RESUMO
OBJECTIVE: To investigate the mutual regulation of hypoxia-inducible factor (HIF)-1α activity and periodontal ligament-associated protein-1 (PLAP-1) expression in human periodontal ligament cells (HPDLs). BACKGROUND: Cellular responses to hypoxia regulate various biological events (e.g., inflammation and tissue regeneration) through activation of HIF-1α. PLAP-1, an extracellular matrix protein preferentially expressed in the periodontal ligament, plays important roles in the functions of HPDLs. Although PLAP-1 expression has been demonstrated in hypoxic regions, the involvement of PLAP-1 in responses to hypoxia has not been revealed. METHODS: HPDLs were cultured under normoxic (20% O2 ) or hypoxic (1% O2 ) conditions with or without deferoxamine mesylate (chemical hypoxia inducer) or chetomin (HIF signaling inhibitor). Expression levels of PLAP-1 and HIF-1α were examined by real-time reverse transcription-polymerase chain reaction and western blot analysis. Luciferase reporter assays of HIF-1α activity were performed using 293T cells stably transfected with a hypoxia response element (HRE)-containing luciferase vector in the presence or absence of recombinant PLAP-1 or PLAP-1 gene transfection. RESULTS: Cultivation under hypoxic conditions elevated the gene and protein expression levels of PLAP-1 in HPDLs. Deferoxamine mesylate treatment also enhanced PLAP-1 expression in HPDLs. Hypoxia-induced PLAP-1 expression was significantly suppressed in the presence of chetomin. PLAP-1-suppressed HPDLs showed increased HIF-1α accumulation in the nucleus during culture under hypoxic conditions, but not in the presence of recombinant PLAP-1. In the presence of recombinant PLAP-1, hypoxia-induced HRE activity of 293T cells was significantly suppressed in a dose-dependent manner. Transfection of the PLAP-1 gene resulted in a significant reduction of HRE activity during culture under hypoxic conditions. CONCLUSION: PLAP-1 expression is upregulated under hypoxic conditions through HIF-1α activation. Moreover, hypoxia-induced PLAP-1 expression regulates HIF-1α signaling.
Assuntos
Desferroxamina , Proteínas da Matriz Extracelular/metabolismo , Hipóxia , Western Blotting , Hipóxia Celular/fisiologia , Desferroxamina/farmacologia , Humanos , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Luciferases/metabolismo , TransfecçãoRESUMO
The recently approved HIV-1 integrase strand transfer inhibitor (INSTI) dolutegravir (DTG) (S/GSK1349572) has overall advantageous activity when tested in vitro against HIV-1 with raltegravir (RAL) and elvitegravir (EVG) resistance signature mutations. We conducted an in vitro resistance selection study using wild-type HIV-1 and mutants with the E92Q, Y143C, Y143R, Q148H, Q148K, Q148R, and N155H substitutions to assess the DTG in vitro barrier to resistance. No viral replication was observed at concentrations of ≥ 32 nM DTG, whereas viral replication was observed at 160 nM RAL or EVG in the mutants. In the Q148H, Q148K, or Q148R mutants, G140S/Q148H, E138K/Q148K, E138K/Q148R, and G140S/Q148R secondary mutations were identified with each INSTI and showed high resistance to RAL or EVG but limited resistance to DTG. E138K and G140S, as secondary substitutions to Q148H, Q148K, or Q148R, were associated with partial recovery in viral infectivity and/or INSTI resistance. In the E92Q, Y143C, Y143R, and N155H mutants, no secondary substitutions were associated with DTG. These in vitro results suggest that DTG has a high barrier to the development of resistance in the presence of RAL or EVG signature mutations other than Q148. One explanation for this high barrier to resistance is that no additional secondary substitution of E92Q, Y143C, Y143R, or N155H simultaneously increased the fold change in 50% effective concentration (EC50) to DTG and infectivity. Although increased DTG resistance via the Q148 pathway and secondary substitutions occurs at low concentrations, a higher starting concentration may reduce or eliminate the development of DTG resistance in this pathway in vitro.
Assuntos
Farmacorresistência Viral/genética , Quinolonas/farmacologia , Raltegravir Potássico/farmacologia , Integrase de HIV/metabolismo , Inibidores de Integrase de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/genética , HIV-1/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacologia , Mutação/genética , Oxazinas , Piperazinas , PiridonasRESUMO
GSK1265744 is a new HIV integrase strand transfer inhibitor (INSTI) engineered to deliver efficient antiviral activity with a once-daily, low-milligram dose that does not require a pharmacokinetic booster. The in vitro antiviral profile and mechanism of action of GSK1265744 were established through integrase enzyme assays, resistance passage experiments, and cellular assays with site-directed molecular (SDM) HIV clones resistant to other classes of anti-HIV-1 agents and earlier INSTIs. GSK1265744 inhibited HIV replication with low or subnanomolar efficacy and with a selectivity index of at least 22,000 under the same culture conditions. The protein-adjusted half-maximal inhibitory concentration (PA-EC50) extrapolated to 100% human serum was 102 nM. When the virus was passaged in the presence of GSK1265744, highly resistant mutants with more than a 10-fold change (FC) in EC50 relative to that of the wild-type were not observed for up to 112 days of culture. GSK1265744 demonstrated activity against SDM clones containing the raltegravir (RAL)-resistant Y143R, Q148K, N155H, and G140S/Q148H signature variants (FC less than 6.1), while these mutants had a high FC in the EC50 for RAL (11 to >130). Either additive or synergistic effects were observed when GSK1265744 was tested in combination with representative anti-HIV agents, and no antagonistic effects were seen. These findings demonstrate that, similar to dolutegravir, GSK1265744 is differentiated as a new INSTI, having a markedly distinct resistance profile compared with earlier INSTIs, RAL, and elvitegravir (EVG). The collective data set supports further clinical development of GSK1265744.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Inibidores de Integrase de HIV/uso terapêutico , HIV-1/efeitos dos fármacos , Piridonas/uso terapêutico , Linhagem Celular , Farmacorresistência Viral/genética , Infecções por HIV/virologia , Integrase de HIV/efeitos dos fármacos , HIV-1/genética , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Humanos , Testes de Sensibilidade Microbiana , Oxazinas , Piperazinas , Quinolonas/uso terapêutico , Raltegravir Potássico/uso terapêutico , Replicação Viral/efeitos dos fármacosRESUMO
Stem and progenitor cells are currently being investigated for their applicability in cell-based therapy for periodontal tissue regeneration. We recently demonstrated that the transplantation of adipose tissue-derived multi-lineage progenitor cells (ADMPCs) enhances periodontal tissue regeneration in beagle dogs. However, the molecular mechanisms by which transplanted ADMPCs induce periodontal tissue regeneration remain to be elucidated. In this study, trophic factors released by ADMPCs were examined for their paracrine effects on human periodontal ligament cell (HPDL) function. ADMPC conditioned medium (ADMPC-CM) up-regulated osteoblastic gene expression, alkaline phosphatase activity and calcified nodule formation in HPDLs, but did not significantly affect their proliferative response. ADMPCs secreted a number of growth factors, including insulin-like growth factor binding protein 6 (IGFBP6), hepatocyte growth factor and vascular endothelial growth factor. Among these, IGFBP6 was most highly expressed. Interestingly, the positive effects of ADMPC-CM on HPDL differentiation were significantly suppressed by transfecting ADMPCs with IGFBP6 siRNA. Our results suggest that ADMPCs transplanted into a defect in periodontal tissue release trophic factors that can stimulate the differentiation of HPDLs to mineralized tissue-forming cells, such as osteoblasts and cementoblasts. IGFBP6 may play crucial roles in ADMPC-induced periodontal regeneration.
Assuntos
Tecido Adiposo/citologia , Meios de Cultivo Condicionados/farmacologia , Cemento Dentário/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Células-Tronco/citologia , Tecido Adiposo/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Células Cultivadas , Cemento Dentário/citologia , Cemento Dentário/metabolismo , Regulação da Expressão Gênica , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/antagonistas & inibidores , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: New cementum forms from existing cementum during periodontal tissue regeneration, indicating that cementoblasts may interact with progenitor cells in the periodontal ligament to enhance cementogenesis. However, the molecular mechanisms of this process are currently unknown. This study aims to clarify the role of cell-cell interactions between cementoblasts and periodontal ligament cells in differentiation into cementoblasts. METHODS: To analyze the role of human cementoblast-like cells (HCEMs) on human periodontal ligament cells (HPDLs), we mixed cell suspensions of enhanced green fluorescent protein-tagged HPDLs and HCEMs, and then seeded and cultured them in single wells (direct co-cultures). We sorted co-cultured HPDLs and analyzed their characteristics, including the expression of cementum-related genes. In addition, we cultured HPDLs and HCEMs in a non-contact environment using a culture system composed of an upper insert and a lower well separated by a semi-permeable membrane (indirect co-cultures), and similar analysis was performed. Gene expression of integrin-binding sialoprotein (IBSP) in cementoblasts was confirmed in mouse periodontal tissues. We also investigated the effect of Wingless-type (Wnt) signaling on the differentiation of HPDLs into cementoblasts. RESULTS: Direct co-culture of HPDLs with HCEMs significantly upregulated the expression of cementoblast-related genes in HPDLs, whereas indirect co-culture exerted no effect. Wnt3A stimulation significantly upregulated IBSP expression in HPDLs, whereas inhibition of canonical Wnt signaling suppressed the effects of co-culture. CONCLUSION: Our results suggest that direct cell interactions with cementoblasts promote periodontal ligament cell differentiation into cementoblasts. Juxtacrine signaling via the canonical Wnt pathway plays a role in this interaction.
Assuntos
Cemento Dentário , Ligamento Periodontal , Camundongos , Humanos , Animais , Cementogênese , Periodonto , Transdução de Sinais , Diferenciação Celular , Sialoproteína de Ligação à Integrina/metabolismo , Sialoproteína de Ligação à Integrina/farmacologiaRESUMO
We have developed an autologous transplantation method using adipose tissue-derived multi-lineage progenitor cells (ADMPCs) as a method of periodontal tissue regeneration that can be adapted to severe periodontal disease. Our previous clinical study confirmed the safety of autologous transplantation of ADMPCs and demonstrated its usefulness in the treatment of severe periodontal disease. However, in the same clinical study, we found that the fibrin gel used as the scaffold material might have caused gingival recession and impaired tissue regeneration in some patients. Carbonate apatite has a high space-making capacity and has been approved in Japan for periodontal tissue regeneration. In this study, we selected carbonate apatite as a candidate scaffold material for ADMPCs and conducted an in vitro examination of its effect on the cellular function of ADMPCs. We further performed autologous ADMPC transplantation with carbonate apatite as the scaffold material in a model of one-wall bone defects in beagles and then analyzed the effect on periodontal tissue regeneration. The findings showed that carbonate apatite did not affect the cell morphology of ADMPCs and that it promoted proliferation. Moreover, no effect on secretor factor transcription was found. The results of the in vivo analysis confirmed the space-making capacity of carbonate apatite, and the acquisition of significant new attachment was observed in the group involving ADMPC transplantation with carbonate apatite compared with the group involving carbonate apatite application alone. Our results demonstrate the usefulness of carbonate apatite as a scaffold material for ADMPC transplantation.
Assuntos
Regeneração Óssea , Doenças Periodontais , Humanos , Animais , Cães , Células-Tronco , Tecido Adiposo , Transplante Autólogo , Doenças Periodontais/terapia , Regeneração Tecidual Guiada Periodontal/métodosRESUMO
Periodontitis is a chronic inflammatory disease that destroys tooth-supporting periodontal tissue. Current periodontal regenerative therapies have unsatisfactory efficacy; therefore, periodontal tissue engineering might be established by developing new cell-based therapies. In this study, we evaluated the safety and efficacy of adipose tissue-derived multi-lineage progenitor cells (ADMPC) autologous transplantation for periodontal tissue regeneration in humans. We conducted an open-label, single-arm exploratory phase I clinical study in which 12 periodontitis patients were transplanted with autologous ADMPCs isolated from subcutaneous adipose tissue. Each patient underwent flap surgery during which autologous ADMPCs were transplanted into the bone defect with a fibrin carrier material. Up to 36 weeks after transplantation, we performed a variety of clinical examinations including periodontal tissue inspection and standardized dental radiographic analysis. A 36-week follow-up demonstrated no severe transplantation-related adverse events in any cases. ADMPC transplantation reduced the probing pocket depth, improved the clinical attachment level, and induced neogenesis of alveolar bone. Therapeutic efficiency was observed in 2- or 3-walled vertical bone defects as well as more severe periodontal bone defects. These results suggest that autologous ADMPC transplantation might be an applicable therapy for severe periodontitis by inducing periodontal regeneration.
Assuntos
Perda do Osso Alveolar , Periodontite , Tecido Adiposo/cirurgia , Perda do Osso Alveolar/cirurgia , Regeneração Óssea , Seguimentos , Regeneração Tecidual Guiada Periodontal/métodos , Humanos , Periodontite/cirurgia , Células-Tronco , Transplante AutólogoRESUMO
Human immunodeficiency virus type-1 (HIV-1) protease is essential for viral propagation, and its inhibitors are key anti-HIV-1 drug candidates. In this study, we discovered a novel HIV-1 protease inhibitor (compound 16) with potent antiviral activity and oral bioavailability using a structure-based drug design approach via X-ray crystal structure analysis and improved metabolic stability, starting from hit macrocyclic peptides identified by mRNA display against HIV-1 protease. We found that the improvement of the proteolytic stability of macrocyclic peptides by introducing a methyl group to the α-position of amino acid is crucial to exhibit strong antiviral activity. In addition, macrocyclic peptides, which have moderate metabolic stability and solubility in solutions containing taurocholic acid, exhibited desirable plasma total clearance and oral bioavailability. These approaches may contribute to the successful discovery and development of orally bioavailable peptide drugs.
RESUMO
A novel strategy for lead identification that we have dubbed the "Pocket-to-Lead" strategy is demonstrated using HIV-1 protease as a model target. Sometimes, it is difficult to obtain hit compounds because of the difficulties in satisfying the complex pharmacophoric features. In this study, a virtual fragment hit which does not match all of the pharmacophore features but has key interactions and vectors that could grow into remaining pharmacophore features was optimized in silico. The designed compound 9 demonstrated weak but evident inhibitory activity (IC50 = 54 µM), and the design concept was proven by the co-crystal structure. Then, structure-based drug design promptly gave compound 14 (IC50 = 0.0071 µM, EC50 = 0.86 µM), an almost 10,000-fold improvement in activity from 9. The structure of the designed molecules proved to be novel with high synthetic feasibility, indicating the usefulness of this strategy to tackle tough targets with complex pharmacophore.
Assuntos
Inibidores da Protease de HIV , HIV-1 , Desenho de Fármacos , Protease de HIV/metabolismo , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , HIV-1/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Inibidores de ProteasesRESUMO
S/GSK1349572 is a next-generation HIV integrase (IN) inhibitor designed to deliver potent antiviral activity with a low-milligram once-daily dose requiring no pharmacokinetic (PK) booster. In addition, S/GSK1349572 demonstrates activity against clinically relevant IN mutant viruses and has potential for a high genetic barrier to resistance. S/GSK1349572 is a two-metal-binding HIV integrase strand transfer inhibitor whose mechanism of action was established through in vitro integrase enzyme assays, resistance passage experiments, activity against viral strains resistant to other classes of anti-HIV agents, and mechanistic cellular assays. In a variety of cellular antiviral assays, S/GSK1349572 inhibited HIV replication with low-nanomolar or subnanomolar potency and with a selectivity index of 9,400. The protein-adjusted half-maximal effective concentration (PA-EC(50)) extrapolated to 100% human serum was 38 nM. When virus was passaged in the presence of S/GSK1349572, highly resistant mutants were not selected, but mutations that effected a low fold change (FC) in the EC(50) (up to 4.1 fold) were identified in the vicinity of the integrase active site. S/GSK1349572 demonstrated activity against site-directed molecular clones containing the raltegravir-resistant signature mutations Y143R, Q148K, N155H, and G140S/Q148H (FCs, 1.4, 1.1, 1.2, and 2.6, respectively), while these mutants led to a high FC in the EC(50) of raltegravir (11- to >130-fold). Either additive or synergistic effects were observed when S/GSK1349572 was tested in combination with representative approved antiretroviral agents; no antagonistic effects were seen. These findings demonstrate that S/GSK1349572 would be classified as a next-generation drug in the integrase inhibitor class, with a resistance profile markedly different from that of first-generation integrase inhibitors.
Assuntos
Fármacos Anti-HIV/farmacologia , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Linhagem Celular , Linhagem Celular Transformada , Farmacorresistência Viral , Inibidores de Integrase de HIV/síntese química , Inibidores de Integrase de HIV/química , HIV-1/enzimologia , HIV-1/fisiologia , Humanos , Testes de Sensibilidade Microbiana/métodos , Mutação , Naftiridinas/síntese química , Naftiridinas/química , Naftiridinas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/virologia , Integração Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Cellular responses to hypoxia regulate various biological events, including angiogenesis and extracellular matrix metabolism. Collagen is a major component of the extracellular matrix in periodontal tissues and its coordinated production is essential for tissue homeostasis. In this study, we investigated the effects of hypoxia on collagen production in human gingival fibroblasts (HGFs) and human periodontal ligament cells (HPDLs). METHODS: HGFs and HPDLs were cultured under either normoxic (20% O2 ) or hypoxic (1% O2 ) conditions. Nuclear expression of hypoxia-inducible factor-1α (HIF-1α) was determined by western blotting. Peri-cellular expression of type I collagen was examined by immunocytochemistry analysis. Synthesis of type I collagen was evaluated by measuring the concentration of procollagen type I C-peptide (PIP) in culture supernatant using enzyme-linked immunosorbent assay. Expression of collagen hydroxylase enzymes prolyl 4-hydroxylase alpha polypeptide 1 (P4HA1) and 2-oxoglutarate 5-dioxygenase 2 (PLOD2) was determined by RT-qPCR and western blotting. The roles of these enzymes were analyzed using siRNA transfection. RESULTS: Cultivation under hypoxic conditions stimulated type I collagen production via HIF-1α in both cell types. Interestingly, hypoxic conditions did not affect collagen 1a1 or 1a2 gene expression but upregulated that of P4HA1 and PLOD2. Additionally, suppressing P4HA1 significantly decreased the levels of hypoxia-induced procollagen type I C-peptide, a product of stable triple helical collagen, in the supernatant. In contrast, PLOD2 suppression decreased cross-linked collagen expression in the pericellular region. CONCLUSION: Our results suggest that hypoxia activates collagen synthesis in HGFs and HPDLs by upregulating hydroxylases P4HA1 and PLOD2 in an HIF-1α-dependent manner.
Assuntos
Fibroblastos , Ligamento Periodontal , Hipóxia Celular , Células Cultivadas , Colágeno , Humanos , Hidroxilação , Hipóxia , Subunidade alfa do Fator 1 Induzível por HipóxiaRESUMO
SKG mice, a newly established model of rheumatoid arthritis (RA), spontaneously develop autoimmune arthritis accompanying extra-articular manifestations, such as interstitial pneumonitis. To examine possible roles of T cells for mediating this systemic autoimmunity, we generated T cell clones from arthritic joints of SKG mice. Two distinct CD8(+) clones were established and both showed in vitro autoreactivity by killing syngeneic synovial cells and a variety of MHC-matched cell lines. Transfer of each clone to histocompatible athymic nude mice elicited joint swelling and histologically evident synovitis accompanying the destruction of adjacent cartilage and bone. Notably, the transfer also produced diffuse severe interstitial pneumonitis. Clone-specific TCR gene messages in the inflamed joints and lungs of the recipients gradually diminished, becoming hardly detectable in 6-11 months; yet, arthritis and pneumonitis continued to progress. Thus, the same CD8(+) T cell clones from arthritic lesions of SKG mice can elicit both synovitis and pneumonitis, which chronically progress and apparently become less T cell dependent in a later phase. The results provide clues to our understanding of how self-reactive T cells cause both articular and extra-articular lesions in RA as a systemic autoimmune disease.
Assuntos
Artrite Experimental/imunologia , Linfócitos T CD8-Positivos/imunologia , Pneumonia/imunologia , Transferência Adotiva , Animais , Artrite Experimental/patologia , Artrite Experimental/fisiopatologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Artrite Reumatoide/fisiopatologia , Autoantígenos/imunologia , Autoimunidade/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Células Clonais/imunologia , Células Clonais/metabolismo , Células Clonais/patologia , Citotoxicidade Imunológica , Modelos Animais de Doenças , Progressão da Doença , Humanos , Articulações/imunologia , Articulações/patologia , Camundongos , Pneumonia/patologia , Pneumonia/fisiopatologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologiaRESUMO
Osteoblastic mineralization occurs during the early stages of bone formation. During this mineralization, hydroxyapatite (HA), a major component of bone, is synthesized, generating hard tissue. Many of the mechanisms driving biomineralization remain unclear because the traditional biochemical assays used to investigate them are destructive techniques incompatible with viable cells. To determine the temporal changes in mineralization-related biomolecules at mineralization spots, we performed time-lapse Raman imaging of mouse osteoblasts at a subcellular resolution throughout the mineralization process. Raman imaging enabled us to analyze the dynamics of the related biomolecules at mineralization spots throughout the entire process of mineralization. Here, we stimulated KUSA-A1 cells to differentiate into osteoblasts and conducted time-lapse Raman imaging on them every 4 hours for 24 hours, beginning 5 days after the stimulation. The HA and cytochrome c Raman bands were used as markers for osteoblastic mineralization and apoptosis. From the Raman images successfully acquired throughout the mineralization process, we found that ß-carotene acts as a biomarker that indicates the initiation of osteoblastic mineralization. A fluctuation of cytochrome c concentration, which indicates cell apoptosis, was also observed during mineralization. We expect time-lapse Raman imaging to help us to further elucidate osteoblastic mineralization mechanisms that have previously been unobservable.
Assuntos
Calcificação Fisiológica/fisiologia , Microscopia/métodos , Osteoblastos/citologia , Osteoblastos/fisiologia , Análise Espectral Raman/métodos , Imagem com Lapso de Tempo/métodos , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Citocromos c/metabolismo , Camundongos , beta Caroteno/metabolismoRESUMO
Specificities of cytotoxic T lymphocyte (CTL) effector cells induced in BALB/c mouse by immunization with the single modified CTL epitope peptide derived from NS3 of dengue virus types 1 and 3, or that of dengue virus types 2 and 4 were examined. The effector cells included CTLs specific for the epitope peptide used for immunization and those cross-reactive to epitope peptides of other flaviviruses. A CTL clone, 2F7, was established from the effector cells. The clone 2F7 was specific for the epitope peptide used for immunization. Recognition by the effector cells was remarkably impaired by amino acid substitutions at positions 3, 5, and 6 of the epitope peptides. These results indicate that immunization with a single CTL epitope peptide of dengue viruses induces serotype-specific CTLs as well as CTLs cross-reactive to the other flaviviruses, and that the a.a. residues at positions 3, 5, and 6 are critical for cross-reaction.
Assuntos
Vírus da Dengue/imunologia , Epitopos de Linfócito T/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas não Estruturais Virais/imunologia , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/imunologia , Animais , Reações Cruzadas , Epitopos de Linfócito T/genética , Feminino , Camundongos , Camundongos Endogâmicos BALB C , RNA Helicases/genética , RNA Helicases/imunologia , Serina Endopeptidases/genética , Serina Endopeptidases/imunologia , Proteínas não Estruturais Virais/genéticaRESUMO
Passage of HIV-1 in the presence of integrase inhibitors (INIs) generates resistant viruses that have mutations in the integrase region. Integrase-resistant mutations Q148K and Q148R were identified as primary mutations with the passage of HIV-1 IIIB in the presence of INIs S-1360 or S/GSK-364735, respectively. Secondary amino acid substitutions E138K or G140S were observed when passage with INI was continued. The role of these mutations was investigated with molecular clones. Relative to Q148K alone, Q148K/E138K had 2- and >6-fold increases in resistance to S-1360 and S/GSK-364735, respectively, and the double mutant had slightly better infectivity and replication kinetics. In contrast, Q148K/G140S and Q148R/E138K had nearly equivalent or slightly reduced fold resistance to the INI compared with their respective Q148 primary mutants, and had increases in infectivity and replication kinetics. Recovery of these surrogates of viral fitness coincided with the recovery of integration efficiency of viral DNA into the host cell chromosome for these double mutants. These data show that recovery of viral integration efficiency can be an important factor for the emergence and maintenance of INI-resistant mutations.
Assuntos
Fármacos Anti-HIV/farmacologia , Integrase de HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Mutação de Sentido Incorreto , Replicação Viral/efeitos dos fármacos , Furanos/farmacologia , Integrase de HIV/genética , HIV-1/genética , Humanos , Células Jurkat , Triazóis/farmacologiaRESUMO
Resistance passage studies were conducted with five INIs (integrase inhibitors) that have been tested in clinical trials to date: a new naphthyridinone-type INI S/GSK-364735, raltegravir, elvitegravir, L-870,810 and S-1360. In establishing the passage system and starting from concentrations several fold above the EC(50) value, resistance mutations against S-1360 and related diketoacid-type compounds could be isolated from infected MT-2 cell cultures from day 14 to 28. Q148R and F121Y were the two main pathways of resistance to S/GSK-364735. Q148R/K and N155H, which were found in patients failing raltegravir treatment in Phase IIb studies, were observed during passage with raltegravir with this method. The fold resistance of 40 mutant molecular clones versus wild type virus was compared with these five INIs. The overall resistance pattern of S/GSK-364735 was similar to that of raltegravir and other INIs. However, different fold resistances of particular mutations were noted among different INIs, reflecting a potential to develop INIs with distinctly different resistant profiles.
Assuntos
Farmacorresistência Viral , Infecções por HIV/virologia , Inibidores de Integrase de HIV/farmacologia , HIV-1/genética , Mutação , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Infecções por HIV/tratamento farmacológico , Integrase de HIV/genética , Integrase de HIV/metabolismo , Inibidores de Integrase de HIV/uso terapêutico , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/isolamento & purificação , Células HeLa , Humanos , Linfócitos T/virologiaRESUMO
The naphthyridinone GSK364735 potently inhibited recombinant human immunodeficiency virus type 1 (HIV-1) integrase in a strand transfer assay (mean 50% inhibitory concentration +/- standard deviation, 8 +/- 2 nM). As expected based on the structure of the drug, it bound competitively with another two-metal binding inhibitor (Kd [binding constant], 6 +/- 4 nM). In a number of different cellular assays, GSK364735 inhibited HIV replication with potency at nanomolar concentrations (e.g., in peripheral blood mononuclear cells and MT-4 cells, 50% effective concentrations were 1.2 +/- 0.4 and 5 +/- 1 nM, respectively), with selectivity indexes of antiviral activity versus in-assay cytotoxicity of at least 2,200. When human serum was added, the antiviral potency decreased (e.g., a 35-fold decrease in the presence of 100% human serum was calculated by extrapolation from the results of the MT-4 cell assay). In cellular assays, GSK364735 blocked viral DNA integration, with a concomitant increase in two-long-terminal-repeat circles. As expected, this integrase inhibitor was equally active against wild-type viruses and mutant viruses resistant to approved drugs targeting either reverse transcriptase or protease. In contrast, some but not all viruses resistant to other integrase inhibitors were resistant to GSK364735. When virus was passaged in the presence of the inhibitor, we identified resistance mutations within the integrase active site that were the same as or similar to mutations arising in response to other two-metal binding inhibitors. Finally, either additive or synergistic effects were observed when GSK364735 was tested in combination with approved antiretrovirals (i.e., no antagonistic effects were seen). Thus, based on all the data, GSK364735 exerted potent antiviral activity through the inhibition of viral DNA integration by interacting at the two-metal binding site within the catalytic center of HIV integrase.