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1.
PLoS One ; 14(8): e0219142, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31393868

RESUMO

BACKGROUND: Additional forms of pre-exposure prophylaxis are needed to prevent HIV-1 infection. 3BNC117 and 10-1074 are broadly neutralizing anti-HIV-1 antibodies that target non-overlapping epitopes on the HIV-1 envelope. We investigated the safety, tolerability, pharmacokinetics, and immunogenicity of the intravenous administration of the combination of 3BNC117 and 10-1074 in healthy adults. METHODS: This randomized, double-blind, placebo-controlled, single center, phase 1 study enrolled healthy adults aged 18-65 years to receive one infusion of 3BNC117 immediately followed by 10-1074 at 10 mg/kg, three infusions of 3BNC117 followed by 10-1074 at 3 mg/kg or 10 mg/kg every 8 weeks, or placebo infusions. The primary outcomes were safety and pharmacokinetics. This trial is registered with ClinicalTrials.gov, number NCT02824536. FINDINGS: Twenty-four participants were enrolled in a 3:1 ratio to receive the study products or placebo. The combination of 3BNC117 and 10-1074 was safe and generally well tolerated. There were no serious adverse events considered related to the infusions. The mean elimination half-lives of 3BNC117 and 10-1074 were 16.4 ± 4.6 days and 23.0 ± 5.4 days, respectively, similar to what was observed in previous studies in which each antibody was administered alone. Anti-drug antibody responses were rare and without evidence of related adverse events or impact on elimination kinetics. INTERPRETATION: Single and repeated doses of the combination of 3BNC117 and 10-1074 were well tolerated in healthy adults. These data support the further development of the combination of 3BNC117 and 10-1074 as a long-acting injectable form of pre-exposure prophylaxis for the prevention of HIV-1 infection.

2.
Nature ; 561(7724): 479-484, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30258136

RESUMO

Individuals infected with HIV-1 require lifelong antiretroviral therapy, because interruption of treatment leads to rapid rebound viraemia. Here we report on a phase 1b clinical trial in which a combination of 3BNC117 and 10-1074, two potent monoclonal anti-HIV-1 broadly neutralizing antibodies that target independent sites on the HIV-1 envelope spike, was administered during analytical treatment interruption. Participants received three infusions of 30 mg kg-1 of each antibody at 0, 3 and 6 weeks. Infusions of the two antibodies were generally well-tolerated. The nine enrolled individuals with antibody-sensitive latent viral reservoirs maintained suppression for between 15 and more than 30 weeks (median of 21 weeks), and none developed viruses that were resistant to both antibodies. We conclude that the combination of the anti-HIV-1 monoclonal antibodies 3BNC117 and 10-1074 can maintain long-term suppression in the absence of antiretroviral therapy in individuals with antibody-sensitive viral reservoirs.

3.
Nat Med ; 24(11): 1701-1707, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30258217

RESUMO

Monotherapy of HIV-1 infection with single antiretroviral agents is ineffective because error-prone HIV-1 replication leads to the production of drug-resistant viral variants1,2. Combinations of drugs can establish long-term control, however, antiretroviral therapy (ART) requires daily dosing, can cause side effects and does not eradicate the infection3,4. Although anti-HIV-1 antibodies constitute a potential alternative to ART5,6, treatment of viremic individuals with a single antibody also results in emergence of resistant viral variants7-9. Moreover, combinations of first-generation anti-HIV-1 broadly neutralizing antibodies (bNAbs) had little measurable effect on the infection10-12. Here we report on a phase 1b clinical trial ( NCT02825797 ) in which two potent bNAbs, 3BNC11713 and 10-107414, were administered in combination to seven HIV-1 viremic individuals. Infusions of 30 mg kg-1 of each of the antibodies were well-tolerated. In the four individuals with dual antibody-sensitive viruses, immunotherapy resulted in an average reduction in HIV-1 viral load of 2.05 log10 copies per ml that remained significantly reduced for three months following the first of up to three infusions. In addition, none of these individuals developed resistance to both antibodies. Larger studies will be necessary to confirm the efficacy of antibody combinations in reducing HIV-1 viremia and limiting the emergence of resistant viral variants.

4.
J Exp Med ; 215(9): 2311-2324, 2018 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-30072495

RESUMO

A clinical trial was performed to evaluate 3BNC117, a potent anti-HIV-1 antibody, in infected individuals during suppressive antiretroviral therapy and subsequent analytical treatment interruption (ATI). The circulating reservoir was evaluated by quantitative and qualitative viral outgrowth assay (Q2VOA) at entry and after 6 mo. There were no significant quantitative changes in the size of the reservoir before ATI, and the composition of circulating reservoir clones varied in a manner that did not correlate with 3BNC117 sensitivity. 3BNC117 binding site amino acid variants found in rebound viruses preexisted in the latent reservoir. However, only 3 of 217 rebound viruses were identical to 868 latent viruses isolated by Q2VOA and near full-length sequencing. Instead, 63% of the rebound viruses appeared to be recombinants, even in individuals with 3BNC117-resistant reservoir viruses. In conclusion, viruses emerging during ATI in individuals treated with 3BNC117 are not the dominant species found in the circulating latent reservoir, but frequently appear to represent recombinants of latent viruses.

5.
J Virol ; 92(5)2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29237833

RESUMO

Recently discovered broadly neutralizing antibodies (bNAbs) against HIV-1 demonstrate extensive breadth and potency against diverse HIV-1 strains and represent a promising approach for the treatment and prevention of HIV-1 infection. The breadth and potency of these antibodies have primarily been evaluated by using panels of HIV-1 Env-pseudotyped viruses produced in 293T cells expressing molecularly cloned Env proteins. Here we report on the ability of five bNAbs currently in clinical development to neutralize circulating primary HIV-1 isolates derived from peripheral blood mononuclear cells (PBMCs) and compare the results to those obtained with the pseudovirus panels used to characterize the bNAbs. The five bNAbs demonstrated significantly less breadth and potency against clinical isolates produced in PBMCs than against Env-pseudotyped viruses. The magnitude of this difference in neutralizing activity varied, depending on the antibody epitope. Glycan-targeting antibodies showed differences of only 3- to 4-fold, while antibody 10E8, which targets the membrane-proximal external region, showed a nearly 100-fold decrease in activity between published Env-pseudotyped virus panels and PBMC-derived primary isolates. Utilizing clonal PBMC-derived primary isolates and molecular clones, we determined that the observed discrepancy in bNAb performance is due to the increased sensitivity to neutralization exhibited by 293T-produced Env-pseudotyped viruses. We also found that while full-length molecularly cloned viruses produced in 293T cells exhibit greater sensitivity to neutralization than PBMC-derived viruses do, Env-pseudotyped viruses produced in 293T cells generally exhibit even greater sensitivity to neutralization. As the clinical development of bNAbs progresses, it will be critical to determine the relevance of each of these in vitro neutralization assays to in vivo antibody performance.IMPORTANCE Novel therapeutic and preventive strategies are needed to contain the HIV-1 epidemic. Antibodies with exceptional neutralizing activity against HIV-1 may provide several advantages to traditional HIV drugs, including an improved side-effect profile, a reduced dosing frequency, and immune enhancement. The activity of these antibodies has been established in vitro by utilizing HIV-1 Env-pseudotyped viruses derived from circulating viruses but produced in 293T cells by pairing Env proteins with a backbone vector. We tested PBMC-produced circulating viruses against five anti-HIV-1 antibodies currently in clinical development. We found that the activity of these antibodies against PBMC isolates is significantly less than that against 293T Env-pseudotyped viruses. This decline varied among the antibodies tested, with some demonstrating moderate reductions in activity and others showing an almost 100-fold reduction. As the development of these antibodies progresses, it will be critical to determine how the results of different in vitro tests correspond to performance in the clinic.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Leucócitos Mononucleares/imunologia , Adolescente , Adulto , Idoso , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Feminino , Células HEK293 , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , Humanos , Concentração Inibidora 50 , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , New York , Adulto Jovem , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia
6.
Nat Med ; 23(2): 185-191, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28092665

RESUMO

Monoclonal antibody 10-1074 targets the V3 glycan supersite on the HIV-1 envelope (Env) protein. It is among the most potent anti-HIV-1 neutralizing antibodies isolated so far. Here we report on its safety and activity in 33 individuals who received a single intravenous infusion of the antibody. 10-1074 was well tolerated and had a half-life of 24.0 d in participants without HIV-1 infection and 12.8 d in individuals with HIV-1 infection. Thirteen individuals with viremia received the highest dose of 30 mg/kg 10-1074. Eleven of these participants were 10-1074-sensitive and showed a rapid decline in viremia by a mean of 1.52 log10 copies/ml. Virologic analysis revealed the emergence of multiple independent 10-1074-resistant viruses in the first weeks after infusion. Emerging escape variants were generally resistant to the related V3-specific antibody PGT121, but remained sensitive to antibodies targeting nonoverlapping epitopes, such as the anti-CD4-binding-site antibodies 3BNC117 and VRC01. The results demonstrate the safety and activity of 10-1074 in humans and support the idea that antibodies targeting the V3 glycan supersite might be useful for the treatment and prevention of HIV-1 infection.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Anti-HIV/administração & dosagem , Infecções por HIV/tratamento farmacológico , Viremia/tratamento farmacológico , Adulto , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Neutralizantes/farmacologia , Contagem de Linfócito CD4 , Células CHO , Cricetulus , Feminino , Anticorpos Anti-HIV/farmacologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/sangue , HIV-1/genética , HIV-1/imunologia , Humanos , Imunoglobulina G/administração & dosagem , Imunoglobulina G/farmacologia , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/imunologia , RNA Viral/sangue , Proteínas Recombinantes , Carga Viral , Viremia/sangue , Adulto Jovem , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética
7.
Nature ; 535(7613): 556-60, 2016 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-27338952

RESUMO

Interruption of combination antiretroviral therapy in HIV-1-infected individuals leads to rapid viral rebound. Here we report the results of a phase IIa open label clinical trial evaluating 3BNC117,a broad and potent neutralizing antibody against the CD4 binding site of the HIV-1 Env protein, during analytical treatment interruption in 13 HIV-1-infected individuals. Participants with 3BNC117-sensitive virus outgrowth cultures were enrolled. Results show that two or four 30 mg kg(-1) 3BNC117 infusions,separated by 3 or 2 weeks, respectively, are generally well tolerated.Infusions are associated with a delay in viral rebound of 5-9 weeks after two infusions, and up to 19 weeks after four infusions, or an average of 6.7 and 9.9 weeks, respectively, compared with 2.6 weeks for historical controls (P < 0.00001). Rebound viruses arise predominantly from a single provirus. In most individuals,emerging viruses show increased resistance, indicating escape.However, 30% of participants remained suppressed until antibody concentrations waned below 20 µg ml(-1), and the viruses emerging in all but one of these individuals showed no apparent resistance to 3BCN117, suggesting failure to escape over a period of 9-19 weeks.We conclude that the administration of 3BNC117 exerts strong selective pressure on HIV-1 emerging from latent reservoirs during analytical treatment interruption in humans.


Assuntos
Fármacos Anti-HIV/administração & dosagem , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Adolescente , Adulto , Idoso , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/uso terapêutico , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/imunologia , Antígenos CD4/metabolismo , Reservatórios de Doenças/virologia , Esquema de Medicação , Feminino , Anticorpos Anti-HIV/administração & dosagem , Anticorpos Anti-HIV/uso terapêutico , Proteína gp160 do Envelope de HIV/antagonistas & inibidores , Proteína gp160 do Envelope de HIV/química , Proteína gp160 do Envelope de HIV/imunologia , Proteína gp160 do Envelope de HIV/metabolismo , Infecções por HIV/imunologia , HIV-1/efeitos dos fármacos , Estudo Historicamente Controlado , Humanos , Masculino , Pessoa de Meia-Idade , Provírus/efeitos dos fármacos , Provírus/crescimento & desenvolvimento , Provírus/imunologia , Fatores de Tempo , Distribuição Tecidual , Carga Viral/efeitos dos fármacos , Carga Viral/imunologia , Adulto Jovem
9.
Nature ; 522(7557): 487-91, 2015 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-25855300

RESUMO

HIV-1 immunotherapy with a combination of first generation monoclonal antibodies was largely ineffective in pre-clinical and clinical settings and was therefore abandoned. However, recently developed single-cell-based antibody cloning methods have uncovered a new generation of far more potent broadly neutralizing antibodies to HIV-1 (refs 4, 5). These antibodies can prevent infection and suppress viraemia in humanized mice and nonhuman primates, but their potential for human HIV-1 immunotherapy has not been evaluated. Here we report the results of a first-in-man dose escalation phase 1 clinical trial of 3BNC117, a potent human CD4 binding site antibody, in uninfected and HIV-1-infected individuals. 3BNC117 infusion was well tolerated and demonstrated favourable pharmacokinetics. A single 30 mg kg(-1) infusion of 3BNC117 reduced the viral load in HIV-1-infected individuals by 0.8-2.5 log10 and viraemia remained significantly reduced for 28 days. Emergence of resistant viral strains was variable, with some individuals remaining sensitive to 3BNC117 for a period of 28 days. We conclude that, as a single agent, 3BNC117 is safe and effective in reducing HIV-1 viraemia, and that immunotherapy should be explored as a new modality for HIV-1 prevention, therapy and cure.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/terapia , HIV-1/imunologia , Carga Viral/imunologia , Viremia/terapia , Adulto , Sequência de Aminoácidos , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/efeitos adversos , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , Sítios de Ligação , Antígenos CD4/metabolismo , Estudos de Casos e Controles , Evolução Molecular , Feminino , Anticorpos Anti-HIV/administração & dosagem , Anticorpos Anti-HIV/efeitos adversos , Anticorpos Anti-HIV/farmacologia , Anticorpos Anti-HIV/uso terapêutico , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/química , HIV-1/efeitos dos fármacos , Humanos , Imunização Passiva/métodos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fatores de Tempo , Carga Viral/efeitos dos fármacos , Viremia/imunologia , Viremia/virologia , Adulto Jovem
10.
J Exp Med ; 208(12): 2357-66, 2011 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-22065672

RESUMO

Adjuvants are critical for the success of vaccines. Agonists of microbial pattern recognition receptors (PRRs) are promising new adjuvant candidates. A mechanism through which adjuvants enhance immune responses is to stimulate innate immunity. We studied the innate immune response in humans to synthetic double-stranded RNA (polyinosinic:polycytidylic acid [poly IC] stabilized with poly-L-lysine [poly ICLC]), an agonist for toll-like receptor (TLR) 3, and the cytosolic RNA helicase MDA-5. Transcriptional analysis of blood samples from eight volunteers, after subcutaneous administration of poly ICLC, showed up-regulation of genes involved in multiple innate immune pathways in all subjects, including interferon (IFN) and inflammasome signaling. Blocking type I IFN receptor ex vivo significantly dampened the response to poly IC. Comparative transcriptional analysis showed that several innate immune pathways were similarly induced in volunteers immunized with the highly efficacious yellow fever vaccine. Therefore, a chemically defined PRR agonist like poly ICLC can be a reliable and authentic microbial mimic for inducing innate immune responses in humans.


Assuntos
Adjuvantes Imunológicos/farmacologia , Carboximetilcelulose Sódica/análogos & derivados , Regulação da Expressão Gênica/imunologia , Imunidade Inata/efeitos dos fármacos , Poli I-C/imunologia , Poli I-C/farmacologia , Polilisina/análogos & derivados , Transdução de Sinais/imunologia , Adjuvantes Imunológicos/administração & dosagem , Carboximetilcelulose Sódica/administração & dosagem , Carboximetilcelulose Sódica/farmacologia , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamassomos/metabolismo , Injeções Subcutâneas , Interferons/metabolismo , Análise em Microsséries , Poli I-C/administração & dosagem , Polilisina/administração & dosagem , Polilisina/imunologia , Polilisina/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Vacinas Virais/imunologia
11.
Eur J Immunol ; 40(1): 36-46, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19830741

RESUMO

DC present exogenous proteins to MHC class I-restricted CD8+ T cells. This function does not require endogenous antigen synthesis within DC, providing the potential to elicit CD8+ T-cell responses to immune complexes, inactivated microbes, dying cells, and proteins such as OVA. In mice, the CD8+ or DEC-205+ DC are specialized for cross-presentation, and this subset can be increased 10-fold in numbers following Fms-like tyrosine kinase 3 ligand (Flt3L) treatment in vivo. Therefore, we studied cross-presentation by abundant Flt3L DC using HIV gag protein. When enriched by positive selection with anti-CD11c beads, cells from Flt3L mice are not only more abundant but are also more highly enriched in CD11chigh DC, particularly the DEC-205+ subset. DC cross-present HIV gag to primed CD8+ T cells, but when the antigen is delivered within an antibody to DEC-205 receptor, cross-presentation becomes 100-fold more efficient than non-targeted antigen. This finding requires gag to be engineered into anti-DEC antibody, not just mixed with antibody. Flt3L DC are a valuable tool to study cross-presentation, since their use overcomes the obstacle posed by the low number of cross-presenting DC in the steady state. These findings support future experiments to use Flt3L to enhance presentation of DC-targeted vaccines.


Assuntos
Anticorpos/imunologia , Antígenos CD/imunologia , Células Dendríticas/imunologia , HIV/imunologia , Lectinas Tipo C/imunologia , Proteínas de Membrana/imunologia , Receptores de Superfície Celular/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Cricetinae , Reações Cruzadas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor
12.
J Immunol ; 176(2): 991-8, 2006 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-16393985

RESUMO

The infection of cultured monocyte-derived dendritic cells (DCs) with HIV-1 involves CD4 and CCR5 receptors, while transmission to T cells is enhanced at least in part by the lectin DC-SIGN/CD209. In the present study, we studied BDCA-1+ myeloid DCs isolated directly from human blood. These cells express CD4 and low levels of CCR5 and CXCR4 coreceptors, but not DC-SIGN. The myeloid DCs replicate two R5 viruses, BaL and YU2, and transfer infection to activated T cells. The virus productively infects a small fraction of the blood DCs that fail to mature in culture, as indicated by the maturation markers CD83 and DC-LAMP/CD208, and the expression of high CD86 and MHC class II, in contrast to many noninfected DCs. A greater proportion of BDCA-1+ DCs are infected when the virus is pseudotyped with the vesicular stomatitis envelope VSV-G (5-15%), as compared with the R5 virus (0.3-3.5%), indicating that HIV-1 coreceptors may limit the susceptibility of DCs to become infected, or the endocytic route of viral entry used by HIV/vesicular stomatitis virus enhances infectivity. When infected and noninfected cells are purified by cell sorting, the former uniformly express HIV p24 gag and are virtually inactive as stimulators of the allogeneic MLR, in contrast to potent stimulation by noninfected DCs from the same cultures. These results point to two roles for a small fraction of blood DCs in HIV-1 pathogenesis: to support productive infection and to evade the direct induction of T cell-mediated immunity.


Assuntos
Células Dendríticas/virologia , HIV-1/patogenicidade , Antígenos de Superfície/metabolismo , Diferenciação Celular , Células Dendríticas/classificação , Células Dendríticas/citologia , Células Dendríticas/imunologia , Infecções por HIV/sangue , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/fisiologia , Humanos , Técnicas In Vitro , Teste de Cultura Mista de Linfócitos , Linfócitos T/virologia , Replicação Viral
13.
J Immunol ; 175(7): 4265-73, 2005 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-16177066

RESUMO

The C-type lectin dendritic cell-specific ICAM 3-grabbing nonintegrin (DC-SIGN)/CD209 efficiently binds several pathogens, including HIV-1. DC-SIGN is expressed on monocyte-derived DCs in culture, and importantly, it is able to sequester HIV-1 within cells and facilitate transmission of virus to CD4+ T cells. To investigate DC-SIGN function, we have generated new mAbs. We report in this study that these and prior anti-DC-SIGN mAbs primarily label macrophages in the medullary sinuses of noninflamed human lymph node. In contrast, expression is not detected on most DCs in the T cell area, except for occasional cells. We also noted that IL-4 alone can induce expression of DC-SIGN in CD14+ monocytes and circulating blood DCs. However, blockade of DC-SIGN with Abs and DC-SIGN small interfering RNA did not result in a major reduction in the capacity of these DCs to transfer HIV to T cells, confirming significant DC-SIGN-independent mechanisms. The blocking approaches did reduce HIV-1 transmission by DC-SIGN-transfected cells by >90%. DC-SIGN blockade also did not reduce the ability of DCs to stimulate T cell proliferation in the MLR. These results indicate that DC-SIGN has the potential to contribute to macrophage function in normal human lymph node, and that DCs do not require DC-SIGN to transmit HIV or to initiate T cell responses.


Assuntos
Moléculas de Adesão Celular/metabolismo , Células Dendríticas/imunologia , Lectinas Tipo C/metabolismo , Linfonodos/metabolismo , Macrófagos/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Anticorpos Monoclonais , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Linhagem Celular , Células Clonais , Células Dendríticas/virologia , Cães , HIV-1/imunologia , Humanos , Lectinas Tipo C/antagonistas & inibidores , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Linfonodos/citologia , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , RNA Interferente Pequeno/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Linfócitos T/virologia , Transfecção
14.
J Control Release ; 95(3): 477-88, 2004 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-15023459

RESUMO

Haptides are 19-21mer cell-binding peptides equivalent to sequences on the C-termini of fibrinogen beta chain (Cbeta), gamma chain (preCgamma) and the extended alphaE chain of fibrinogen (CalphaE). In solution, Haptides accumulated in cells by non-saturable kinetics [Exp. Cell Res. 287 (2003) 116]. This study describes Haptide interactions with liposomes and Haptide-mediated liposome uptake by cells. Haptides became incorporated into negatively charged liposomes, changing their zeta potential. Atomic force microscopy and particle sizing by light scattering showed that the liposomes dissolved Haptide nanoparticles and absorbed them from solution. Pre-mixing fluorescent rhodamine-containing liposomes or "stealth" doxorubicin (DOX)-containing liposomes (Doxil) with Cbeta, preCgamma or to a lesser degree CalphaE, significantly enhanced their uptake by fibroblasts and endothelial cells. Confocal microscopy showed Haptide-induced liposome uptake saturated above approximately 40 microM Haptide. Cytotoxicity tests with lower concentrations of Doxil liposomes indicated that premixing with approximately 40 microM Cbeta or preCgamma increased their toxicity by one order of magnitude. It was evident that the liposomes complexed with an amphiphilic Haptide are transduced through cell membranes, probably by a non-receptor-mediated process. These results suggest that Cbeta or pre-Cgamma could be employed to augment the cellular uptake of drugs in liposomal formulations.


Assuntos
Fibrinogênio/análogos & derivados , Fibrinogênio/metabolismo , Lipossomos/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Aorta/citologia , Bovinos , Células Cultivadas , Química Farmacêutica , Doxorrubicina/química , Portadores de Fármacos , Fibrinogênio/química , Fibroblastos/citologia , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Humanos , Lipossomos/administração & dosagem , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Fragmentos de Peptídeos/química , Rodaminas/química
15.
Exp Cell Res ; 287(1): 116-29, 2003 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-12799188

RESUMO

Fibrinogen seems to contribute significantly to cell binding and recruitment into wounds besides its major role in clot formation. We describe 19- to 21-mer cell-binding (haptotactic) peptides from the C-termini of fibrinogen beta-chain (Cbeta), the extended alphaE chain, and near the C-terminal of the gamma-chain. When these peptides were covalently bound to a biologically inert matrix such as Sepharose beads (SB), they elicited beads attachment to cells, mostly of mesenchymal origin (including fibroblasts, endothelial cells, and smooth muscle cells) as well as some transformed cell lines. Based on such haptotactic activity, these peptides were termed "haptides." By contrast, peptides homologous to fibrinogen C-termini alpha- and gamma-chains elicited no such activity. The haptide Cbeta could not block the interaction of fibroblasts with antibodies directed against integrins beta(1), alpha(v), alpha(v)beta(1), alpha(v)beta(3), and alphaIIbeta(3). Moreover, GRGDS peptide could not inhibit enhanced cell binding to SB-Cbeta, as expected from an integrin-mediated process. In soluble form the haptides were accumulated in cells with nonsaturable kinetics without any toxic or proproliferative effects in concentrations up to 80 microM. These findings suggest that the conserved haptidic sequences within fibrin(ogen) can be associated with the adhesion and migration of cells into fibrin clots and may have a significant role in normal wound healing and in various pathological conditions.


Assuntos
Coagulação Sanguínea/fisiologia , Adesão Celular/fisiologia , Epitopos/química , Células Eucarióticas/metabolismo , Fibrinogênio/química , Peptídeos/química , Cicatrização/fisiologia , Animais , Bovinos , Linhagem Celular , Quimiotaxia/fisiologia , Humanos , Camundongos , Estrutura Terciária de Proteína/fisiologia , Ratos , Homologia de Sequência de Aminoácidos
16.
Tissue Eng ; 8(4): 661-72, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12202005

RESUMO

It has been demonstrated that bone marrow (BM)-derived pluripotent stem cells can be incorporated into muscle, bone, nerve, lung, stomach, intestine, and skin. Fibrin-based biodegradable microbeads (FMB) were developed for culturing, in suspension, a high density of cells, mostly of mesenchymal origin. In the current study, FMB were used to isolate and expand mesenchymal progenitor cells from BM of mice and rats. Cells from BM isolated on FMB (FMB-BM cells) were visualized by fluorescent confocal microscopy and quantified by a modified MTS colorimetric assay. Downloading the BM cells from FMB onto plastic induced their differentiation into islets of cells with osteogenic phenotype that secreted mineralized extracellular matrix. This was augmented by inducers of osteogenesis, such as ascorbic acid, beta-glycerophosphate, and dexamethasone, or osteoblast-growth peptides (OGP). Implanting FMB-BM cells under the kidney capsule in mouse tested the osteogenic potential of these cells in vivo. Thirty days after implantation, bone structures with typical BM elements were seen in 8/53 kidneys in 6-Gy-irradiated mice and in 1/10 kidneys in nonirradiated recipients; bone formation was verified by soft x-ray imaging and elemental analysis that showed elevated Ca and Fe in the implant region. FMB-BM cells - downloaded onto plastic flasks, cultured for 2 weeks, mechanically harvested and then implanted - induced 100% bone formation in both irradiated (6/6) and nonirradiated (3/3) mice. Histology revealed well-organized bone structures under the kidney capsule, including osteoblasts and typical elements of BM. Our findings demonstrate that FMB are capable of isolating and expanding progenitor cells from BM for osteogenesis and possibly for regenerating other mesenchymal tissues.


Assuntos
Osso e Ossos/citologia , Técnicas de Cultura de Células/métodos , Fibrina , Células-Tronco Pluripotentes/citologia , Animais , Substitutos Ósseos , Feminino , Rim/citologia , Camundongos , Camundongos Endogâmicos C57BL , Microesferas , Osteogênese , Ratos , Transplante de Células-Tronco
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