RESUMO
The mammalian liver possesses a remarkable regenerative ability. Two modes of damage response have been described: (1) The "oval cell" response emanates from the biliary tree when all hepatocytes are affected by chronic liver disease. (2) A massive, proliferative response of mature hepatocytes occurs upon acute liver damage such as partial hepatectomy (PHx). While the oval cell response has been captured in vitro by growing organoids from cholangiocytes, the hepatocyte proliferative response has not been recapitulated in culture. Here, we describe the establishment of a long-term 3D organoid culture system for mouse and human primary hepatocytes. Organoids can be established from single hepatocytes and grown for multiple months, while retaining key morphological, functional and gene expression features. Transcriptional profiles of the organoids resemble those of proliferating hepatocytes after PHx. Human hepatocyte organoids proliferate extensively after engraftment into mice and thus recapitulate the proliferative damage-response of hepatocytes.
Assuntos
Proliferação de Células , Hepatócitos/metabolismo , Organoides/metabolismo , Animais , Técnicas de Cultura de Células , Células Cultivadas , Hepatócitos/citologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Organoides/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de TempoRESUMO
OBJECTIVES: To elucidate the mechanism of tigecycline resistance in Escherichia coli that is mediated by the tet(A) variant gene. METHODS: E. coli strain 573 carried a plasmid-borne tet(A) variant gene, tentatively designated tet(A)TIG, that conferred decreased tigecycline susceptibility (MIC 0.5â mg/L). When exposed to increasing concentrations of tigecycline (0.25-8â mg/L), mutants growing at 2, 4 and 8â mg/L were obtained and sequenced. Copies of plasmid and tet(A)TIG relative to the chromosomal DNA in the mutants were determined by WGS and quantitative PCR (qPCR). Expression of tet(A)TIG in the mutants was evaluated by RT-qPCR. The tet(A)TIG-carrying plasmids were visualized by S1-PFGE and Southern blot hybridization. PCR served for the detection of a tet(A)TIG-carrying unconventional circularizable structure (UCS). RESULTS: Tigecycline resistance with maximum MICs of 16â mg/L was seen in E. coli mutants selected in the presence of tigecycline. Compared with the parental strain, the relative copy number and transcription level of tet(A)TIG in the mutants increased significantly in the presence of 2, 4 and 8â mg/L tigecycline, respectively. With increasing tigecycline selection pressure, the tet(A)TIG-carrying plasmids in the mutants increased in size, correlating with the number of tandem amplificates of a ΔTnAs1-flanked UCS harbouring tet(A)TIG. These tandem amplificates were not stable in the absence of tigecycline. CONCLUSIONS: Tigecycline resistance is due to the tandem amplification of a ΔTnAs1-flanked tet(A)TIG-carrying plasmid-borne segment in E. coli. The gain/loss of the tandem amplificates in the presence/absence of tigecycline represents an economic way for the bacteria to survive in the presence of tigecycline.
Assuntos
Antibacterianos , Escherichia coli , Testes de Sensibilidade Microbiana , Plasmídeos , Tigeciclina , Tigeciclina/farmacologia , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Plasmídeos/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Minociclina/farmacologia , Minociclina/análogos & derivados , Amplificação de Genes , Farmacorresistência Bacteriana/genética , Sequenciamento Completo do Genoma , AntiportersRESUMO
The Ni-catalyzed enantioselective addition reaction of aryl halides to aldehydes was studied with cyanobis(oxazoline) as chiral ligands and Mn as reductant. Aryl and heteroaryl bromides reacted with phenyl aldehyde at room temperature to produce dibenzyl alcohols in 16-99 % yields with 53-92 % ees. Moreover, the coupling of phenyl chloride with a variety of aryl, heteroaryl and alkyl aldehydes was demonstrated in the presence of cyanobis(oxazoline)/Ni(II) at 60 °C in generally high yields with moderate enantioselectivities.
RESUMO
Hemophilia A gene therapy targets hepatocytes to express B domain deleted (BDD) clotting factor VIII (FVIII) to permit viral encapsidation. Since BDD is prone to misfolding in the endoplasmic reticulum (ER) and ER protein misfolding in hepatocytes followed by high-fat diet (HFD) can cause hepatocellular carcinoma (HCC), we studied how FVIII misfolding impacts HCC development using hepatocyte DNA delivery to express three proteins from the same parental vector: (1) well-folded cytosolic dihydrofolate reductase (DHFR); (2) BDD-FVIII, which is prone to misfolding in the ER; and (3) N6-FVIII, which folds more efficiently than BDD-FVIII. One week after DNA delivery, when FVIII expression was undetectable, mice were fed HFD for 65 weeks. Remarkably, all mice that received BDD-FVIII vector developed liver tumors, whereas only 58% of mice that received N6 and no mice that received DHFR vector developed liver tumors, suggesting that the degree of protein misfolding in the ER increases predisposition to HCC in the context of an HFD and in the absence of viral transduction. Our findings raise concerns of ectopic BDD-FVIII expression in hepatocytes in the clinic, which poses risks independent of viral vector integration. Limited expression per hepatocyte and/or use of proteins that avoid misfolding may enhance safety.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Camundongos , Animais , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Hepatócitos , DNA , Fatores de Coagulação SanguíneaRESUMO
Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.
Assuntos
Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatopatias/genética , Alcaloides de Pirrolizidina/farmacologia , Animais , Transplante de Células , Quimera , Modelos Animais de Doenças , Feminino , Terapia Genética , Hepatite B , Vírus da Hepatite B , Hepatócitos/transplante , Proteínas de Homeodomínio/genética , Humanos , Hidrolases/genética , Subunidade gama Comum de Receptores de Interleucina/genética , Fígado/patologia , Hepatopatias/patologia , Malária , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Plasmodium falciparumRESUMO
Myocardial infarction and coagulation disorders are leading causes of disability and death in the world. An important role of the lectin complement pathway in myocardial infarction and coagulation has been demonstrated in mice genetically deficient in lectin complement pathway proteins. However, these studies are limited to comparisons between wild-type and deficient mice and lack the ability to examine reversal/inhibition of injury after disease establishment. We developed a novel mouse that expresses functional human mannose-binding lectin (MBL) 2 under the control of Mbl1 promoter. Serum MBL2 concentrations averaged approximately 3 µg/mL in MBL2(+/+)Mbl1(-/-)Mbl2(-/-) [MBL2 knock in (KI)] mice. Serum MBL2 level in MBL2 KI mice significantly increased after 7 (8 µg/mL) or 14 (9 µg/mL) days of hyperglycemia compared to normoglycemic mice (P < 0.001). Monoclonal antibody 3F8 inhibited C3 deposition on mannan-coated plates in MBL2 KI, but not wild-type, mice. Myocardial ischemia/reperfusion in MBL2 KI mice revealed that 3F8 preserved cardiac function and decreased infarct size and fibrin deposition in a time-dependent manner. Furthermore, 3F8 prevented ferric chloride-induced occlusive arterial thrombogenesis in vivo. MBL2 KI mice represent a novel animal model that can be used to study the lectin complement pathway in acute and chronic models of human disease. Furthermore, these novel mice demonstrate the therapeutic window for MBL2 inhibition for effective treatment of disease and its complications.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Neutralizantes/farmacologia , Modelos Animais de Doenças , Lectina de Ligação a Manose/antagonistas & inibidores , Infarto do Miocárdio/tratamento farmacológico , Trombose/tratamento farmacológico , Animais , Técnicas de Introdução de Genes , Humanos , Lectina de Ligação a Manose/sangue , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/metabolismo , Camundongos , Camundongos Knockout , Infarto do Miocárdio/sangue , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Regiões Promotoras Genéticas , Trombose/sangue , Trombose/genética , Trombose/patologiaRESUMO
Bleeding disorders and thrombotic complications constitute a major cause of death and disability worldwide. Although it is known that the complement and coagulation systems interact, no studies have investigated the specific role or mechanisms of lectin-mediated coagulation in vivo. FeCl(3) treatment resulted in intra-arterial occlusive thrombogenesis within 10 min in wild-type (WT) and C2/factor B-null mice. In contrast, mannose-binding lectin (MBL)-null and MBL-associated serine protease (MASP)-1/-3 knockout (KO) mice had significantly decreased FeCl(3)-induced thrombogenesis. Reconstitution with recombinant human (rh) MBL restored FeCl(3)-induced thrombogenesis in MBL-null mice to levels comparable to WT mice, suggesting a significant role of the MBL/MASP complex for in vivo coagulation. Additionally, whole blood aggregation demonstrated increased MBL/MASP complex-dependent platelet aggregation. In vitro, MBL/MASP complexes were captured on mannan-coated plates, and cleavage of a chromogenic thrombin substrate (S2238) was measured. We observed no significant differences in S2238 cleavage between WT, C2/factor B-null, MBL-A(-/-), or MBL-C(-/-) sera; however, MBL-null or MASP-1/-3 KO mouse sera demonstrated significantly decreased S2238 cleavage. rhMBL alone failed to cleave S2238, but cleavage was restored when rMASP-1 was added to either MASP-1/-3 KO sera or rhMBL. Taken together, these findings indicate that MBL/MASP complexes, and specifically MASP-1, play a key role in thrombus formation in vitro and in vivo.
Assuntos
Coagulação Sanguínea , Trombose das Artérias Carótidas/enzimologia , Lectina de Ligação a Manose da Via do Complemento , Serina Proteases Associadas a Proteína de Ligação a Manose/fisiologia , Animais , Coagulação Sanguínea/imunologia , Trombose das Artérias Carótidas/induzido quimicamente , Trombose das Artérias Carótidas/genética , Cloretos/toxicidade , Lectina de Ligação a Manose da Via do Complemento/genética , Modelos Animais de Doenças , Compostos Férricos/toxicidade , Humanos , Imunidade Inata/genética , Lectinas de Ligação a Manose/deficiência , Lectinas de Ligação a Manose/genética , Serina Proteases Associadas a Proteína de Ligação a Manose/efeitos adversos , Serina Proteases Associadas a Proteína de Ligação a Manose/deficiência , Serina Proteases Associadas a Proteína de Ligação a Manose/genética , Camundongos , Trombina/fisiologiaRESUMO
Capsid engineering of adeno-associated virus (AAV) can surmount current limitations to gene therapy such as broad tissue tropism, low transduction efficiency, or pre-existing neutralizing antibodies (NAb) that restrict patient eligibility. We previously generated an AAV3B combinatorial capsid library by integrating rational design and directed evolution with the aim of improving hepatotropism. A potential isolate, AAV3B-DE5, gained a selective proliferative advantage over five rounds of iterative selection in hepatocyte spheroid cultures. In this study, we reanalyzed our original dataset derived from the AAV3B combinatorial library and isolated variants from earlier (one to three) rounds of selection, with the assumption that variants with faster replication kinetics are not necessarily the most efficient transducers. We identified a potential candidate, AAV3B-V04, which demonstrated significantly enhanced transduction in mouse-passaged primary human hepatocytes as well as in humanized liver chimeric mice, compared to the parental AAV3B or the previously described isolate, AAV3B-DE5. Interestingly, the AAV3B-V04 capsid variant exhibited significantly reduced seroreactivity to pooled or individual human serum samples. Forty-four percent of serum samples with pre-existing NAbs to AAV3B had 5- to 20-fold lower reciprocal NAb titers to AAV3B-V04. AAV3B-V04 has only nine amino acid substitutions, clustered in variable region IV compared to AAV3B, indicating the importance of the loops at the top of the three-fold protrusions in determining both transduction efficiency and immunogenicity. This study highlights the effectiveness of rational design combined with targeted selection for enhanced AAV transduction via molecular evolution approaches. Our findings support the concept of limiting selection rounds to isolate the best transducing AAV3B variant without outgrowth of faster replicating candidates. We conclude that AAV3B-V04 provides advantages such as improved human hepatocyte tropism and immune evasion and propose its utility as a superior candidate for liver gene therapy.
Assuntos
Capsídeo , Evasão da Resposta Imune , Humanos , Animais , Camundongos , Capsídeo/metabolismo , Evasão da Resposta Imune/genética , Transdução Genética , Hepatócitos/metabolismo , Proteínas do Capsídeo/genética , Anticorpos Neutralizantes , Tropismo/genética , Dependovirus , Vetores Genéticos/genéticaRESUMO
OBJECTIVE: To investigate the prevalence of a tet(A) gene variant and its role in developing high-level tigecycline resistance among carbapenem-resistant Klebsiella pneumoniae (CRKP) clinical isolates. METHODS: The mechanism of high-level tigecycline resistance in CRKP mediated by a tet(A) variant was explored by induction experiments, antimicrobial susceptibility testing, whole-genome sequencing and bioinformatics analysis. The amplification and overexpression of the tet(A) variant were measured by the determination of sequencing depth, gene copy numbers, and qRT-PCR. RESULTS: A high rate (62.1%, 998/1607) of tet(A) variant carriage was observed among 1607 CRKP clinical isolates from Henan Province, China. High-level tigecycline resistance could rapidly develop by the amplification of the tet(A) variant in these isolates. The analysis of the raw sequencing data and the plasmid mapping depth revealed that the ΔtnpA homologous sequence of Tn1721 supports the amplification of the region that harbours the tet(A) variant by forming a large number of repeat arrays through translocatable units (TUs). Moreover, the epidemiological analysis of tet(A) variant-carrying structures among 1607 clinical CRKPs showed that the TU structure is widely present. CONCLUSION: The presence of a tigecycline resistance-mediating tet(A) variant in CRKP clinical isolates represents a greater health concern than initially thought and should be monitored consistently.
RESUMO
The aim of this study was to investigate the transferability of acquired linezolid resistance genes and associated mobile genetic elements in an Enterococcus faecalis isolate QZ076, cocarrying optrA, cfr, cfr(D), and poxtA2 genes. MICs were determined by broth microdilution. Whole-genome sequencing (WGS) was performed using the Illumina and Nanopore platforms. The transfer of linezolid resistance genes was investigated by conjugation, using E. faecalis JH2-2 and clinical methicillin-resistant Staphylococcus aureus (MRSA) 109 as recipients. E. faecalis QZ076 harbors four plasmids, designated pQZ076-1 to pQZ076-4, with optrA located in the chromosomal DNA. The gene cfr was located on a novel pseudocompound transposon, designated Tn7515, integrated into the 65,961-bp pCF10-like pheromone-responsive conjugative plasmid pQZ076-1. Tn7515 generated 8-bp direct target duplications (5'-GATACGTA-3'). The genes cfr(D) and poxtA2 were colocated on the 16,397-bp mobilizable broad-host-range Inc18 plasmid pQZ076-4. The cfr-carrying plasmid pQZ076-1 could transfer from E. faecalis QZ076 to E. faecalis JH2-2, along with the cfr(D)- and poxtA2-cocarrying plasmid pQZ076-4, conferring the corresponding resistant phenotype to the recipient. Moreover, pQZ076-4 could also transfer to MRSA 109. To the best of our knowledge, this study presented the first report of four acquired linezolid resistance genes [optrA, cfr, cfr(D), and poxtA2] being simultaneously present in the same E. faecalis isolate. The location of the cfr gene on a pseudocompound transposon in a pheromone-responsive conjugative plasmid will accelerate its rapid dissemination. In addition, the cfr-carrying pheromone-responsive conjugative plasmid in E. faecalis was also able to mobilize the interspecies transfer of the cfr(D)- and poxtA2-cocarrying plasmid between enterococci and staphylococci. IMPORTANCE In this study, the simultaneous occurrence of four acquired oxazolidinone resistance genes [optrA, cfr, cfr(D), and poxtA2] was identified in an E. faecalis isolate of chicken origin. The association of the cfr gene with a novel pseudocompound transposon Tn7515 integrated into a pCF10-like pheromone-responsive conjugative plasmid will accelerate its dissemination. Moreover, the location of the resistance genes cfr(D) and poxtA2 on a mobilizable broad-host-range Inc18 family plasmid represents the basis for their intra- and interspecies dissemination with the aid of a conjugative plasmid and further accelerates the spreading of acquired oxazolidinone resistance genes, such as cfr, cfr(D), and poxtA2, among Gram-positive pathogens.
Assuntos
Infecções por Bactérias Gram-Positivas , Staphylococcus aureus Resistente à Meticilina , Oxazolidinonas , Animais , Linezolida/farmacologia , Antibacterianos/farmacologia , Enterococcus faecalis/genética , Galinhas , Farmacorresistência Bacteriana/genética , Genes Bacterianos/genética , Plasmídeos/genética , Testes de Sensibilidade Microbiana , Cromossomos , Infecções por Bactérias Gram-Positivas/epidemiologiaRESUMO
Hepatitis B virus (HBV) chronically infects an estimated 300 million people, and standard treatments are rarely curative. Infection increases the risk of liver cirrhosis and hepatocellular carcinoma, and consequently, nearly 1 million people die each year from chronic hepatitis B. Tools and approaches that bring insights into HBV biology and facilitate the discovery and evaluation of antiviral drugs are in demand. Here, we describe a method to initiate the replication of HBV, a DNA virus, using synthetic RNA. This approach eliminates contaminating background signals from input virus or plasmid DNA that plagues existing systems and can be used to study multiple stages of HBV replication. We further demonstrate that this method can be uniquely applied to identify sequence variants that confer resistance to antiviral drugs.
Assuntos
Hepatite B Crônica , Neoplasias Hepáticas , Humanos , Vírus da Hepatite B/genética , Antivirais/farmacologia , Antivirais/uso terapêutico , RNA , Hepatite B Crônica/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/tratamento farmacológico , Replicação ViralRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) patient-derived xenograft (PDX) models hold potential to advance knowledge in HCC biology to help improve systemic therapies. Beside hepatitis B virus-associated tumors, HCC is poorly established in PDX. METHODS: PDX formation from fresh HCC biopsies were obtained and implanted intrahepatically or in subrenal capsule (SRC). Mouse liver injury was induced in immunodeficient Fah-/- mice through cycling off nitisinone after HCC biopsy implantation, versus continuous nitisinone as non-liver injury controls. Mice with macroscopically detectable PDX showed rising human alpha1-antitrypsin (hAAT) serum levels, and conversely, no PDX was observed in mice with undetectable hAAT. RESULTS: Using rising hAAT as a marker for PDX formation, 20 PDX were established out of 45 HCC biopsy specimens (44%) reflecting the four major HCC etiologies most commonly identified at Memorial SloanKettering similar to many other institutions in the United States. PDX was established only in severely immunodeficient mice lacking lymphocytes and NK cells. Implantation under the renal capsule improved PDX formation two-fold compared to intrahepatic implantation. Two out of 18 biopsies required murine liver injury to establish PDX, one associated with hepatitis C virus and one with alcoholic liver disease. PDX tumors were histologically comparable to biopsy specimens and 75% of PDX lines could be passaged. CONCLUSIONS: Using cycling off nitisinone-induced liver injury, HCC biopsies implanted under the renal capsule of severely immunodeficient mice formed PDX with 57% efficiency as determined by rising hAAT levels. These findings facilitate a more efficient make-up of PDX for research into subset-specific HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Biópsia , Carcinoma Hepatocelular/patologia , Modelos Animais de Doenças , Xenoenxertos , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Estados Unidos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Advanced non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging global health problem associated with pre-disposing genetic polymorphisms, most strikingly an isoleucine to methionine substitution in patatin-like phospholipase domain-containing protein 3 (PNPLA3-I148M). Here, we study how human hepatocytes with PNPLA3 148I and 148M variants engrafted in the livers of broadly immunodeficient chimeric mice respond to hypercaloric diets. As early as four weeks, mice developed dyslipidemia, impaired glucose tolerance, and steatosis with ballooning degeneration selectively in the human graft, followed by pericellular fibrosis after eight weeks of hypercaloric feeding. Hepatocytes with the PNPLA3-148M variant, either from a homozygous 148M donor or overexpressed in a 148I donor background, developed microvesicular and severe steatosis with frequent ballooning degeneration, resulting in more active steatohepatitis than 148I hepatocytes. We conclude that PNPLA3-148M in human hepatocytes exacerbates NAFLD. These models will facilitate mechanistic studies into human genetic variant contributions to advanced fatty liver diseases.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Aciltransferases , Animais , Hepatócitos/metabolismo , Humanos , Lipase/genética , Lipase/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Fosfolipases A2 Independentes de CálcioRESUMO
Adeno-associated virus (AAV) vector serotypes vary in their ability to transduce hepatocytes from different species. Chimeric mouse models harboring human hepatocytes have shown translational promise for liver-directed gene therapies. However, many variables that influence human hepatocyte transduction and transgene expression in such models remain poorly defined. Here, we aimed to test whether three experimental conditions influence AAV transgene expression in immunodeficient, fumaryl-acetoactetate-hydrolase-deficient (Fah -/-) chimeric mice repopulated with primary human hepatocytes. We examined the effects of the murine liver injury cycle, human donor variability, and vector doses on hepatocyte transduction with various AAV serotypes expressing a green fluorescent protein (GFP). We determined that the timing of AAV vector challenge in the liver injury cycle resulted in up to 7-fold differences in the percentage of GFP expressing human hepatocytes. The GFP+ hepatocyte frequency varied 7-fold between human donors without, however, changing the relative transduction efficiency between serotypes for an individual donor. There was also a clear relationship between AAV vector doses and human hepatocyte transduction and transgene expression. We conclude that several experimental variables substantially affect human hepatocyte transduction in the Fah -/- chimera model, attention to which may improve reproducibility between findings from different laboratories.
RESUMO
Limitations to successful gene therapy with adeno-associated virus (AAV) can comprise pre-existing neutralizing antibodies to the vector capsid that can block cellular entry, or inefficient transduction of target cells that can lead to sub-optimal expression of the therapeutic transgene. Recombinant serotype 3 AAV (AAV3) is an emerging candidate for liver-directed gene therapy. In this study, we integrated rational design by using a combinatorial library derived from AAV3B capsids with directed evolution by in vitro selection for liver-targeted AAV variants. The AAV3B-DE5 variant described herein was undetectable in the original viral library but gained a selective advantage upon in vitro passaging in human hepatocarcinoma spheroid cultures. AAV3B-DE5 contains 24 capsid amino acid substitutions compared with AAV3B, distributed among all five variable regions, with strong selective pressure on VR-IV, VR-V, and VR-VII. In vivo, AAV3B-DE5 demonstrated improved human hepatocyte tropism in a liver chimeric mouse model. Importantly, this variant exhibited reduced seroreactivity to human intravenous immunoglobulin (i.v. Ig), as well as individual serum samples from 100 healthy human donors. Therefore, molecular evolution using a combinatorial library platform generated a viral capsid with high hepatocyte tropism and enhanced evasion of pre-existing AAV neutralizing antibodies.
RESUMO
Although there is no effective cure for chronic hepatitis B virus (HBV) infection, antibodies are protective and correlate with recovery from infection. To examine the human antibody response to HBV, we screened 124 vaccinated and 20 infected, spontaneously recovered individuals. The selected individuals produced shared clones of broadly neutralizing antibodies (bNAbs) that targeted 3 non-overlapping epitopes on the HBV S antigen (HBsAg). Single bNAbs protected humanized mice against infection but selected for resistance mutations in mice with prior established infection. In contrast, infection was controlled by a combination of bNAbs targeting non-overlapping epitopes with complementary sensitivity to mutations that commonly emerge during human infection. The co-crystal structure of one of the bNAbs with an HBsAg peptide epitope revealed a stabilized hairpin loop. This structure, which contains residues frequently mutated in clinical immune escape variants, provides a molecular explanation for why immunotherapy for HBV infection may require combinations of complementary bNAbs.
Assuntos
Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Anti-Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Pré-Escolar , Modelos Animais de Doenças , Epitopos/imunologia , Feminino , Células HEK293 , Células Hep G2 , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/imunologia , Humanos , Lactente , Camundongos , Camundongos Knockout , Conformação ProteicaRESUMO
OBJECTIVE: To investigate the influence of modified Qianjin Huanglian Pill (QJHL), a Chinese herbal compound, on pancreas in mice with monosodium L-glutamate (MSG) induced insulin resistance (IR) and its molecular mechanism. METHODS: Controlled by rosiglitazone (Ros), the MSG indiced IR mice were treated with QJHL for 28 days. The laboratory indices were examined including fasting serum glucose (FSG), fasting serum insulin (FSI), insulin sensitivity index (ISI), and morphological changes of pancreas, and levels of insulin receptor (InsR), insulin receptor substrate (IRS1/2) and glucose transporter (GLUT2) mRNA expression in pancreas tissue were determined by the reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: As compared with the model group, the level of FSG was lower (P < 0.01) and ISI was higher (P < 0.05) after treatment in the QJHL treated group, with pancreatic islet hyperplasia and hypertrophy ameliorated significantly (P < 0.01). And these changes were similar to those in the Ros treated group (P > 0.05). Moreover, the level of GLUT2 mRNA expression in pancreas of the QJHL group increased significantly (P < 0.01), while it was unchanged in the Ros group. CONCLUSION: QJHL could reduce IR, ameliorate pathological changes of pancreas, which is possibly related with its action on increasing GLUT2 mRNA expression in the pancreas tissue.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Resistência à Insulina , Insulina/sangue , Pâncreas/patologia , Animais , Animais Recém-Nascidos , Glicemia/metabolismo , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Pâncreas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Glutamato de SódioRESUMO
OBJECTIVE: To investigate the effect of qianjin huanglian pill on kidney in monosodium L-glutamate (MSG)-treated insulin resistance (IR) mice. METHOD: The ameliorative effect of qianjin huanglian pill on IR in MSG mice was evaluated in comparison with rosiglitazone (Ros). The fasting serum glucose, fasting serum insulin, insulin sensitivity index, urinary albumin excretion, glomerular diameter and pathological changes of kidney were investigated in the evaluation. RESULT: After 2 weeks of qianjin huanglian pill treatment, the urinary albumin excretion (UAE) was reduced in low-dose group (P < 0.05) as compared with the model group. After 4 weeks of qianjin huanglian pill treatment, the fasting serum glucose was reduced in high-dose group (P < 0.001 compared with the model group). ISI of mice was ameliorated in high-dose group (P < 0.05 compared with the model group). The glomerular diameter was decreased, the hyperplasia of glomerulus was ameliorated in high-dose and low-dose groups (P < 0.01 compared with model group). CONCLUSION: In MSG mice, we found qianjin huanglian pill could increase insulin sensitivity, decrease the urinary albumin excretion, ameliorate the pathological changes of kidney due to insulin resistance.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Resistência à Insulina , Rim/efeitos dos fármacos , Animais , Glicemia/metabolismo , Química Farmacêutica , Relação Dose-Resposta a Droga , Feminino , Rim/patologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Masculino , Camundongos , Glutamato de Sódio/toxicidadeRESUMO
This paper reports on the effect of GCP-02, a dual activator of the peroxisome proliferator-activated receptors alpha/gamma (PPARalpha/gamma), on glucose and lipid metabolism in insulin-resistant obese mice induced by monosodium glutamate. The mice were divided into four groups on the basis of treatment: control group, rosiglitazone (positive control) (7 micromol/kg), and low- and high-dosage GCP-02 (7 micromol/kg and 3.5 micromol/kg, respectively). Drugs were given orally once a day for 19 days, and mice underwent testing for insulin tolerance, oral glucose tolerance and gluconeogenesis, and plasma cholesterol, triglyceride and free fatty acid levels. Mice were sacrificed, and body length and weight were measured; intraperitoneal adipose, heart and liver weighed; and plasma alanine aminotransferase (ALT) level and aspartate aminotransferase (AST) activity measured. Liver, soleus muscle and myocardium were assayed for glycogen, triglyceride and free fatty acid content and myocardia tested for superoxide dismutase (SOD) activity and malonaldehyde content. RT-PCR revealed expression of insulin receptor substrate 1 and 2 (IRS1, IRS2) and related genes in liver. GCP-02 had a more powerful effect than rosiglitazone on improving insulin sensitivity, ameliorating glucose tolerance, suppressing L-alanine-induced gluconeogenesis, and decreasing plasma levels of cholesterol, triglyceride and free fatty acid. It reduced body weight in control mice, significantly lowered hepatic content of glycogen, triglyceride and free fatty acid and myocardial content of triglyceride, and increased myocardial SOD activity. IRS2 mRNA was down-regulated in control mice but up-regulated by GCP-02. Thus, GCP-02 is a potential candidate for the prevention and therapy of diseases associated with insulin resistance such as type 2 diabetes mellitus and cardiovascular disease.
Assuntos
Sulfonatos de Arila/farmacologia , Hipoglicemiantes/farmacologia , PPAR alfa/agonistas , PPAR gama/agonistas , Fenilpropionatos/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Administração Oral , Animais , Sulfonatos de Arila/administração & dosagem , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Teste de Tolerância a Glucose , Hipoglicemiantes/administração & dosagem , Proteínas Substratos do Receptor de Insulina , Resistência à Insulina , Peptídeos e Proteínas de Sinalização Intracelular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lipídeos/sangue , Camundongos , Camundongos Endogâmicos ICR , Fenilpropionatos/administração & dosagem , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/genética , RNA Mensageiro/metabolismo , Rosiglitazona , Tiazolidinedionas/farmacologiaRESUMO
The rapid increase of obese population in the United States has made obesity into epidemic proportion. Obesity is a strong risk factor for metabolic syndrome, type 2 diabetes mellitus, cardiovascular diseases, cancer and other diseases. Compelling evidence has demonstrated that increased adipose tissue mass is not only the consequence of obesity, but also plays a central role in the development of obesity-associated diseases. Recent studies have profoundly changed the concept of adipose tissue from being an energy depot to an active endocrine organ. The development of obesity alters adipocyte-derived hormones or cytokines expression, which provide a link between obesity and impaired insulin sensitivity and metabolic defects in other tissues. This review summarizes the current knowledge on how major adipose-derived hormones or adipocytokines influence insulin sensitivity.