RESUMEN
The innate immune-signaling kinase, TBK1, couples pathogen surveillance to induction of host defense mechanisms. Pathological activation of TBK1 in cancer can overcome programmed cell death cues, enabling cells to survive oncogenic stress. The mechanistic basis of TBK1 prosurvival signaling, however, has been enigmatic. Here, we show that TBK1 directly activates AKT by phosphorylation of the canonical activation loop and hydrophobic motif sites independently of PDK1 and mTORC2. Upon mitogen stimulation, triggering of the innate immune response, re-exposure to glucose, or oncogene activation, TBK1 is recruited to the exocyst, where it activates AKT. In cells lacking TBK1, insulin activates AKT normally, but AKT activation by exocyst-dependent mechanisms is impaired. Discovery and characterization of a 6-aminopyrazolopyrimidine derivative, as a selective low-nanomolar TBK1 inhibitor, indicates that this regulatory arm can be pharmacologically perturbed independently of canonical PI3K/PDK1 signaling. Thus, AKT is a direct TBK1 substrate that connects TBK1 to prosurvival signaling.
Asunto(s)
Neoplasias/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Animales , Supervivencia Celular , Transformación Celular Neoplásica , Células Cultivadas , Células HCT116 , Humanos , Inmunidad Innata , Ratones , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , TransfecciónRESUMEN
The closely related TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) serve in the generation and maintenance of mature B-lymphocytes. Both BAFF and APRIL assemble as homotrimers that bind and activate several receptors that they partially share. However, heteromers of BAFF and APRIL that occur in patients with autoimmune diseases are incompletely characterized. The N and C termini of adjacent BAFF or APRIL monomers are spatially close and can be linked to create single-chain homo- or hetero-ligands of defined stoichiometry. Similar to APRIL, heteromers consisting of one BAFF and two APRILs (BAA) bind to the receptors B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) but not to the BAFF receptor (BAFFR). Heteromers consisting of one APRIL and two BAFF (ABB) bind to TACI and BCMA and weakly to BAFFR in accordance with the analysis of the receptor interaction sites in the crystallographic structure of ABB. Receptor binding correlated with activity in reporter cell line assays specific for BAFFR, TACI, or BCMA. Single-chain BAFF (BBB) and to a lesser extent single-chain ABB, but not APRIL or single-chain BAA, rescued BAFFR-dependent B cell maturation in BAFF-deficient mice. In conclusion, BAFF-APRIL heteromers of different stoichiometries have distinct receptor-binding properties and activities. Based on the observation that heteromers are less active than BAFF, we speculate that their physiological role might be to down-regulate BAFF activity.
Asunto(s)
Factor Activador de Células B/metabolismo , Antígeno de Maduración de Linfocitos B/metabolismo , Proteína Activadora Transmembrana y Interactiva del CAML/metabolismo , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Animales , Factor Activador de Células B/química , Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/metabolismo , Antígeno de Maduración de Linfocitos B/genética , Dimerización , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Unión Proteica , Transducción de Señal , Proteína Activadora Transmembrana y Interactiva del CAML/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/química , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genéticaRESUMEN
Tumor-associated inflammation drives cancer progression and therapy resistance, often linked to the infiltration of monocyte-derived tumor-associated macrophages (TAMs), which are associated with poor prognosis in various cancers. To advance immunotherapies, testing on immunocompetent pre-clinical models of human tissue is crucial. We have developed an in vitro model of microvascular networks with tumor spheroids or patient tissues to assess monocyte trafficking into tumors and evaluate immunotherapies targeting the human tumor microenvironment. Our findings demonstrate that macrophages in vascularized breast and lung tumor models can enhance monocyte recruitment via CCL7 and CCL2, mediated by CSF-1R. Additionally, a multispecific antibody targeting CSF-1R, CCR2, and neutralizing TGF-ß (CSF1R/CCR2/TGF-ß Ab) repolarizes TAMs towards an anti-tumoral M1-like phenotype, reduces monocyte chemoattractant protein secretion, and blocks monocyte migration. This antibody also inhibits monocyte recruitment in patient-specific vascularized tumor models. In summary, this vascularized tumor model recapitulates the monocyte recruitment cascade, enabling functional testing of innovative therapeutic antibodies targeting TAMs in the tumor microenvironment.
RESUMEN
Tumor-associated inflammation drives cancer progression and therapy resistance, with the infiltration of monocyte-derived tumor-associated macrophages (TAMs) associated with poor prognosis in diverse cancers. Targeting TAMs holds potential against solid tumors, but effective immunotherapies require testing on immunocompetent human models prior to clinical trials. Here, we develop an in vitro model of microvascular networks that incorporates tumor spheroids or patient tissues. By perfusing the vasculature with human monocytes, we investigate monocyte trafficking into the tumor and evaluate immunotherapies targeting the human tumor microenvironment. Our findings demonstrate that macrophages in vascularized breast and lung tumor models can enhance monocyte recruitment via TAM-produced CCL7 and CCL2, mediated by CSF-1R. Additionally, we assess a novel multispecific antibody targeting CCR2, CSF-1R, and neutralizing TGF-ß, referred to as CSF1R/CCR2/TGF-ß Ab, on monocytes and macrophages using our 3D models. This antibody repolarizes TAMs towards an anti-tumoral M1-like phenotype, reduces monocyte chemoattractant protein secretion, and effectively blocks monocyte migration. Finally, we show that the CSF1R/CCR2/TGF-ß Ab inhibits monocyte recruitment in patient-specific vascularized tumor models. Overall, this vascularized tumor model offers valuable insights into monocyte recruitment and enables functional testing of innovative therapeutic antibodies targeting TAMs in the tumor microenvironment (TME).
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The EGFR and TGFß signaling pathways are important mediators of tumorigenesis, and cross-talk between them contributes to cancer progression and drug resistance. Therapies capable of simultaneously targeting EGFR and TGFß could help improve patient outcomes across various cancer types. Here, we developed BCA101, an anti-EGFR IgG1 mAb linked to an extracellular domain of human TGFßRII. The TGFß "trap" fused to the light chain in BCA101 did not sterically interfere with its ability to bind EGFR, inhibit cell proliferation, or mediate antibody-dependent cellular cytotoxicity. Functional neutralization of TGFß by BCA101 was demonstrated by several in vitro assays. BCA101 increased production of proinflammatory cytokines and key markers associated with T-cell and natural killer-cell activation, while suppressing VEGF secretion. In addition, BCA101 inhibited differentiation of naïve CD4+ T cells to inducible regulatory T cells (iTreg) more strongly than the anti-EGFR antibody cetuximab. BCA101 localized to tumor tissues in xenograft mouse models with comparable kinetics to cetuximab, both having better tumor tissue retention over TGFß "trap." TGFß in tumors was neutralized by approximately 90% in animals dosed with 10 mg/kg of BCA101 compared with 54% in animals dosed with equimolar TGFßRII-Fc. In patient-derived xenograft mouse models of head and neck squamous cell carcinoma, BCA101 showed durable response after dose cessation. The combination of BCA101 and anti-PD1 antibody improved tumor inhibition in both B16-hEGFR-expressing syngeneic mouse models and in humanized HuNOG-EXL mice bearing human PC-3 xenografts. Together, these results support the clinical development of BCA101 as a monotherapy and in combination with immune checkpoint therapy. SIGNIFICANCE: The bifunctional mAb fusion design of BCA101 targets it to the tumor microenvironment where it inhibits EGFR and neutralizes TGFß to induce immune activation and to suppress tumor growth.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Neoplasias , Animales , Humanos , Ratones , Anticuerpos Monoclonales Humanizados/uso terapéutico , Carcinoma de Células Escamosas/terapia , Línea Celular Tumoral , Cetuximab/farmacología , Cetuximab/uso terapéutico , Receptores ErbB/metabolismo , Neoplasias de Cabeza y Cuello/terapia , Factor de Crecimiento Transformador beta , Microambiente Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias/terapiaRESUMEN
The transcription factor interferon regulatory factor 5 (IRF5) plays essential roles in pathogen-induced immunity downstream of Toll-, nucleotide-binding oligomerization domain-, and retinoic acid-inducible gene I-like receptors and is an autoimmune susceptibility gene. Normally, inactive in the cytoplasm, upon stimulation, IRF5 undergoes posttranslational modification(s), homodimerization, and nuclear translocation, where dimers mediate proinflammatory gene transcription. Here, we report the rational design of cell-penetrating peptides (CPPs) that disrupt IRF5 homodimerization. Biochemical and imaging analysis shows that IRF5-CPPs are cell permeable, noncytotoxic, and directly bind to endogenous IRF5. IRF5-CPPs were selective and afforded cell type- and species-specific inhibition. In plasmacytoid dendritic cells, inhibition of IRF5-mediated interferon-α production corresponded to a dose-dependent reduction in nuclear phosphorylated IRF5 [p(Ser462)IRF5], with no effect on pIRF5 levels. These data support that IRF5-CPPs function downstream of phosphorylation. Together, data support the utility of IRF5-CPPs as novel tools to probe IRF5 activation and function in disease.
Asunto(s)
Péptidos de Penetración Celular , Péptidos de Penetración Celular/genética , Péptidos de Penetración Celular/metabolismo , Péptidos de Penetración Celular/farmacología , Células Dendríticas/metabolismo , Regulación de la Expresión Génica , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , FosforilaciónRESUMEN
Type 2 diabetes is a polygenic disorder characterized by multiple biochemical defects including transcriptional, translational, and posttranslational abnormalities. Although major progress has been made in elucidation of factors at the transcriptional and posttranslational levels, defects at the translational level remain elusive. Mutation of a kinase that regulates translation initiation has been implicated in the etiology of a monogenic form of diabetes known as Wolcott-Rallison syndrome. Characterization of mice rendered deficient in eukaryotic initiation factors has provided model systems to study the involvement of translation in regulating insulin synthesis and secretion, hepatic function, peripheral insulin resistance, and diabetic complications. Recent progress in the understanding of endoplasmic reticulum overload by unfolded proteins has begun to uncover mechanisms leading to pancreatic beta-cell exhaustion. Future advances in this area may lead to identification of the missing links in the pathogenesis of beta-cell failures due to conditions such as hyperinsulinemia, hyperglycemia, and long-term treatment with sulfonylureas, and thus may identify novel therapeutic targets for diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Animales , Arteriosclerosis/genética , Antígenos CD36/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 4E Eucariótico de Iniciación , Glucosa/metabolismo , Humanos , Resistencia a la Insulina , Islotes Pancreáticos/fisiopatología , Hígado/metabolismo , Fosforilación , Proteínas Quinasas S6 Ribosómicas , eIF-2 QuinasaRESUMEN
Chronic hepatitis C virus (HCV) infection is a major burden on humanity. The current HCV therapy has limited efficacy, and there is pressing need for new and more effective therapies. Host cell factors that are required for HCV infection, replication and/or pathogenesis represent potential therapeutic targets. Of particular interest are cellular receptors that mediate HCV entry, factors that facilitate HCV replication and assembly, and intracellular pathways involving lipid biosynthesis, oxidative stress and innate immune response. A crucial challenge now is to manipulate such cellular targets pharmacologically for chronic HCV treatment, without being limited by side effects.
Asunto(s)
Antivirales/farmacología , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Hepatitis C Crónica/tratamiento farmacológico , Antivirales/uso terapéutico , Hepatitis C Crónica/genética , Hepatitis C Crónica/inmunología , Humanos , Lípidos/biosíntesis , Estrés Oxidativo , Ensamble de Virus/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
PRK1 is a lipid- and Rho GTPase-activated serine/threonine protein kinase implicated in the regulation of receptor trafficking, cytoskeletal dynamics and tumorigenesis. Although Rho binding has been mapped to the HR1 region in the regulatory domain of PRK1, the mechanism involved in the control of PRK1 activation following Rho binding is poorly understood. We now provide the first evidence that the very C-terminus beyond the hydrophobic motif in PRK1 is essential for the activation of this kinase by RhoA. Deletion of the HR1 region did not completely abolish the binding of PRK1-DeltaHR1 to GTPgammaS-RhoA nor the activation of this mutant by GTPgammaS-RhoA in vitro. In contrast, removing of the last six amino acid residues from the C-terminus of PRK1 or truncating of a single C-terminal residue from PRK1-DeltaHR1 completely abrogated the activation of these mutants by RhoA both in vitro and in vivo. The critical dependence of the very C-terminus of PRK1 on the signaling downstream of RhoA was further demonstrated by the failure of the PRK1 mutant lacking its six C-terminal residues to augment lisophosphatidic acid-elicited neurite retraction in neuronal cells. Thus, we show that the HR1 region is necessary but not sufficient in eliciting a full activation of PRK1 upon binding of RhoA. Instead, such activation is controlled by the very C-terminus of PRK1. Our results also suggest that the very C-terminus of PRK1, which is the least conserved among members of the protein kinase C superfamily, is a potential drug target for pharmacological intervention of RhoA-mediated signaling pathways.
Asunto(s)
Proteína Quinasa C/metabolismo , Transducción de Señal/fisiología , Proteína de Unión al GTP rhoA/metabolismo , Animales , Línea Celular , Activación Enzimática , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Lisofosfolípidos/metabolismo , Ratones , Contracción Muscular/fisiología , Mutación , Proteína Quinasa C/química , Proteína Quinasa C/genética , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Unión al GTP rhoA/genéticaRESUMEN
In this article, we explore the role of the C-terminus (V5 domain) of PKCepsilon plays in the catalytic competence of the kinase using serial truncations followed by immune-complex kinase assays. Surprisingly, removal of the last seven amino acid residues at the C-terminus of PKCepsilon resulted in a PKCepsilon-Delta731 mutant with greatly reduced intrinsic catalytic activity while truncation of eight amino acid residues at the C-terminus resulted in a catalytically inactive PKCepsilon mutant. Computer modeling and molecular dynamics simulations showed that the last seven and/or eight amino acid residues of PKCepsilon were involved in interactions with residues in the catalytic core. Further truncation analyses revealed that the hydrophobic phosphorylation motif was dispensable for the physical interaction between PKCepsilon and 3-phosphoinositide-dependent kinase-1 (PDK-1) as the PKCepsilon mutant lacking both the turn and the hydrophobic motifs could still be co-immunoprecipitated with PDK-1. These results provide fresh insights into the biochemical and structural basis underlying the isozyme-specific regulation of PKC and suggest that the very C-termini of PKCs constitute a promising new target for the development of novel isozyme-specific inhibitors of PKC.
Asunto(s)
Proteína Quinasa C-epsilon/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos , Animales , Catálisis , Dominio Catalítico , Simulación por Computador , Eliminación de Gen , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Fosforilación , Unión Proteica , Proteína Quinasa C-epsilon/genéticaRESUMEN
Chronic infection with hepatitis C virus (HCV) is an emerging global epidemic. The development of effective HCV antiviral therapeutics continues to be a daunting challenge owing to the absence of adequate animal models and tissue-culture systems for analysis and propagation of the virus. Despite these obstacles, inhibitors of the replicative elements of HCV, immune modulators and non-specific hepatoprotective agents are being pursued and exciting progress has been made. Successful therapeutic intervention of HCV will probably require combination approaches and new approaches, including host drug discovery targets.
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Hepatitis C Crónica/tratamiento farmacológico , Tecnología Farmacéutica/métodos , Vacunas contra Hepatitis Viral/uso terapéutico , Animales , Secuencia de Bases , Hepatitis C Crónica/genética , Hepatitis C Crónica/metabolismo , Humanos , Datos de Secuencia Molecular , Vacunas contra Hepatitis Viral/química , Vacunas contra Hepatitis Viral/genéticaRESUMEN
Hepatitis C virus (HCV)-specific impairments in host immunity have been described at multiple levels of the innate and adaptive response, which may lead to viral persistence in the majority of infections. Understanding of HCV-associated immune defects could lead to novel therapeutic advances. Natural killer (NK) cells, the major effector cells of the innate immune system, are functionally impaired in chronic HCV infection. It has been suggested that this phenotype is a result of virus-specific defects in antigen-presenting cells (APCs) that regulate NK cell activity, as normal NK function is restored when they are stimulated ex vivo. In this study, we used human NK cell cytotoxicity assays to evaluate the activation-induced effects of NK cells on the HCV replicon-containing hepatic cells. We found that cytokine-activated NK cells were capable of inducing an HCV-associated, perforin/granzyme-dependent lysis of human hepatoma cells and that this required direct cellular contact and was independent of MHC class I expression levels. In contrast, on removal of cytokine stimulation, NK cells failed to exert any direct cytolytic effect on replicon targets. These findings suggest an important underlying mechanism by which NK cells control HCV infection and, with appropriate understanding of HCV-associated immune defects, could lead to novel therapeutic advances.
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Citotoxicidad Inmunológica , Hepacivirus/inmunología , Hepatocitos/inmunología , Células Asesinas Naturales/inmunología , Replicón , Replicación Viral , Antígenos CD/inmunología , Carcinoma Hepatocelular , Citocinas/farmacología , Hepacivirus/genética , Hepacivirus/fisiología , Hepatocitos/virología , Humanos , Células Asesinas Naturales/efectos de los fármacos , Activación de Linfocitos , Tetraspanina 28 , Células Tumorales CultivadasRESUMEN
Cross-linking of the B cell antigen receptor (BCR) results in the activation of several protein tyrosine kinases leading to phospholipase C-gamma2-dependent phospholipid hydrolysis and Ca2+ mobilization, followed by activation of the protein kinase C (PKC) family members. Sustained Ca2+ release in B lymphocytes is dependent on the membrane localization and activation of the protein tyrosine kinase BTK. Ca2+ release is a tightly regulated process involving BTK membrane localization through its phosphorylation by PKCbeta. A selective role of PKCbeta in B cell signaling was first revealed by the characterization of PKCbeta knockout mice, which displayed decreased B cell proliferation in response to various mitogenic stimuli. However, it is not clear whether the B cell defects displayed by the PKCbeta knockout mice are due a B cell developmental defect or the scaffolding function of PKCbeta, resulting in a defect in the recruitment or formation of signal transducing complex molecules. Thus, in this report we investigated the effects of pharmacologic inhibition of the catalytic function of PKCbeta on B cell survival and growth. Treatment of Daudi B lymphoma cell line with a selective PKCbeta inhibitor, LY333531, inhibited anti-IgM-induced phosphorylation of BTK on Ser180 in a concentration-dependent manner, which was concomitant with an increase in BTK activation, and Ca2+ mobilization. In primary splenic B cells, LY333531 inhibited BCR-induced B cell proliferation, but did not affect basal or LPS-induced proliferation. Finally, LY333531 treatment resulted in the induction of apoptosis of anti-IgM-activated B cells, which corroborated with their inability to up-regulate pro-survival factors, Bcl-X(L) and Bcl-2. These results support the important and selective role of the PKCbeta enzymatic function in controlling Ca2+ release during BCR signaling leading to B lymphocyte survival and growth.
Asunto(s)
Linfocitos B/enzimología , Linfocitos B/inmunología , Proteína Quinasa C/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Agammaglobulinemia Tirosina Quinasa , Animales , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Supervivencia Celular , Reactivos de Enlaces Cruzados , Femenino , Técnicas In Vitro , Indoles/farmacología , Maleimidas/farmacología , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/inmunología , Proteína Quinasa C beta , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/metabolismoRESUMEN
The TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) modulate B cell function by forming homotrimers and heterotrimers. To determine the structure of a heterotrimer of BAFF and APRIL, these ligands were expressed as a single chain protein in HEK 293 cells, purified by affinity and size exclusion chromatographies, and crystallized. Crystals belonging to the orthorhombic crystal system with a space group of C2221 diffracted to 2.43 Å. Initial structural solution was obtained by the molecular replacement method, and the structure was further refined to an R factor of 0.179 and free R factor of 0.234. The atomic coordinates and structure factors have been deposited into the Protein Data Bank (accession code 4ZCH).
RESUMEN
Chronic infection with hepatitis C virus (HCV) is associated with liver cirrhosis that often leads to hepatic failure and hepatocellular carcinoma (HCC). HCV infection has become a global health threat and the main cause of adult liver transplants in developed nations. Current approved anti-HCV therapies (interferon and pegylated interferon alone or in combination with ribavirin) are not effective in eliminating the viral infection in a significant population of patients (e.g., those infected with HCV genotype 1). Furthermore, these therapies are plagued with many undesirable side effects. Therefore, the HCV epidemic represents a huge unmet medical need that has triggered intensive research efforts towards the development of more effective drugs. Given its essential role in the process of HCV replication, the viral NS3/4A serine protease is arguably the most thoroughly characterized HCV enzyme and the most intensively pursued anti-HCV target for drug development. This is further fueled by the successful use of small-molecule inhibitors of the human immunodeficiency virus (HIV) viral protease, which have had an impressive effect on HIV-related morbidity and mortality, offering hope that analogous drugs might also have a similar impact against HCV. Here, we review the recent progress and development of small-molecule inhibitors of the HCV NS3/4A protease. In particular, we focus on the discovery of VX-950, the latest HCV NS3-4A protease inhibitor to be advanced to clinical studies. While the challenges of designing potent inhibitors of the viral protease have been solved, as highlighted by BILN 2061 and VX-950, it is still too early to determine whether these efforts will eventually yield promising drug candidates. For the emerging small-molecule HCV inhibitors, viral resistance will likely be a big problem. Thus, combination therapy of different drugs with different targets/mechanisms will be necessary to effectively inhibit HCV replication. It is also hoped that a detail characterization of how the resistance mutations that affect NS3 inhibitor binding may provide useful information for the design of inhibitors with the potential to treat resistant viruses that may arise during chronic HCV infection.
Asunto(s)
Antivirales/uso terapéutico , Proteínas Portadoras/antagonistas & inhibidores , Hepatitis C Crónica/tratamiento farmacológico , Inhibidores de Serina Proteinasa/uso terapéutico , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Animales , Proteínas Portadoras/metabolismo , Hepacivirus/enzimología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Serina Endopeptidasas , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales/metabolismoRESUMEN
Interferon-alpha-based therapies are the mainstay of hepatitis C treatment. However, these broad immunomodulatory agents, already limited by their side effects, expense and route of administration, are not efficacious in all cases. Therefore, research efforts have focused on identifying additional drugs and therapeutic modalities to benefit those individuals refractive to current therapy, and which might be more affordable, such as orally active small-molecule alternatives and preventive or therapeutic vaccines. It is likely that successful therapeutic intervention of hepatitis C will require a combination of approaches targeting different aspects of the viral life-cycle.
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Antivirales/uso terapéutico , Hepatitis C/tratamiento farmacológico , Animales , Quimioterapia Combinada , Hepacivirus/efectos de los fármacos , Hepacivirus/crecimiento & desarrollo , Hepatitis C/inmunología , Hepatitis C/prevención & control , Humanos , Interferones/uso terapéutico , Prenilación de Proteína/efectos de los fármacos , Ribavirina/uso terapéutico , Vacunas contra Hepatitis Viral/inmunología , Replicación Viral/efectos de los fármacosRESUMEN
Members of the protein kinase C (PKC) family are expressed in many different cell types, where they are known to regulate a wide variety of cellular processes that impact on cell growth and differentiation, cytoskeletal remodelling and gene expression in the response to diverse stimuli. The broad tissue distribution and redundancy of in vitro function have often hampered the identification of definitive roles for each PKC family member. However, recent in vivo studies of PKC isoenzyme-selective knockout and transgenic mice have highlighted distinct functions of individual PKCs in the immune system. These genetic analyses, along with biochemical studies utilizing PKC isoenzyme-specific cDNA (wild-type, constitutively active and dominant-negative), antisense oligonucleotides (ASO), RNA interference (RNAi), and pharmacological inhibitors, indicate that PKC-regulated signalling pathways play a significant role in many aspects of immune responses, from development, differentiation, activation and survival of lymphocytes to macrophage activation. The importance of PKCs in cellular immune responses suggests that improved understanding of the molecular events that govern their actions could point to new avenues for development of treatments for immune disorders.
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Sistema Inmunológico/enzimología , Proteína Quinasa C/fisiología , Animales , Linfocitos B/enzimología , Linfocitos B/inmunología , Sistema Inmunológico/citología , Inmunidad Innata , Isoenzimas/fisiología , Macrófagos/enzimología , Macrófagos/inmunología , Mastocitos/enzimología , Mastocitos/inmunología , Ratones , Transducción de Señal , Linfocitos T/enzimología , Linfocitos T/inmunología , Timo/citología , Timo/crecimiento & desarrollo , Timo/inmunologíaRESUMEN
Pro-inflammatory cytokines play a major role in the initiation and maintenance of joint inflammation and destruction in rheumatoid arthritis (RA). The therapeutic success of biologics targeting tumour necrosis factor-alpha (TNF-α), interleukin-1 (IL-1) and interleukin (IL)-6 receptor (IL-6R) has broadened the treatment options for RA. These agents have potential overlapping and discriminating biologic effects, as well as different pharmacological features. Tocilizumab (TCZ) is a humanized monoclonal antibody that binds and neutralizes IL-6R, resulting in the inhibition of various IL-6-mediated biological activities, including inflammation-related, immunomodulatory and tissue/matrix remodelling effects. Randomized, double-blind, controlled phase III studies and a number of early clinical observational studies have shown that treatment with TCZ results in rapid and sustained improvement in the signs and symptoms of RA among different patient populations. These studies have established the efficacy and safety of TCZ. Here, we review the pleiotropic functions of IL-6 and how it impinges on many aspects of RA pathogenesis, and highlight the clinical experience to date with TCZ as an emerging new treatment option for RA.
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Antiinflamatorios/inmunología , Antiinflamatorios/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Artritis Reumatoide/terapia , Inmunoterapia , Interleucina-6/metabolismo , Animales , Artritis Reumatoide/inmunología , Matriz Extracelular/efectos de los fármacos , Humanos , Inmunomodulación , Inmunoterapia/tendencias , Ensayos Clínicos Controlados Aleatorios como Asunto , Receptores de Interleucina-6/inmunologíaRESUMEN
OBJECTIVE: Rheumatoid arthritis therapies that are based on inhibition of a single cytokine, e.g., tumor necrosis factor α (TNFα) or interleukin-6 (IL-6), produce clinically meaningful responses in only about half of the treated patients. This study was undertaken to investigate whether combined inhibition of TNFα and IL-17 has additive or synergistic effects in the suppression of mesenchymal cell activation in vitro and inflammation and tissue destruction in arthritis in vivo. METHODS: Cultures of human fibroblast-like synoviocytes (FLS) were stimulated with TNFα, IL-17, or a combination of both. Single/combined neutralizing antibodies against TNFα and IL-17 were used to examine in vitro cytokine responses and in vivo development of arthritis and bone and cartilage destruction in TNFα-transgenic mice. Bispecific anti-TNFα/IL-17 antibodies were designed, and their potential to block cytokine responses in human FLS was tested. RESULTS: TNFα and IL-17 had additive/synergistic effects in promoting production of IL-6, IL-8, and granulocyte colony-stimulating factor, as well as matrix metalloproteinases, in FLS. Bispecific anti-TNFα/IL-17 antibodies showed superior efficacy in blocking cytokine and chemokine responses in vitro. Furthermore, dual versus single inhibition of both cytokines using neutralizing antibodies was more effective in inhibiting the development of inflammation and bone and cartilage destruction in arthritic mice. CONCLUSION: Combined blockade of TNFα and IL-17 was more effective than single blockade in inhibiting cytokine, chemokine, and matrix enzyme responses from human mesenchymal cells and in blocking tissue destruction associated with arthritis, and additionally showed a positive impact on rebalance of bone homeostasis. Bispecific anti-TNFα/IL-17 antibodies may have superior efficacy in the treatment of arthritis and may overcome the limited therapeutic responses obtained with single cytokine neutralization.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Interleucina-17/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Anticuerpos Biespecíficos/inmunología , Antirreumáticos/inmunología , Artritis Reumatoide/patología , Células Cultivadas , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Factor Estimulante de Colonias de Granulocitos/metabolismo , Humanos , Técnicas In Vitro , Interleucina-17/inmunología , Interleucina-17/farmacología , Interleucina-8/metabolismo , Metaloproteasas/metabolismo , Ratones , Ratones Transgénicos , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The use of type I interferon (IFN), in combination with ribvirin, to treat chronic hepatitis C virus (HCV) infection has many drawbacks that prevent widespread application, ultimately leading to a significant unmet clinical need. Potential improvements in IFN therapy through targeted delivery, molecular alteration, and combination with other agents are ongoing in an attempt to decrease adverse effects and increase efficacy. In this report, the HCV replicon cell culture system was used to assess potential synergistic antiviral effects of multiple IFN species when administered in combination. Quantitative analysis of HCV replicon RNA by TaqMan (PE Applied Biosystems, Foster City, CA) and qualitative analysis of HCV protein expression were used to measure the antiviral efficacy of individual and combination IFN treatments, and synergistic responses of IFN combinations were determined through statistical analysis of the TaqMan results. We found that when administered simultaneously, type I/II IFN combinations (IFN-alpha2b + IFN-gamma or IFN-beta + IFN-gamma) resulted in dramatic antiviral synergy, whereas a type I/I combination (IFN-alpha2b + IFN-beta) demonstrated a slightly antagonistic profile. The synergistic effect is likely due to differential cell surface receptors and signaling pathways employed by types I and II IFNs. Conversely, all type I IFN species bind the same receptor and signal through similar pathways, possibly accounting for the nearly additive response observed. In support of this hypothesis, IFN treatment resulted in differential induction of Stat1 phosphorylation at Tyr 701. In conclusion, simultaneous type I/II IFN combination treatment may allow an overall decreased effective IFN dose, which may reduce the side effect profiles that hinder current therapy.