RESUMO
OBJECTIVES: Chronic hepatitis B (CHB) caused by HBV infection greatly increases the risk of liver cirrhosis and hepatocellular carcinoma. Hepatitis B surface antigen (HBsAg) plays critical roles in the pathogenesis of CHB. HBsAg loss is the key indicator for cure of CHB, but is rarely achieved by current approved anti-HBV drugs. Therefore, novel anti-HBV strategies are urgently needed to achieve sustained HBsAg loss. DESIGN: We developed multiple chimeric antigen receptors (CARs) based on single-chain variable fragments (scFvs, namely MA18/7-scFv and G12-scFv), respectively, targeting HBV large and small envelope proteins. Their impacts on HBsAg secretion and HBV infection, and the underlying mechanisms, were extensively investigated using various cell culture models and HBV mouse models. RESULTS: After secretory signal peptide mediated translocation into endoplasmic reticulum (ER) and secretory pathway, MA18/7-scFv and CARs blocked HBV infection and virion secretion. G12-scFv preferentially inhibited virion secretion, while both its CAR formats and crystallisable fragment (Fc)-attached versions blocked HBsAg secretion. G12-scFv and G12-CAR arrested HBV envelope proteins mainly in ER and potently inhibited HBV budding. Furthermore, G12-scFv-Fc and G12-CAR-Fc strongly suppressed serum HBsAg up to 130-fold in HBV mouse models. The inhibitory effect lasted for at least 8 weeks when delivered by an adeno-associated virus vector. CONCLUSION: CARs possess direct antiviral activity, besides the well-known application in T-cell therapy. Fc attached G12-scFv and G12-CARs could provide a novel approach for reducing circulating HBsAg.
Assuntos
Hepatite B Crônica , Hepatite B , Neoplasias Hepáticas , Receptores de Antígenos Quiméricos , Camundongos , Animais , Antígenos de Superfície da Hepatite B , Vírus da Hepatite B/genética , Retículo Endoplasmático/metabolismoRESUMO
BACKGROUND & AIMS: Non-cytolytic cure of HBV-infected hepatocytes by cytokines, including type I interferons (IFNs), is of importance for resolving acute and chronic infection. However, as IFNs stimulate hundreds of genes, those most relevant for HBV suppression remain largely unknown. Amongst them are the large myxovirus resistance (Mx) GTPases. Human MX1 (or MxA) is active against many RNA viruses, while MX2 (or MxB) was recently found to restrict HIV-1, HCV, and herpesviruses. Herein, we investigated the anti-HBV activity of MX2. METHODS: The potential anti-HBV activity of MX2 and functional variants were assessed in transfected and HBV-infected hepatoma cells and primary human hepatocytes, employing multiple assays to analyze the synthesis and decay of HBV nucleic acids. The specific roles of MX2 in IFN-α-driven inhibition of HBV transcription and replication were assessed by MX2-specific shRNA interference (RNAi). RESULTS: Both MX2 alone and IFN-α substantially inhibited HBV replication, due to significant deceleration of the synthesis and slight acceleration of the turnover of viral RNA. RNAi knockdown of MX2 significantly reduced the inhibitory effects of IFN-α. Strikingly, MX2 inhibited HBV infection by reducing covalently closed circular DNA (cccDNA), most likely by indirectly impairing the conversion of relaxed circular DNA to cccDNA rather than by destabilizing existing cccDNA. Various mutations affecting the GTPase activity and oligomerization status reduced MX2's anti-HBV activity. CONCLUSION: MX2 is an important IFN-α inducible effector that decreases HBV RNA levels but can also potently inhibit HBV infection by indirectly impairing cccDNA formation. MX2 likely has the potential for therapeutic applications aimed at curing HBV infection by eliminating cccDNA. LAY SUMMARY: This study shows that the protein MX2, which is induced by interferon-α, has important anti-hepatitis B virus (HBV) effector functions. MX2 can reduce the amount of covalently closed circular DNA, which is the form of DNA that HBV uses to maintain viral persistence within hepatocytes. MX2 also reduces HBV RNA levels by downregulating synthesis of viral RNA. MX2 likely represents a novel intrinsic HBV inhibitor that could have therapeutic potential, as well as being useful for improving our understanding of the complex biology of HBV and the antiviral mechanisms of interferon-α.
Assuntos
Antivirais/farmacologia , Vírus da Hepatite B/fisiologia , Hepatite B/metabolismo , Interferon-alfa/farmacologia , Proteínas de Resistência a Myxovirus/deficiência , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética , DNA Circular/metabolismo , DNA Viral/metabolismo , Técnicas de Silenciamento de Genes , Células Hep G2 , Hepatite B/imunologia , Hepatite B/virologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Proteínas de Resistência a Myxovirus/genética , Interferência de RNA , RNA Viral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , TransfecçãoRESUMO
Antibody/antigen binding results in immune complexes (IC) that have a variety of regulatory functions. One important feature is the enhanced host immune activation against antigen contained in the complex. ICs play important roles at several critical steps that lead to B and T cell activation, including antigen targeting/retention, facilitated antigen uptake, antigen presenting cell activation and proper balancing of positive and negative stimulatory signals. In both poultry industry and clinical health care, ICs have been used as preventive and therapeutic vaccines. With our deepening understanding of antibody biology, particularly in light of new revelations of regulatory functions of Fc receptors, mechanistically more precise engineering has spearheaded tailored use of this tool for infection control and cancer therapy. IC-based treatment and prophylaxis have been tested to different extents in HBV, HIV and influenza viral infection control and are actively examined as an alternative treatment for several forms of tumor. As a part of this book series, this chapter aims to discuss the mechanistic aspects of IC signaling and their impact on immune cells. We give samples how this old technology has been used by practitioners over the last several decades and suggest potential paths for future development of IC-based immune therapy.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Vacinação , Vacinas/imunologia , Vacinas/uso terapêutico , Células Apresentadoras de Antígenos/imunologia , Antígenos/imunologia , Humanos , Ativação LinfocitáriaRESUMO
Inflammatory cells in atherosclerotic plaque exclusively originate from hematopoietic stem/progenitor cells (HSPCs). In this study, we investigated whether circulating HSPCs frequency related to coronary stenosis in patients with coronary heart disease (CHD). Coronary angiography was performed in 468 participants who were recruited at Cardiology Centre in LuHe Hospital from March 2016 to May 2017. Among these subjects, 344 underwent echocardiography. Mononuclear cells isolated from peripheral blood were stained with an antibody cocktail containing anti-human CD34, anti-human lineage, anti-human CD38, and anti-human CD45RA. Lineage-CD38-CD45RAdimCD34+HSPCs were quantified by flow cytometry. CHD was defined as coronary stenosis ≥50% and the extent of CHD was further categorised by coronary stenosis ≥70%. A p < 0.0031 was regarded statistically significant by the Bonferroni correction. Circulating HSPCs frequency was 1.8-fold higher in CHD patients than non-CHD participants (p = 0.047). Multivariate-adjusted logistic analysis demonstrated that HSPCs was the only marker that was associated with the odds ratio of having mild vs. severe coronary stenosis (2.08 (95% CI, 1.35-3.21), p = 0.0009). Left ventricular ejection fraction was inversely correlated with HSPCs frequency and CRP in CHD patients (p < 0.05 for both). In conclusion, HSPCs frequency in circulation is intimately related to coronary stenoses in CHD patients.
Assuntos
Estenose Coronária/diagnóstico por imagem , Células-Tronco Hematopoéticas/citologia , Monócitos/citologia , ADP-Ribosil Ciclase 1/metabolismo , Idoso , Antígenos CD34/metabolismo , Angiografia Coronária , Estenose Coronária/imunologia , Feminino , Citometria de Fluxo , Células-Tronco Hematopoéticas/imunologia , Humanos , Antígenos Comuns de Leucócito/metabolismo , Modelos Logísticos , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Monócitos/imunologiaRESUMO
In recent years, therapeutic monoclonal antibodies have made impressive progress, providing great benefit by successfully treating malignant and chronic inflammatory diseases. Monoclonal antibodies with broadly neutralizing effects against specific antigens, or that target specific immune regulators, manifest therapeutic effects via their Fab fragment specificities. Subsequently therapeutic efficacy is mediated mostly by interactions of the Fc fragments of the antibodies with their receptors (FcR) displayed on cells of the immune system. These interactions can trigger a series of immunoregulatory responses, involving both innate and adaptive immune systems and including cross-presentation of antigens, activation of CD8 + T cells and CD4 + T cells, phagocytosis, complement-mediated antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The nature of the triggered effector functions of the antibodies is markedly affected by the glycosylation patterns of the Fc fragments. These can cause differences in the conformation of the heavy chains of antibodies, with resultant changes in antibody binding affinity and activation of the complement system. Studies of the Fc glycosylation profiles together with the associated Fc effector functions and FcR/CR interactions promoted interest and progress in engineering therapeutic antibodies. Furthermore, because antigen-antibody immune complexes (ICs) have shown similar actions, in addition to certain novel immunoregulatory mechanisms that also reshape immune responses, the properties of ICs are being explored in new approaches for prevention and therapy of diseases. In this review, both basic studies and experimental/clinical applications of ICs leading to the development of preventive and therapeutic vaccines are presented.
RESUMO
Hepatitis B virus (HBV) envelopes as well as empty subviral particles carry in their lipid membranes the small (S), middle (M), and large (L) surface proteins, collectively known as hepatitis B surface antigen (HBsAg). Due to their common S domain all three proteins share a surface-exposed hydrophilic antigenic loop (AGL) with a complex disulfide bridge-dependent structure. The AGL is critical for HBV infectivity and virion secretion, and thus represents a major target for neutralizing antibodies. Previously, a human monoclonal antibody (mAb) targeting a conformational epitope in the AGL, IgG12, exhibited 1000-fold higher neutralizing activity than hepatitis B immune globulin (HBIG). Here we designed a single-chain variable fragment (scFv) homolog of IgG12, G12-scFv, which could be efficiently produced in soluble form in the cytoplasm of E. coli SHuffle cells. Independent in vitro assays verified specific binding of G12-scFv to a conformational S epitope shared with IgG12. Despite 20-fold lower affinity, G12-scFv but not an irrelevant scFv potently neutralized HBV infection of susceptible hepatoma cells (IC50â¯=â¯1.8â¯nM). Strikingly, low concentrations of G12-scFv blocked virion secretion from HBV producing cells (IC50â¯=â¯1.25â¯nM) without disturbing intracellular viral replication, whereas extracellular HBsAg was reduced only at >100-fold higher though still nontoxic concentration. The inhibitory effects correlated with S binding specificity and presumably also G12-scFv internalization into cells. Together these data suggest G12-scFv as a highly specific yet easily accessible novel tool for basic, diagnostic, and possibly future therapeutic applications.
Assuntos
Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/efeitos dos fármacos , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologia , Vírion/efeitos dos fármacos , Anticorpos Neutralizantes , Escherichia coli , Células Hep G2 , Vírus da Hepatite B/fisiologia , Humanos , Concentração Inibidora 50 , Anticorpos de Cadeia Única/biossíntese , Vírion/fisiologia , Replicação Viral/efeitos dos fármacosAssuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Antivirais/uso terapêutico , Antivirais/uso terapêutico , Hepatite B Crônica/tratamento farmacológico , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Produtos do Gene pol/genética , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos de Superfície da Hepatite B/sangue , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/genética , Humanos , Interferon-alfa/uso terapêutico , Polietilenoglicóis/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Transativadores/genética , Carga Viral , Proteínas Virais Reguladoras e AcessóriasRESUMO
An HBsAg-HBIG therapeutic vaccine (Yeast-derived Immune Complexes, YIC) for chronic hepatitis B (CHB) patients has undergone a series of clinical trials. The HBeAg sero-conversion rate of YIC varied from 21.9% to 14% depending on the immunization protocols from 6 to 12 injections. To analyze the immunological mechanisms exerted by 6 injections of YIC, 44 CHB patients were separately immunized with YIC, alum as adjuvant control or normal saline as blank control, with add on of antiviral drug Adefovir in all groups. Kinetic increase in Th1 and Th2 cells CD4+ T cell sub-populations with association in decrease in Treg cells and increase of Tc1 and Tc17 cells in CD8+ T cells were observed in YIC immunized group. No such changes were found in the other groups. By multifunctional analysis of cytokine profiles, significant increase of IL-2 levels was observed, both in CD4+ and CD8+ T cells in the YIC immunized group, accompanied by increase in IFN-gamma and decrease of inhibitory factors (IL-10, TGF-ß and Foxp3) in CD4+ T cells. In the alum immunized group, slight increase of IL-10, TGF-ß and Foxp3 in CD4+ T cells was found after the second injection, but decreased after more injections, suggesting that alum induced early inflammatory responses to a certain extent. Similar patterns of responses of IL-17A and TNF-α in CD8+T cells were shown between YIC and the saline group. Results indicate that add on of Adefovir, did not affect host specific immune responses.
Assuntos
Adenina/análogos & derivados , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/uso terapêutico , Hepatite B Crônica/tratamento farmacológico , Organofosfonatos/uso terapêutico , Adenina/efeitos adversos , Adenina/imunologia , Adenina/uso terapêutico , Adjuvantes Imunológicos , Adulto , Complexo Antígeno-Anticorpo , Terapia Combinada , Feminino , Vacinas contra Hepatite B/efeitos adversos , Vacinas contra Hepatite B/imunologia , Hepatite B Crônica/imunologia , Humanos , Interleucina-10/imunologia , Interleucina-17/imunologia , Interleucina-2/imunologia , Masculino , Organofosfonatos/efeitos adversos , Organofosfonatos/imunologia , Células Th1/imunologia , Células Th2/imunologia , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologia , Adulto JovemRESUMO
Clinical and experimental preparations of IgG/soluble antigen complexes, as well as those formed following antibody therapy in vivo, are multifaceted immune regulators. These immune complexes (ICs) have been tested in humans and animal models, mostly in forms of experimental or clinical vaccination, for at least a century. With intensified research on Fcγ receptor-mediated immune modulation, as well as with immune complex-directed antigen processing, presentation, and inflammatory responses, there are renewed interests of using ICs in vaccines and immunotherapies. Currently, IC-based immune therapy has been broadly experimented in HBV and HIV viral infection control and antitumor treatments. However, mechanistic insights of IC-based treatments are relatively recent subjects of study; strong efforts are needed to establish links to connect laboratory findings with clinical practices. This review covers the history, mechanisms, and in vivo outcomes of this safe and effective therapeutic tool, with a clear aim to bridge laboratory findings with evolving clinical applications.
Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Fatores Imunológicos/metabolismo , Imunoterapia/métodos , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Infecções por HIV/terapia , Hepatite B Crônica/terapia , Humanos , Neoplasias/terapiaRESUMO
Hepatitis B virus (HBV) causes a chronic infection in 350 million people worldwide and greatly increases the risk of liver cirrhosis and hepatocellular carcinoma. The majority of chronic HBV carriers live in Asia. HBV can be divided into eight genotypes with unique geographic distributions. Mutations accumulate during chronic infection or in response to external pressure. Because HBV is an RNA-DNA virus the emergence of drug resistance and vaccine escape mutants has become an important clinical and public health concern. Here, we provide an overview of the molecular biology of the HBV life cycle and an evaluation of the changing role of hepatitis B e antigen (HBeAg) at different stages of infection. The impact of viral genotypes and mutations/deletions in the precore, core promoter, preS, and S gene on the establishment of chronic infection, development of fulminant hepatitis and liver cancer is discussed. Because HBV is prone to mutations, the biological properties of drug-resistant and vaccine escape mutants are also explored.
RESUMO
New effects and mechanisms of alum on innate immunity have emerged in recent years. A number of cellular and molecular mechanisms induced by aluminum adjuvant have been reported, while the role of NALP3 and inflammasome in the cellular pathway induced by alum is still controversial. The effect of injection of alum alone without vaccine antigen into human has not been reported so far. Recently, in a phase IIIa double-blinded placebo controlled clinical trial testing the therapeutic HBsAg-anti-HBs vaccine formulated with alum against chronic viral hepatitis B patients, the placebo group receiving alum only showed substantial therapeutic effects. To explore possible underlying therapeutic mechanisms, mice were treated either with multiple injections of alum alone or with alum adsorbed to proteins (HBsAg-anti-HBs). After 4 injections Gr1(+)/CD11b(+) cells in the spleen were increased in both alum alone and alum adsorbed in proteins groups. Increased Gr1(+)/CD11b(+) cells in spleens remained consistently high in the alum alone treated group, while Gr1(+)/CD11b(+)cells decreased in the alum adsorbed to proteins group after 6 injections. Both treatments triggered increased levels of TNF-alpha measured in the plasma, but only the alum alone treated mice showed increased levels of IL-10. Histology of the liver tissues revealed a higher number of spotty necrotic foci in the alum alone immunized group. Taken together, potent inflammatory responses were induced in the alum alone immunized mice, which suggests that the substantial therapeutic effects observed in chronic hepatitis B patients immunized with alum alone might be attributed to inflammatory responses.
Assuntos
Adjuvantes Imunológicos/farmacologia , Compostos de Alúmen/farmacologia , Antígenos de Superfície da Hepatite B/farmacologia , Vacinas contra Hepatite B/farmacologia , Fígado/imunologia , Baço/imunologia , Adjuvantes Imunológicos/efeitos adversos , Compostos de Alúmen/efeitos adversos , Animais , Feminino , Antígenos de Superfície da Hepatite B/efeitos adversos , Vacinas contra Hepatite B/efeitos adversos , Hepatite B Crônica/sangue , Hepatite B Crônica/imunologia , Hepatite B Crônica/patologia , Hepatite B Crônica/terapia , Humanos , Inflamação/sangue , Inflamação/induzido quimicamente , Inflamação/imunologia , Interleucina-10/sangue , Interleucina-10/imunologia , Fígado/metabolismo , Fígado/patologia , Camundongos , Baço/metabolismo , Baço/patologia , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: The Fc receptor associated pathway might improve the immune responses against hepatitis B virus (HBV) as previously described by us. In addition, the Flt3 ligand (FL) has been reported to potentiate antigen presenting cells in vivo and may act as a potential adjuvant to boost antigen-specific immune responses. In this study, the immune efficacies of a set of fusion proteins of HBsAg and Fc and/or FL were evaluated in HBsAg transgenic mice. METHODS: The fusion proteins composed of HBsAg and the Fc domain of murine IgG1 (HBsAg-Fc) and/or the Flt3 ligand, and yeast-derived recombinant HBsAg were used as immunogen to immunize HBsAg transgenic mice, respectively. Serum and liver HBsAg levels, serum anti-HBsAg and cytokine profile, and the activities of alanine aminotransferase (ALT)/AST were investigated after immunization. RESULTS: After six injections, the most pronounced decrease in serum and liver HBsAg levels was observed in the HBsAg-Fc immunized group. In addition, serum Th1 cytokines and ALT/AST activities were highest in this group, indicating an effective induction of a favorable cellular immune response. Interestingly, the fusion protein containing HBsAg-Fc and the Flt3 ligand stimulated an alternative Th1-type immune response featured with high level productions of tumor necrosis factor α (TNF-α) and monocyte chemoabstractant protein 1 (MCP-1), causing a more severe cytotoxicity in hepatocytes while showed less effective in reducing serum HBsAg level. CONCLUSION: HBsAg-Fc is effective in eliciting both the humoral and cellular immune responses against HBsAg in HBsAg transgenic mice, which makes it a potential immunogen for the immunotherapy of chronic hepatitis B.
Assuntos
Citocinas/metabolismo , Antígenos de Superfície da Hepatite B/imunologia , Antígenos de Superfície da Hepatite B/metabolismo , Receptores Fc/imunologia , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Animais , Quimiocina CCL2/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Antígenos de Superfície da Hepatite B/genética , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Fc/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Current treatments for chronic hepatitis B are effective in only a fraction of patients. All approved directly antiviral agents are nucleos(t)ide analogs (NAs) that target the DNA polymerase activity of the hepatitis B virus (HBV) P protein; resistance and cross-resistance may limit their long-term applicability. P protein is an unusual reverse transcriptase that initiates reverse transcription by protein priming, by which a Tyr residue in the unique terminal protein domain acts as an acceptor of the first DNA nucleotide. Priming requires P protein binding to the ε stem-loop on the pregenomic RNA (pgRNA) template. This interaction also mediates pgRNA encapsidation and thus provides a particularly attractive target for intervention. Exploiting in vitro priming systems available for duck HBV (DHBV) but not HBV, we demonstrate that naphthylureas of the carbonyl J acid family, in particular KM-1, potently suppress protein priming by targeting P protein and interfering with the formation of P-DHBV ε initiation complexes. Quantitative evaluation revealed a significant increase in complex stability during maturation, yet even primed complexes remained sensitive to KM-1 concentrations below 10 µM. Furthermore, KM-1 inhibited the DNA-dependent DNA polymerase activity of both DHBV and HBV nucleocapsids, including from a lamivudine-resistant variant, directly demonstrating the sensitivity of human HBV to the compound. Activity against viral replication in cells was low, likely due to low intracellular availability. KM-1 is thus not yet a drug candidate, but its distinct mechanism of action suggests that it is a highly useful lead for developing improved, therapeutically applicable derivatives.
Assuntos
Antivirais/farmacologia , Cinamatos/farmacologia , Produtos do Gene pol/metabolismo , Hepadnaviridae/efeitos dos fármacos , Hepadnaviridae/metabolismo , Naftalenossulfonatos/farmacologia , Animais , Antivirais/química , Sítios de Ligação , Cinamatos/química , DNA Viral/biossíntese , Farmacorresistência Viral , Produtos do Gene pol/química , Vírus da Hepatite B do Pato/efeitos dos fármacos , Vírus da Hepatite B do Pato/metabolismo , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/metabolismo , Humanos , Substâncias Macromoleculares , Modelos Moleculares , Naftalenossulfonatos/química , Nucleocapsídeo/efeitos dos fármacos , Nucleocapsídeo/metabolismo , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismoRESUMO
Bacterial biofilms can be viewed as a specific type of persistent bacterial infection. After initial invasion, microbes can attach to living and non-living surfaces, such as prosthetics and indwelling medical devices, and form a biofilm composed of extracellular polysaccharides, proteins, and other components. In hosts, biofilm formation may trigger drug resistance and inflammation, resulting in persistent infections. The clinical aspects of biofilm formation and leading strategies for biofilm inhibitors will be discussed in this mini-review.
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Adesinas Bacterianas/fisiologia , Aminoaciltransferases/genética , Infecções Bacterianas/microbiologia , Proteínas de Bactérias/genética , Biofilmes , Cisteína Endopeptidases/genética , Inflamação/microbiologia , Percepção de Quorum , Adesinas Bacterianas/efeitos dos fármacos , Aminoaciltransferases/antagonistas & inibidores , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Infecções Bacterianas/cirurgia , Proteínas de Bactérias/antagonistas & inibidores , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Doença Crônica , Inibidores de Cisteína Proteinase/farmacologia , Humanos , Percepção de Quorum/efeitos dos fármacos , Percepção de Quorum/fisiologia , Infecção dos Ferimentos/microbiologia , Infecção dos Ferimentos/cirurgiaRESUMO
Cyclophilin A (CypA), predominantly located intracellularly, is a multifunctional protein. We previously reported decreased CypA levels in hepatocytes of transgenic mice expressing hepatitis B virus (HBV) surface antigen (HBsAg). In this study, we found that expression of HBV small surface protein (SHBs) in human hepatoma cell lines specifically triggered CypA secretion, whereas SHBs added extracellularly to culture medium did not. Moreover, CypA secretion was not promoted by the expression of a secretion deficient SHBs mutant, suggesting a close association between secretion of CypA and SHBs. Interaction between CypA and SHBs was observed by using coimmunoprecipitation and glutathione S-transferase pull-down assays. Hydrodynamic injection of the SHBs expression construct into C57BL/6J mice resulted in increased serum CypA levels and ALT/AST levels, as well as the infiltration of inflammatory cells surrounding SHBs-positive hepatocytes. The inflammatory response and serum ALT/AST level were reduced when the chemotactic effect of CypA was inhibited by cyclosporine and anti-CD147 antibody. Furthermore, higher serum CypA levels were detected in chronic hepatitis B patients than in healthy individuals. In HBV patients who had received liver transplantation, serum CypA levels declined dramatically after the loss of HBsAg as a consequence of liver transplantation. Taken together, these results indicate that expression and secretion of SHBs can promote CypA secretion, which may contribute to the pathogenesis of HBV infection.
Assuntos
Ciclofilina A/metabolismo , Antígenos de Superfície da Hepatite B/fisiologia , Hepatite B Crônica/etiologia , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Células Cultivadas , Ciclosporina/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Replicação ViralRESUMO
BACKGROUND: There are around 350 million of hepatitis B surface antigen (HBsAg) carriers worldwide, and among them, high risk of developing hepatocellular carcinoma (HCC) has been identified by epidemiological studies. To date, the molecular role of HBsAg in HCC development has not been fully studied. We have previously reported that in cell cultures, HBsAg up-regulated the expression of lymphoid enhancer-binding factor 1 (LEF-1), a key component of the Wnt pathway. In this study we aimed to study this effect of HBsAg on LEF-1 in the development of HCC. METHODS: Expression of HBsAg, LEF-1 and its downstream effector genes were compared among 30 HCCs, their peritumor tissue counterparts and 9 normal control liver tissues by quantitative real-time PCR. In addition, immunohistochemical staining studies on HBsAg and LEF-1 expression were conducted among these samples. RESULTS: The expression of LEF-1 was compared between 13 HBsAg positive HCC tissues and 17 HBsAg negative HCC tissues. Simultaneous detection of LEF-1 and HBsAg was observed in HBsAg positive HCC tissues and, additionally, the simultaneous detection of HBsAg and LEF-1 was more pronounced in peritumor tissues, compared to that in the tumor tissues. The distribution of cellular LEF-1 in peritumor tissues was predominantly in the cytoplasm; while LEF-1 in the tumor tissues was located either exclusively in the nucleus or both in the nucleus and cytoplasm. By real-time PCR, the expression levels of LEF-1 downstream effector genes cyclin D1 and c-myc were higher in peritumor cells compared to that of the tumor cells. However, a 38 kDa truncated isoform of LEF-1, rather than the 55 kDa wild-type LEF-1, was significantly elevated in the HBsAg positive tumor cells. CONCLUSION: Data indicate that deregulation of the Wnt pathway by HBsAg occurred in HBV-associated HCCs, but was more pronounced in the peritumor cells. It is speculated that HBsAg could stimulate proliferation and functional modification of hepatocytes via LEF-1 through the Wnt pathway at the pre-malignant stage.
Assuntos
Carcinoma Hepatocelular/metabolismo , Antígenos de Superfície da Hepatite B/metabolismo , Neoplasias Hepáticas/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Carcinoma Hepatocelular/imunologia , Ciclina D1/metabolismo , Antígenos de Superfície da Hepatite B/imunologia , Humanos , Neoplasias Hepáticas/imunologia , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Regulação para Cima , Proteínas Wnt/metabolismoRESUMO
To reveal the putative cellular factors involved in SARS coronavirus replication, the helicase (Hel, nsp13) of SARS coronavirus was used to screen the cDNA library of rat pulmonary epithelial cells using the yeast two-hybrid system. Positively interacting proteins were further tested using a mammalian cell hybrid system and co-immunoprecipitation in the human A549 cell line, which has been shown to support SARS coronavirus replication. Out of the seven positive clones observed by yeast two-hybrid assay, only the Ddx5 (Asp-Glu-Ala-Asp box polypeptide 5) protein showed specific interaction with SARS-CoV helicase. When expression of DdX5 was knocked down by small interfering RNA (siRNA), SARS coronavirus replication was significantly inhibited in fetal rhesus kidney (FRhK-4) cells. Since Ddx5 is a multifunctional protein that plays important roles in transcriptional regulation, its interaction with SARS coronavirus helicase provides interesting clues for studying virus-host cell interactions in SARS-CoV infections.
Assuntos
RNA Helicases DEAD-box/metabolismo , Metiltransferases/metabolismo , Mapeamento de Interação de Proteínas , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Animais , Linhagem Celular , Técnicas de Silenciamento de Genes , Humanos , Macaca mulatta , Ligação Proteica , RNA Helicases , Ratos , Técnicas do Sistema de Duplo-Híbrido , Proteínas não Estruturais Virais , Replicação ViralRESUMO
The mechanisms for HBV persistence and the pathogenesis of chronic HB have been shown mainly due to defects in host immune responses. However, HBV isolates with different biological features may also contribute to different clinical outcomes and epidemiological implications in viral hepatitis B (HB). This review presents interesting biological features of HBV isolates based on the structural and functional analysis of full-length HBV isolates from various patients. Among isolates from children after failure of HB vaccination, 129L mutant at the 'a' determinant was found with normal binding efficiency to anti-HBs, but with reduced immunogenicity, which could initiate persistent HBV infections. Isolates from fulminant hepatitis (FH) B patients were not all highly replicative, but differences in capacities of anti-HBs induction could be involved in the pathogenesis of FH. The high replicative competency of isolates from hepatocellular carcinoma (HCC) patients could result in enhanced immune-mediated cytopathic effects against HBV viral proteins, and increased transactivating activity by the X protein. The mechanism of a double-spliced variant in enhancing replication of the wild-type virus is presented. The importance of integrating structural and functional analysis to reveal biological features of HBV isolates in viral pathogenesis is discussed.
Assuntos
Genoma Viral , Vírus da Hepatite B/genética , Vírus da Hepatite B/isolamento & purificação , Hepatite B/virologia , Criança , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus da Hepatite B/imunologia , Humanos , Virulência , Replicação ViralRESUMO
In hepatitis B virus (HBV) replication cycle, pregenomic RNA undergoes splicing and the reverse transcribed defective genomes can be packaged and released. Various types of spliced defective HBV genomes have been isolated from the sera and liver tissues of viral hepatitis B patients. To explore the functions of a 2.2 kb double spliced HBV variant, a 3.2 kb full-length HBV isolate (#97-34) and its 2.2 kb double-spliced HBV variant (#AP-12) from the tumor tissue of a patient with hepatocellular carcinoma (HCC) were amplified and cloned. Sequencing results showed that #AP12 had deletions in pre-S2, part of pre-S1, S genes, part of the spacer, and part of the reverse transcriptase gene, while the X gene was intact. When this defective double-spliced genome and its full-length counterpart genome were co-transfected into HepG2 cells, the former was shown to enhance the replication of the latter, both by real-time PCR and Southern blotting. When a replication incompetent clone 97-34G1881A was used to co-transfect with #AP12, #AP12 DNA was increased, indicating that replication of the wild-type virus was not the only factor involved in this observation. However, the replication enhancing competency of #AP12 was shown to require an intact HBV X expression cassette. The double-spliced defective variant might contribute to persistent HBV replication in a subpopulation of HCC patients.
Assuntos
Carcinoma Hepatocelular/virologia , Vírus Defeituosos/genética , Genoma Viral/genética , Vírus da Hepatite B/genética , Neoplasias Hepáticas/virologia , Splicing de RNA , Replicação Viral , Linhagem Celular , Meios de Cultura/química , DNA Viral/análise , DNA Viral/genética , Genótipo , Hepatite B/sangue , Hepatite B/genética , Hepatite B/virologia , Antígenos de Superfície da Hepatite B/análise , Antígenos E da Hepatite B/análise , Vírus da Hepatite B/isolamento & purificação , Humanos , Masculino , Dados de Sequência Molecular , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
The genome of hepatitis B virus (HBV) consists of four open reading frames, encoding the envelope proteins (Pre-S/S), the core proteins (Pre-C/C), the polymerase (P) and the transactivating X protein (X). In the sera of HBV-infected patients, hepatitis B surface antigen (HBsAg) particles without the viral genome can outnumber virions by more than 1000-fold. To analyse the interactions between HBsAg and host cells, global gene-expression profiles of a small HBsAg (SHBs)-secreting stable cell line (HepG2-S-G2) and its counterpart control cell line (HepG2-Neo-F4) were compared. Marked upregulation of lymphoid enhancer-binding factor 1 (LEF-1), a transcription factor in the Wnt pathway, was found in SHBs-expressing cells and was confirmed by interference experiments with small interfering RNA. However, compared with the control cells, HepG2-S-G2 did not show higher proliferative competence in culture or increased tumorigenesis in nude mice. A possible mechanism to explain the discrepancy between the upregulation of LEF-1 and the lack of increased tumorigenesis is SHBs expression resulting in altered expression and distribution of LEF-1 protein in cell compartments and upregulation of LEF-1 isoforms that could suppress, rather than enhance, the Wnt pathway.