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1.
J Gene Med ; 11(4): 326-34, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19219895

RESUMO

BACKGROUND: Developing vectors that target specifically to disease sites after systemic injection is an important goal in gene therapy research. METHODS: We prepared fluorescent DNA polyplexes (< or =150 nm in diameter) comprising plasmid DNA condensed with poly(L-lysine) and coated with a multivalent reactive copolymer based on poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). These polyplexes were then surface modified with a recombinant P-selectin glycoprotein ligand-1 immunoglobulin chimera (rPSGL-Ig) previously investigated as a selectin antagonist in clinical studies. RESULTS: Five minutes after jugular vein injection of these polyplexes, fluorescence accumulation in inflamed cremasteric venules of C57BL6 mice was more than eight-fold higher than that observed after injection of Fc-blocked control polyplexes. Fluorescence above background was not observed in P-selectin deficient mice, confirming the specificity for P-selectin in this model. CONCLUSIONS: These data provide encouragement for the further development of rPSGL-Ig-coated polyplexes as potential nonviral vectors for targeted gene therapy in inflammatory conditions, such as ischaemia reperfusion injury, unstable atherosclerotic plaques and myocarditis. This approach may also be transferable to the use of other targeting ligands whose cognate partner is specifically upregulated on the vascular endothelium in individual pathological situations.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Endotélio/patologia , Inflamação/tratamento farmacológico , Glicoproteínas de Membrana/administração & dosagem , Selectina-P/metabolismo , Polímeros/química , Animais , Corantes Fluorescentes , Imunoglobulinas , Glicoproteínas de Membrana/farmacocinética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia , Plasmídeos , Polilisina , Polímeros/farmacocinética , Proteínas Recombinantes
2.
J Leukoc Biol ; 85(1): 55-63, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18927400

RESUMO

Ly-6G is a member of the Ly-6 family of GPI-linked proteins, which is expressed on murine neutrophils. Antibodies against Ly-6G cause neutropenia, and fatal reactions also develop if mice are primed with TNF-alpha prior to antibody treatment. We have investigated the mechanisms behind these responses to Ly-6G ligation in the belief that similar mechanisms may be involved in neutropenia and respiratory disorders associated with alloantibody ligation of the related Ly-6 family member, NB1, in humans. Neutrophil adhesion, microvascular obstruction, breathing difficulties, and death initiated by anti-Ly-6G antibodies in TNF-alpha-primed mice were shown to be highly complement-dependent, partly mediated by CD11b, CD18, and FcgammaR and associated with clustering of Ly-6G. Neutrophil depletion, on the other hand, was only partly complement-dependent and was not altered by blockade of CD11b, CD18, or FcgammaR. Unlike other neutrophil-activating agents, Ly-6G ligation did not induce neutropenia via sequestration in the lungs. Cross-linking Ly-6G mimicked the responses seen with whole antibody in vivo and also activated murine neutrophils in vitro. Although this suggests that the responses are, in part, mediated by nonspecific properties of antibody ligation, neutrophil depletion requires an additional mechanism possibly specific to the natural function of Ly-6G.


Assuntos
Antígenos Ly/imunologia , Proteínas do Sistema Complemento/imunologia , Neutropenia/imunologia , Neutrófilos/imunologia , Insuficiência Respiratória/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Antígeno CD11b/imunologia , Antígenos CD18/imunologia , Reagentes de Ligações Cruzadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microcirculação/imunologia , Neutropenia/mortalidade , Neutropenia/fisiopatologia , Receptores de IgG/genética , Receptores de IgG/imunologia , Respiração , Insuficiência Respiratória/mortalidade , Insuficiência Respiratória/fisiopatologia , Fator de Necrose Tumoral alfa/imunologia
3.
Traffic ; 7(6): 647-62, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16683915

RESUMO

The biogenesis of endothelial-specific Weibel-Palade bodies (WPB) is poorly understood, despite their key role in both haemostasis and inflammation. Biogenesis of specialized organelles of haemopoietic cells is often adaptor protein complex 3-dependent (AP-3-dependent), and AP-3 has previously been shown to play a role in the trafficking of both WPB membrane proteins, P-selectin and CD63. However, WPB are thought to form at the trans Golgi network (TGN), which is inconsistent with a role for AP-3, which operates in post-Golgi trafficking. We have therefore investigated in detail the mechanisms of delivery of these two membrane proteins to WPB. We find that P-selectin is recruited to forming WPB in the trans-Golgi by AP-3-independent mechanisms that use sorting information within both the cytoplasmic tail and the lumenal domain of the receptor. In contrast, CD63 is recruited to already-budded WPB by an AP-3-dependent route. These different mechanisms of recruitment lead to the presence of distinct immature and mature populations of WPB in human umbilical vein endothelial cells (HUVEC).


Assuntos
Antígenos CD/metabolismo , Selectina-P/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Corpos de Weibel-Palade/metabolismo , Complexo 3 de Proteínas Adaptadoras , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/ultraestrutura , Humanos , Migração e Rolagem de Leucócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia Eletrônica , Modelos Biológicos , Selectina-P/química , Selectina-P/genética , Sinais Direcionadores de Proteínas/genética , Estrutura Terciária de Proteína , Transporte Proteico , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tetraspanina 30 , Fatores de Transcrição/metabolismo , Corpos de Weibel-Palade/ultraestrutura , Rede trans-Golgi/metabolismo
4.
Dev Cell ; 10(2): 223-32, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16459301

RESUMO

Weibel-Palade bodies are the 1-5 microm long rod-shaped storage organelles of endothelial cells. We have investigated the determinants and functional significance of this shape. We find that the folding of the hemostatic protein von Willebrand's factor (VWF) into tubules underpins the rod-like shape of Weibel-Palade bodies. Further, while the propeptide and the N-terminal domains of mature VWF are sufficient to form tubules, their maintenance relies on a pH-dependent interaction between the two. We show that the tubular conformation of VWF is essential for a rapid unfurling of 100 microm long, platelet-catching VWF filaments when exposed to neutral pH after exocytosis in cell culture and in living blood vessels. If tubules are disassembled prior to exocytosis, then short or tangled filaments are released and platelet recruitment is reduced. Thus, a 100-fold compaction of VWF into tubules determines the unique shape of Weibel-Palade bodies and is critical to this protein's hemostatic function.


Assuntos
Endotélio Vascular/metabolismo , Endotélio Vascular/ultraestrutura , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/fisiologia , Animais , Sequência de Bases , Células Cultivadas , DNA/genética , Exocitose , Hemostasia/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monensin/farmacologia , Adesividade Plaquetária , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Vênulas/efeitos dos fármacos , Vênulas/fisiologia , Fator de von Willebrand/química , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo
5.
Am J Pathol ; 166(3): 945-52, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15743805

RESUMO

P-selectin glycoprotein ligand-1 (PSGL-1) binding to P-selectin controls early leukocyte rolling during inflammation. Interestingly, antibodies and pharmacological inhibitors (eg, rPSGL-Ig) that target the N-terminus of PSGL-1 reduce but do not abolish P-selectin-dependent leukocyte rolling in vivo whereas PSGL-1-deficient mice have almost no P-selectin-dependent rolling. We have investigated mechanisms of P-selectin-dependent, PSGL-1-independent rolling using intravital microscopy. Initially we used fluorescent microspheres to study the potential of L-selectin and the minimal selectin ligand sialyl Lewis(x) (sLe(x)) to interact with postcapillary venules in the absence of PSGL-1. Microspheres coated with combinations of L-selectin and sLe(x) interacted with surgically stimulated cremaster venules in a P-selectin-dependent manner. Microspheres coated with either L-selectin or sLe(x) alone showed less evidence of interaction. We also investigated leukocyte rolling in the presence of PSGL-1 antibody or inhibitor (rPSGL-Ig), both of which partially inhibited P-selectin-dependent leukocyte rolling. Residual rolling was substantially inhibited by L-selectin-blocking antibody or a previously described sLe(x) mimetic (CGP69669A). Together these data suggest that leukocytes can continue to roll in the absence of optimal P-selectin/PSGL-1 interaction using an alternative mechanism that involves P-selectin-, L-selectin-, and sLe(x)-bearing ligands.


Assuntos
Selectina L/metabolismo , Migração e Rolagem de Leucócitos , Leucócitos/citologia , Glicoproteínas de Membrana/metabolismo , Selectina-P/metabolismo , Animais , Biotinilação , Adesão Celular , Técnicas de Cultura , Glicoproteínas/metabolismo , Leucócitos/metabolismo , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia , Microesferas , Modelos Biológicos , Oligossacarídeos/metabolismo , Ligação Proteica , Antígeno Sialil Lewis X , Fatores de Tempo
6.
J Leukoc Biol ; 77(1): 59-66, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15466915

RESUMO

Selectins and their ligands support leukocyte rolling, facilitating the subsequent firm adhesion and migration that occur during inflammation. TBC-1269 (Bimosiamose), a structural mimetic of natural selectin ligands, inhibits P-, E-, and L-selectin in vitro, has anti-inflammatory effects in vivo, and recently underwent phase II clinical trials for childhood asthma and psoriasis. We studied whether the anti-inflammatory effects of TBC-1269 could be related to leukocyte rolling in vivo. Although TBC-1269 inhibited rolling of a murine leukocyte cell line on murine P-selectin in vitro and thioglycollate-induced peritonitis in vivo, it did not alter leukocyte rolling in mouse cremaster venules. TBC-1269 reduced neutrophil recruitment in thioglycollate-induced peritonitis in wild-type and P-selectin-/- mice but not in E-selectin-/- mice. We suggest that the in vivo effects of TBC-1269 may be mediated through E-selectin but do not appear to involve leukocyte rolling.


Assuntos
Compostos de Bifenilo/uso terapêutico , Selectina E/fisiologia , Inflamação/patologia , Leucócitos/metabolismo , Manosídeos/uso terapêutico , Mimetismo Molecular , Selectina-P/fisiologia , Animais , Ligação Competitiva , Selectina E/genética , Selectina E/imunologia , Leucócitos/imunologia , Leucócitos/patologia , Ligantes , Masculino , Manose/análogos & derivados , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Selectina-P/genética , Selectina-P/imunologia , Peptoides/química , Peptoides/farmacologia , Peritonite/induzido quimicamente , Peritonite/patologia , Tioglicolatos/toxicidade , Vênulas/citologia
7.
Timely Top Med Cardiovasc Dis ; 8: E6, 2004 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-15543251

RESUMO

The acronym DIC is commonly interpreted as "death is coming." This pessimistic view emphasizes the deficiency of available treatment options following diagnosis of disseminated intravascular coagulation. Clinically, DIC manifests as a systemic hemorrhagic disorder associated with widespread activation and eventual exhaustion of the coagulation system, although events underlying DIC also involve effectors of inflammation. DIC can be associated with diverse conditions including sepsis and major trauma and, when identified, signifies a significant worsening in prognosis and expected mortality. Although recent clinical studies have shown that activated protein C reduces mortality in patients with severe sepsis, there is a need for further investigation and a better understanding of the underlying mechanisms.


Assuntos
Coagulação Intravascular Disseminada/terapia , Coagulação Intravascular Disseminada/complicações , Coagulação Intravascular Disseminada/diagnóstico , Coagulação Intravascular Disseminada/etiologia , Humanos
8.
Drug News Perspect ; 17(4): 243-50, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15334173

RESUMO

The acronym DIC is commonly interpreted as "death is coming". This pessimistic view emphasizes the deficiency of available treatment options following diagnosis of disseminated intravascular coagulation. Clinically, DIC manifests as a systemic hemorrhagic disorder associated with widespread activation and eventual exhaustion of the coagulation system, although events underlying DIC also involve effectors of inflammation. DIC can be associated with diverse conditions including sepsis and major trauma and, when identified, signifies a significant worsening in prognosis and expected mortality. Although recent clinical studies have shown that activated protein C reduces mortality in patients with severe sepsis, there is a need for further investigation and a better understanding of the underlying mechanisms.


Assuntos
Coagulação Intravascular Disseminada/terapia , Animais , Coagulação Intravascular Disseminada/complicações , Coagulação Intravascular Disseminada/diagnóstico , Coagulação Intravascular Disseminada/etiologia , Humanos , Inflamação/patologia , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/fisiologia
9.
FASEB J ; 18(1): 152-4, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14597557

RESUMO

Selectins mediate leukocyte rolling and may represent good anti-inflammatory drug targets. Detailed knowledge regarding the structure of selectin ligands has permitted development of selectin antagonists with varying specificities and activity. Efficacy of monovalent selectin antagonists may be increased by presenting them on a polymer backbone. We have synthesized a range of multivalent selectin antagonists and characterized their activity by using intravital microscopy of the mouse cremaster muscle. The monovalent inhibitor CGP77175A inhibited E-selectin-dependent leukocyte rolling at a dose of 3 mg/kg. Multivalent presentation of CGP77175A on a modified polylysine backbone (degree of polymerization = 1200; 50% of the polylysines carry the inhibitor) greatly enhanced in vivo activity giving an inhibitor that produced an equivalent effect at 0.1 mg/kg. The polylysine conjugate was also longer acting than the monovalent antagonist. In spite of greatly enhanced activity against E-selectin compared with monovalent inhibitor, the multivalent inhibitor had no measurable effect on P- or L-selectin-dependent leukocyte rolling.


Assuntos
Selectina E/metabolismo , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Oligossacarídeos/química , Polímeros/farmacologia , Animais , Cinética , Camundongos , Oligossacarídeos/farmacologia , Polilisina/química , Polilisina/farmacologia , Polímeros/química , Antígeno Sialil Lewis X
10.
Blood ; 101(3): 921-8, 2003 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-12393622

RESUMO

Widespread microvascular injury followed by vessel obstruction may lead to disseminated intravascular coagulation (DIC). We describe a murine model wherein leukocytes interacting with inflamed microvessels in vivo are activated by antibodies. Treatment of tumor necrosis factor alpha (TNF-alpha)-primed mice with anti-Ly-6G antibodies reproduced many of the features of septic or traumatic shock including microvessel obstruction and coagulation, severe vasculitis, respiratory difficulties, and vascular leakage. Mice lacking either E-selectin or P-selectin were protected from this reaction as were animals treated with a combination of either selectin-blocking antibodies and heparin or a selectin antagonist plus heparin. Combined blockade of leukocyte/platelet adhesion and coagulation may provide convincing protection in DIC.


Assuntos
Anticorpos/farmacologia , Anticoagulantes/farmacologia , Coagulação Intravascular Disseminada/prevenção & controle , Selectinas/imunologia , Animais , Anticorpos/administração & dosagem , Anticoagulantes/administração & dosagem , Antígenos Ly/imunologia , Adesão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Coagulação Intravascular Disseminada/tratamento farmacológico , Coagulação Intravascular Disseminada/etiologia , Quimioterapia Combinada , Heparina/administração & dosagem , Heparina/farmacologia , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Camundongos , Camundongos Knockout , Microcirculação/patologia , Selectinas/genética , Taxa de Sobrevida , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/efeitos adversos
11.
Blood ; 101(8): 3249-56, 2003 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-12480716

RESUMO

Selectin-dependent leukocyte rolling is one of the earliest steps of an acute inflammatory response and, as such, contributes to many inflammatory diseases. Although inhibiting leukocyte rolling with selectin antagonists is a strategy that promises far-reaching clinical benefit, the perceived value of this strategy has been limited by studies using inactive, weak, or poorly characterized antagonists. Recombinant P-selectin glycoprotein ligand-1-immunoglobulin (rPSGL-Ig) is a recombinant form of the best-characterized selectin ligand (PSGL-1) fused to IgG, and is one of the best prospects in the search for effective selectin antagonists. We have used intravital microscopy to investigate the ability of rPSGL-Ig to influence leukocyte rolling in living blood vessels and find that it can reduce rolling dependent on each of the selectins in vivo. Interestingly, doses of rPSGL-Ig required to reverse pre-existing leukocyte rolling are 30-fold higher than those required to limit inflammation, suggesting additional properties of this molecule. In support of this, we find that rPSGL-Ig can bind the murine chemokine KC and inhibit neutrophil migration toward this chemoattractant in vitro.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Quimiocinas CXC , Quimiocinas/antagonistas & inibidores , Fatores Quimiotáticos/antagonistas & inibidores , Selectina E/fisiologia , Selectina L/fisiologia , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Glicoproteínas de Membrana/farmacologia , Selectina-P/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Estenose da Valva Aórtica/complicações , Estenose da Valva Aórtica/patologia , Quimiocina CXCL1 , Quimiotaxia de Leucócito , Modelos Animais de Doenças , Selectina E/genética , Endotélio Vascular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular , Isquemia/etiologia , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Peritonite/induzido quimicamente , Peritonite/patologia , Proteínas Recombinantes de Fusão/farmacologia , Traumatismo por Reperfusão/patologia , Tioglicolatos/toxicidade , Fator de Necrose Tumoral alfa/farmacologia
12.
FASEB J ; 16(11): 1461-2, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12205048

RESUMO

Leukocytic inflammation can be limited by inhibiting selectin-dependent leukocyte rolling. In spite of intensive efforts to develop small molecule selectin inhibitors with defined structure-activity profiles, inhibition of P-selectin-dependent leukocyte rolling in vivo by such a compound has yet to be described. We recently reported that glycosulfopeptides (GSP), modeled on the high affinity selectin ligand PSGL-1, inhibit leukocyte binding to P-selectin in vitro. Here, we have used intravital microscopy to investigate whether GSP can inhibit P-selectin-dependent leukocyte rolling in vivo. Surgical preparation of the mouse cremaster muscle for intravital microscopy induced P-selectin-dependent leukocyte rolling. Baseline rolling was recorded for 1 min followed by i.v. injection of GSP. 2-GSP-6 and 4-GSP-6 substantially reversed P-selectin-dependent leukocyte rolling, whereas control GSP, which are not fully glycosylated, did not. Inhibition of leukocyte rolling by 2- and 4-GSP-6 lasted 2-4 min. Clearance studies with 125I-labeled 4-GSP-6 demonstrated rapid reduction in its circulating levels concurrent with accumulation in urine. These data represent the first demonstration that a precisely defined structure based on a natural P-selectin ligand can inhibit P-selectin-dependent leukocyte rolling in vivo.


Assuntos
Movimento Celular/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Glicoproteínas de Membrana/química , Selectina-P/fisiologia , Peptídeos/farmacologia , Animais , Técnicas de Cultura , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Glicoproteínas/farmacologia , Cinética , Leucócitos/imunologia , Camundongos , Modelos Biológicos , Enxofre
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