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1.
Clin Vaccine Immunol ; 19(10): 1641-50, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22896688

RESUMO

Staphylococcus aureus is a Gram-positive pathogen that causes devastating disease and whose pathogenesis is dependent on interactions with host cell factors. Staphylococcal clumping factor A (ClfA) is a highly conserved fibrinogen (Fg)-binding protein and virulence factor that contributes to host tissue adhesion and initiation of infection. ClfA is being investigated as a possible component of a staphylococcal vaccine. We report the development of an Fg-binding assay that is specific for ClfA-mediated binding. Using the assay, we show that despite the presence of anti-ClfA antibodies, human sera from unvaccinated subjects are unable to prevent the binding of S. aureus to an Fg-coated surface. In contrast, antibodies elicited by a recombinant ClfA-containing vaccine were capable of blocking the ClfA-dependent binding of a diverse and clinically relevant collection of staphylococcal strains to Fg. These functional antibodies were also able to displace S. aureus already bound to Fg, suggesting that the ligand-binding activity of ClfA can be effectively neutralized through vaccination.


Assuntos
Aderência Bacteriana , Coagulase/imunologia , Fibrinogênio/metabolismo , Vacinas Antiestafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Anticorpos Antibacterianos/sangue , Coagulase/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/patogenicidade
2.
Antimicrob Agents Chemother ; 55(5): 1843-51, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21357300

RESUMO

INX-08189 is an aryl-phosphoramidate of 6-O-methyl-2'-C-methyl guanosine. INX-08189 was highly potent in replicon assays, with a 50% effective concentration of 10±6 nM against hepatitis C genotype 1b at 72 h. The inhibitory effect on viral replication was rapid, with a 50% effective concentration (EC50) of 35±8 nM at 24 h. An intracellular 2'-C-methyl guanosine triphosphate (2'-C-MeGTP) concentration of 2.43±0.42 pmol/10(6) cells was sufficient to achieve 90% inhibition of viral replication. In vitro resistance studies confirmed that the S282T mutation in the NS5b gene conferred an approximately 10-fold reduction in sensitivity to INX-08189. However, the complete inhibition of S282T mutant replicons still could be achieved with an EC90 of 344±170 nM. Drug combination studies of INX-08189 and ribavirin indicated significant synergy in antiviral potency both in wild-type and S282T-expressing replicons. Genotype 1b replicons could be cleared after 14 days of culture when exposed to as little as 20 nM INX-08189. No evidence of mitochondrial toxicity was observed after 14 days of INX-08189 exposure in both HepG2 and CEM human cell lines. In vivo studies of rats and cynomolgus monkeys demonstrated that 2'-C-MeGTP concentrations in liver equivalent to the EC90 could be attained after a single oral dose of INX-08189. Rat liver 2'-C-MeGTP concentrations were proportional to dose, sustained for greater than 24 h, and correlated with plasma concentrations of the nucleoside metabolite 2'-C-methyl guanosine. The characteristics displayed by INX-08189 support its continued development as a clinical candidate for the treatment of chronic HCV infection.


Assuntos
Amidas/química , Antivirais/farmacologia , Antivirais/farmacocinética , Guanosina/farmacologia , Guanosina/farmacocinética , Hepacivirus/efeitos dos fármacos , Ácidos Fosfóricos/química , Pró-Fármacos/farmacologia , Pró-Fármacos/farmacocinética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Guanosina/análogos & derivados , Guanosina/química , Humanos , Macaca fascicularis , Masculino , Pró-Fármacos/química , Ratos , Ratos Sprague-Dawley , Replicação Viral/efeitos dos fármacos
3.
J Microbiol Methods ; 78(1): 71-8, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19427882

RESUMO

Two monoclonal antibodies (MAbs) were raised against the Candida albicans cell-surface glycoprotein Als3 using the N-terminal domain of the protein as the immunogen. ELISA was used to demonstrate the specificity of the MAbs for the Als3 fragment, but not for the corresponding N-terminal domain fragments from other proteins in the Als family. The anti-Als3 MAbs immunolabeled the surface of germ tubes from a diverse collection of wild-type C. albicans isolates, but did not label yeast cells, an als3Delta/als3Delta deletion mutant strain, nor isolates of other Candida species associated with human disease. Als3 was visualized readily in fresh and formalin-fixed, paraffin-embedded kidney tissue from a murine model of candidiasis. The anti-Als3 MAbs were also useful for immunogold electron microscopy and Western blotting. Both MAbs blocked C. albicans adhesion to vascular endothelial cells and buccal epithelial cells. These versatile MAbs are a valuable addition to the reagents available to study C. albicans cell surface dynamics and interaction of the fungus with host cells.


Assuntos
Anticorpos Antifúngicos/imunologia , Anticorpos Monoclonais/imunologia , Candida albicans/fisiologia , Candidíase/microbiologia , Proteínas Fúngicas/imunologia , Interações Hospedeiro-Patógeno , Animais , Candida albicans/genética , Candida albicans/imunologia , Candidíase/imunologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C
4.
Microb Pathog ; 43(2-3): 55-66, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17521860

RESUMO

Enterococci are opportunistic pathogens known to cause numerous clinical infections and complications in humans. Adhesin-mediated binding to extracellular matrix (ECM) proteins of the host is thought to be a crucial step in the pathogenesis of these bacterial infections. Adhesin of collagen from Enterococcus faecalis (Ace) is a cell-wall anchored protein of E. faecalis that has been shown to be important for bacterial binding to the ECM. In this report, we characterize the conditions for Ace expression and demonstrate Ace binding to mammalian epithelial and endothelial cells as well as to collagens found in the ECM. To further characterize Ace expression and function, we report the generation of a panel of monoclonal antibodies (mAbs) directed against this important E. faecalis virulence factor. Through the use of multiple in vitro assays, surface plasmon resonance and flow cytometry, we have characterized this panel of mAbs which may prove to be not only beneficial in studies that address the precise biological role of adhesion of E. faecalis, but may also serve as beneficial therapeutic agents against E. faecalis infections.


Assuntos
Adesinas Bacterianas/metabolismo , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Colágeno/metabolismo , Enterococcus faecalis/imunologia , Adesinas Bacterianas/imunologia , Animais , Anticorpos Antibacterianos/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Proteínas de Bactérias/imunologia , Proteínas de Transporte/imunologia , Linhagem Celular , Enterococcus faecalis/metabolismo , Feminino , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Ligação Proteica , Ressonância de Plasmônio de Superfície
5.
Hybridoma (Larchmt) ; 26(1): 28-34, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17316083

RESUMO

Staphylococcus epidermidis is an important opportunistic human pathogen that has recently emerged as a major cause of foreign-body infections. The most important stage contributing to the pathogenesis of this bacteria is the initial adherence to host tissue. SdrG is a cell-wall-anchored fibrinogen-binding adhesin of S. epidermidis that has been shown to be necessary for bacterial binding to fibrinogen-coated foreign bodies, such as catheters. Here we report the generation and characterization of a panel of monoclonal antibodies (MAbs) directed against this S. epidermidis virulence factor. Through the use of multiple in vitro assays, surface plasmon resonance, and flow cytometry, we have characterized a diverse array of MAbs that may prove to be beneficial in studies that address the precise biologic role of SdrG.


Assuntos
Adesinas Bacterianas/imunologia , Anticorpos Monoclonais/metabolismo , Proteínas de Bactérias/imunologia , Proteínas de Transporte/imunologia , Fibrinogênio/metabolismo , Staphylococcus epidermidis/imunologia , Adesinas Bacterianas/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Feminino , Lactococcus lactis/genética , Lactococcus lactis/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica/imunologia , Staphylococcus epidermidis/genética
6.
Antimicrob Agents Chemother ; 50(2): 511-8, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16436704

RESUMO

A human donor-selected immunoglobulin G for intravenous injection (IGIV) product with elevated titers against the staphylococcal fibrinogen-binding MSCRAMM proteins ClfA and SdrG (INH-A21) was tested in vitro and in vivo. INH-A21 contained a significantly increased ability to inhibit the fibrinogen-binding activity of recombinant forms of both ClfA and SdrG. Evaluation of the opsonizing potential of INH-A21 was evaluated using fluorescently labeled bacteria; this assay indicated an increase in phagocytic activity compared to normal IGIV. The prophylactic efficacy of INH-A21 against an intraperitoneal challenge of methicillin-resistant Staphylococcus epidermidis (MRSE) was evaluated in a neonatal rat model. INH-A21 was also evaluated for prophylactic and therapeutic efficacy in a rabbit model of catheter-induced aortic valve infective endocarditis caused by either MRSE or methicillin-resistant Staphylococcus aureus (MRSA). Results from the in vivo models demonstrated potent prophylactic and therapeutic efficacy against both MRSE and MRSA. These data suggest that INH-A21 may be an important tool for the prevention and treatment of staphylococcal infections, especially in high-risk populations.


Assuntos
Anticorpos Antibacterianos/uso terapêutico , Proteínas de Bactérias/imunologia , Proteínas de Transporte/imunologia , Coagulase/imunologia , Imunoglobulina G/uso terapêutico , Imunoglobulinas Intravenosas/uso terapêutico , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/imunologia , Staphylococcus epidermidis/imunologia , Animais , Feminino , Humanos , Coelhos , Ratos , Ratos Sprague-Dawley
7.
Infect Immun ; 73(8): 5229-32, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16041045

RESUMO

We report the humanization and characterization of monoclonal antibody (MAb) T1-2 or tefibazumab, a monoclonal antibody that recognizes clumping factor A expressed on the surface of Staphylococcus aureus. We demonstrate that the binding kinetics of MAb T1-2 is indistinguishable compared to that of its murine parent. Furthermore, MAb T1-2 is shown to enhance the opsonophagocytic uptake of ClfA-coated latex beads, protect against an intravenous challenge in a prophylactic model of rabbit infective endocarditis, and enhance the efficacy of vancomycin therapy in a therapeutic model of established infective endocarditis.


Assuntos
Anticorpos Monoclonais/imunologia , Coagulase/imunologia , Staphylococcus aureus/imunologia , Animais , Anticorpos Monoclonais/sangue , Linhagem Celular , Humanos , Coelhos
8.
Infect Immun ; 72(11): 6237-44, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15501749

RESUMO

Staphylococcus capitis (S. capitis) has been implicated in a large proportion of coagulase-negative staphylococcal infections in very-low-birth-weight infants. To identify potential therapeutic targets, the S. capitis genome was probed for the presence of genes encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMM). By using Southern blot analysis, an S. capitis gene, designated sdrX, that contained sequence motifs consistent with the Sdr family of MSCRAMM proteins was identified. By using monospecific antisera in Western blot and flow cytometry, SdrX was demonstrated to be expressed on the surface of S. capitis. Human collagen type VI was found to bind both the recombinant A domain of SdrX and viable S. capitis expressing SdrX. SdrX is the first collagen-binding Sdr protein described and is the first MSCRAMM protein identified in S. capitis.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Colágeno/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Staphylococcus/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/metabolismo , Sequência de Aminoácidos , Ácido Aspártico , Proteínas de Bactérias/genética , Clonagem Molecular , Humanos , Recém-Nascido , Proteínas de Membrana/genética , Dados de Sequência Molecular , Serina , Staphylococcus/genética , Staphylococcus/crescimento & desenvolvimento
9.
Infect Immun ; 71(12): 6864-70, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14638774

RESUMO

The Staphylococcus aureus MSCRAMM (microbial surface components recognizing adhesive matrix molecules) protein clumping factor A (ClfA) has been shown to be a critical virulence factor in several experimental models of infection. This report describes the generation, characterization, and in vivo evaluation of a murine monoclonal antibody (MAb) against ClfA. Flow cytometric analysis revealed that MAb 12-9 recognized ClfA protein expressed by all of the clinical S. aureus strains obtained from a variety of sources. In assays measuring whole-cell S. aureus binding to human fibrinogen, MAb 12-9 inhibited S. aureus binding by over 90% and displaced up to 35% of the previously adherent S. aureus bacteria. Furthermore, a single infusion of MAb 12-9 was protective against an intravenous challenge with a methicillin-resistant strain of S. aureus in a murine sepsis model (P < 0.0001). These data suggest that anti-ClfA MAb 12-9 should be further investigated as a novel immunotherapy for the treatment and prevention of life-threatening S. aureus infections.


Assuntos
Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Coagulase/imunologia , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/imunologia , Adesinas Bacterianas/genética , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/uso terapêutico , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/uso terapêutico , Aderência Bacteriana , Coagulase/genética , Humanos , Hibridomas , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/mortalidade , Staphylococcus aureus/patogenicidade , Ressonância de Plasmônio de Superfície
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