus X (HBx) protein contributes to the progression of hepatitis B virus (HBV)-related hepatic injury and diseases, but the exact mechanism remains unclear. Protein phosphatase 2 A (PP2A) is a major serine/threonine phosphatase involved in regulating many cellular phosphorylation signals that are important for regulation of cell cycle and apoptosis. Does HBx target to PP2A-B56γ and therefore affect HBx-induced hepatotoxicity? In the present study, the expression of B56γ positively correlated with the level of HBx in HBV-infected primary human hepatocytes in human-liver-chimeric mice, HBx-transgenic mice, HBV-infected cells, and HBx-expressing hepatic cells. B56γ promoted p53/p21-dependent cell cycle arrest and apoptosis. Mechanistically, B56γ was transactivated by AP-1, which was under the regulation of endoplasmic reticulum (ER) stress induced CREBH signaling in HBx-expressing hepatic cells. B56γ dephosphorylated p-Thr55-p53 to trigger p53/p21 pathway-dependent cell cycle G1 phase arrest, resulting in apoptosis of hepatic cells. In conclusion, this study provides a novel insight into a mechanism of B56γ mediating cell cycle arrest and apoptosis of HBx-expressing hepatic cells and a basis for B56γ being a potential therapeutic target for HBV-infected hepatic cells.


Assuntos
Proteína Fosfatase 2/metabolismo , Transativadores/metabolismo , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Doxiciclina/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Fase G1/fisiologia , Células Hep G2 , Hepatite B/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo , Proteínas Virais Reguladoras e Acessórias
15.
Front Microbiol ; 9: 908, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29867819

RESUMO

Humanized mouse model generated by grafting primary human hepatocytes (PHHs) to immunodeficient mouse has contributed invaluably to understanding the pathogenesis of hepatitis B virus (HBV). However, the source of PHHs is limited, which necessitates the search for alternatives. Recently, hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells (hiPSCs) have been used for in vitro HBV infection. Herein, we developed a robust human liver chimeric animal model to study in vivo HBV infection by engrafting the hiPSC-HLCs to Fah-/-Rag2-/-IL-2Rγc-/-SCID (FRGS) mice. After being optimized by a small molecule, XMU-MP-1, the hiPSC-HLCs engrafted FRGS (hHLC-FRGS) mice displayed approximately 40% liver chimerism at week 6 after engraftment and maintained at this level for at least 14 weeks. Viremia and HBV infection markers include antigens, RNA, DNA, and covalently closed circular DNA were detectable in HBV infected hHLC-FRGS mice. Furthermore, hiPSC-HLCs and hHLC-FRGS mice were successfully used to evaluate different antivirals. Therefore, we established a humanized mouse model for not only investigating HBV pathogenesis but also testing the effects of the anti-HBV drugs. Highlights:    (1) The implanted hiPSC-HLCs established a long-term chimerism in FRGS mice liver.    (2) hHLC-FRGS mice are adequate to support chronic HBV infection with a full viral life cycle.    (3) hiPSC-HLCs and hHLC-FRGS mice are useful tools for evaluation of antivirals against HBV infection in vitro and in vivo. Research in Context  To overcome the disadvantages of using primary human hepatocytes, we induced human pluripotent stem cells to hepatocyte-like cells (hiPSC-HLCs) that developed the capability to express important liver functional markers and critical host factors for HBV infection. The hiPSC-HLCs were permissive for the HBV infection and supported a full HBV replication. The hiPSC-HLCs were then engrafted to immunodeficient mouse to establish a chimeric liver mouse model, which was capable of supporting HBV infection in vivo and evaluating the effects of antiviral drugs. Our results shed light into improving the cellular and animal models for studying HBV and other hepatotropic viruses.

16.
Sci Rep ; 7: 40199, 2017 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-28079152

RESUMO

Hepatitis D virus (HDV) is a defective RNA virus that requires the presence of hepatitis B virus (HBV) for its life cycle. The in vitro HDV infection system is widely used as a surrogate model to study cellular infection with both viruses owing to its practical feasibility. However, previous methods for running this system were less efficient for high-throughput screening and large-scale studies. Here, we developed a novel method for the production of infectious HDV by adenoviral vector (AdV)-mediated transduction. We demonstrated that the AdV-based method yields 10-fold higher viral titers than the transient-transfection approach. The HDV-containing supernatant derived from AdV-infected Huh7 cells can be used as the inoculum in infectivity assays without requiring further concentration prior to use. Furthermore, we devloped a chemiluminescent immunoassay (HDV-CLEIA) to quantitatively determine intracellular HDAg with a dynamic range of 5-11,000 pg/mL. HDV-CLEIA can be used as an alternative approach to assess HDV infection. The advantages of our updated methodology were demonstrated through in vitro HDV infection of HepaRG cells and by evaluating the neutralization activity using antibodies that target various regions of the HBV/HDV envelope proteins. Together, the methods presented here comprise a novel toolbox of in vitro assays for studying HDV infection.


Assuntos
Técnicas Citológicas/métodos , Hepatite D/patologia , Vírus Delta da Hepatite/crescimento & desenvolvimento , Modelos Biológicos , Adenoviridae/genética , Linhagem Celular , Vetores Genéticos , Hepatócitos/virologia , Humanos , Transdução Genética
17.
Sci Transl Med ; 8(352): 352ra108, 2016 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-27535619

RESUMO

Tissue repair and regenerative medicine address the important medical needs to replace damaged tissue with functional tissue. Most regenerative medicine strategies have focused on delivering biomaterials and cells, yet there is the untapped potential for drug-induced regeneration with good specificity and safety profiles. The Hippo pathway is a key regulator of organ size and regeneration by inhibiting cell proliferation and promoting apoptosis. Kinases MST1 and MST2 (MST1/2), the mammalian Hippo orthologs, are central components of this pathway and are, therefore, strong target candidates for pharmacologically induced tissue regeneration. We report the discovery of a reversible and selective MST1/2 inhibitor, 4-((5,10-dimethyl-6-oxo-6,10-dihydro-5H-pyrimido[5,4-b]thieno[3,2-e][1,4]diazepin-2-yl)amino)benzenesulfonamide (XMU-MP-1), using an enzyme-linked immunosorbent assay-based high-throughput biochemical assay. The cocrystal structure and the structure-activity relationship confirmed that XMU-MP-1 is on-target to MST1/2. XMU-MP-1 blocked MST1/2 kinase activities, thereby activating the downstream effector Yes-associated protein and promoting cell growth. XMU-MP-1 displayed excellent in vivo pharmacokinetics and was able to augment mouse intestinal repair, as well as liver repair and regeneration, in both acute and chronic liver injury mouse models at a dose of 1 to 3 mg/kg via intraperitoneal injection. XMU-MP-1 treatment exhibited substantially greater repopulation rate of human hepatocytes in the Fah-deficient mouse model than in the vehicle-treated control, indicating that XMU-MP-1 treatment might facilitate human liver regeneration. Thus, the pharmacological modulation of MST1/2 kinase activities provides a novel approach to potentiate tissue repair and regeneration, with XMU-MP-1 as the first lead for the development of targeted regenerative therapeutics.


Assuntos
Fator de Crescimento de Hepatócito/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Regeneração/efeitos dos fármacos , Sulfonamidas/farmacologia , Acetaminofen/toxicidade , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Colite/induzido quimicamente , Colite/prevenção & controle , Cristalização , Fator de Crescimento de Hepatócito/química , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/transplante , Ensaios de Triagem em Larga Escala , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lesão Pulmonar/tratamento farmacológico , Camundongos , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas/química , Medicina Regenerativa , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/química
18.
Exp Anim ; 65(4): 373-382, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27264142

RESUMO

Hepatitis B virus (HBV) is the leading cause of liver disease and hepatic carcinoma (HCC). Approximately 350 million people worldwide are infected with HBV and at risk of chronicity. An efficient HBV-tolerant murine model that mimics HBV infection in humans is desirable for HBV-related research. In this study, we investigated and established a murine model by hydrodynamic injection (HDI) of pAAV/HBV into the tail vein of AAVS1 site element-transgenic mice. In 80% of the injected mice, the serum level of HBsAg reached 103-4 IU/ml and persisted for more than half a year. Next, the model was used to evaluate RNA interference (RNAi)-based antiviral therapy. Data obtained using the model demonstrated that this model will facilitate the elucidation of the mechanisms underlying chronic HBV infection and will also be useful for evaluating new antiviral drugs.


Assuntos
Vírus da Hepatite B/fisiologia , Hepatite B Crônica/etiologia , Animais , Antivirais/farmacologia , Modelos Animais de Doenças , Feminino , Antígenos de Superfície da Hepatite B/sangue , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/genética , Humanos , Hidrodinâmica , Injeções , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Interferência de RNA
19.
Exp Anim ; 65(2): 117-23, 2016 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-26597317

RESUMO

On the basis of its close phylogenetic relationship with primates, the development of Tupaia belangeri as an infection animal model and drug metabolism model could provide a new option for preclinical studies, especially in hepatitis virus research. As a replacement for primary human hepatocytes (PHHs), primary tupaia hepatocytes (PTHs) have been widely used. Similar to human serum albumin, tupaia serum albumin (TSA) is the most common liver synthesis protein and is an important biomarker for PTHs and liver function. However, no detection or quantitative method for TSA has been reported. In this study, mouse monoclonal antibodies (mAbs) 4G5 and 9H3 against TSA were developed to recognize PTHs, and they did not show cross-reactivity with serum albumin from common experimental animals, such as the mouse, rat, cow, rabbit, goat, monkey, and chicken. The two mAbs also exhibited good performance in fluorescence activated cell sorting (FACS) analysis and immunofluorescence (IF) detection of PTHs. A chemiluminescent enzyme immune assay method using the two mAbs, with a linear range from 96.89 pg/ml to 49,609.38 pg/ml, was developed for the quantitative detection of TSA. The mAbs and the CLEIA method provide useful tools for research on TSA and PTHs.


Assuntos
Anticorpos Monoclonais , Hepatócitos , Técnicas Imunoenzimáticas/métodos , Medições Luminescentes/métodos , Albumina Sérica/análise , Albumina Sérica/imunologia , Tupaia , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Camundongos , Modelos Animais
20.
Gut ; 65(4): 658-71, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26423112

RESUMO

OBJECTIVE: This study aimed to investigate the therapeutic potential of monoclonal antibody (mAb) against HBV as a novel treatment approach to chronic hepatitis B (CHB) in mouse models. METHODS: Therapeutic effects of mAbs against various epitopes on viral surface protein were evaluated in mice mimicking persistent HBV infection. The immunological mechanisms of mAb-mediated viral clearance were systematically investigated. RESULTS: Among 11 tested mAbs, a novel mAb E6F6 exhibited the most striking therapeutic effects in several HBV-persistent mice. Single-dose administration of E6F6 could profoundly suppress the levels of hepatitis B surface antigen (HBsAg) and HBV DNA for several weeks in HBV-transgenic mice. E6F6 regimen efficiently prevented initial HBV infection, and reduced viral dissemination from infected hepatocytes in human-liver-chimeric mice. E6F6-based immunotherapy facilitated the restoration of anti-HBV T-cell response in hydrodynamic injection (HDI)-based HBV carrier mice. Immunological analyses suggested that the Fcγ receptor-dependent phagocytosis plays a predominant role in E6F6-mediated viral suppression. Molecular analyses suggested that E6F6 recognises an evolutionarily conserved epitope (GPCK(R)TCT) and only forms a smaller antibody-viral particle immune complex with limited interparticle crosslinking when it binds to viral particles. This unique binding characteristic of E6F6 to HBV was possibly associated with its effective in vivo opsonophagocytosis for viral clearance. CONCLUSIONS: These results provided new insight into understanding the therapeutic role and mechanism of antibody against persistent viral infection. The E6F6-like mAbs may provide a novel immunotherapeutic agent against human chronic HBV infection.


Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Superfície da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Imunoterapia/métodos , Animais , DNA Viral/efeitos dos fármacos , Modelos Animais de Doenças , Epitopos , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Hepatócitos/virologia , Camundongos , Camundongos Transgênicos , Fagocitose , Replicação Viral/efeitos dos fármacos
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