RESUMO
SUMMARY BACKGROUND: BO2C11 is a human monoclonal factor (F) VIII inhibitor. When bound to the C2 domain of FVIII, the Fab fragment of BO2C11 (Fab(BO2C11)) buries a surface of C2 that contains residues participating in a binding site for von Willebrand factor (VWF). BO2C11 has thus been proposed to neutralize FVIII by steric hindrance. OBJECTIVES: The BO2C11 epitope on C2 overlaps with residues located at the periphery of the putative VWF binding site; hence, most of the residues that constitute the VWF binding site on C2 and a3 remain accessible for VWF interaction following BO2C11/FVIII complex formation. We thus investigated the contribution of alternative molecular mechanisms to FVIII inactivation by BO2C11. METHODS: Continuum electrostatic calculations were applied to the crystal structure of C2, free or Fab(BO2C11)-complexed. In silico predictions were confirmed by site-directed mutagenesis and VWF-binding assays of the mutated FVIII. RESULTS: Binding of Fab(BO2C11) to C2 induced perturbations in the electrostatic potential of C2 and in the local electrostatic parameters of 18 charged residues in C2, which are distant from the BO2C11 epitope. Nine of the predicted electrostatic hotspots clustered on the VWF-binding site of C2. Mutation of some of the predicted electrostatic hotspots has been associated with hemophilia A and reduced VWF binding in vitro. CONCLUSIONS: Inhibitors may neutralize FVIII by alteration of protein surface electrostatics at a long distance from their epitope. Perturbation of the electrostatic environment of C2, either upon binding by anti-FVIII antibodies or consecutive to missense mutations in the F8 gene, may lead to hampered VWF binding and reduced FVIII residence time in circulation.
Assuntos
Anticorpos Monoclonais/farmacologia , Epitopos/imunologia , Fator VIII/imunologia , Eletricidade Estática , Fator de von Willebrand/metabolismo , Anticorpos Monoclonais/imunologia , Sítios de Ligação/efeitos dos fármacos , Fator VIII/antagonistas & inibidores , Fator VIII/química , Fator VIII/genética , Hemofilia A , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação de Sentido IncorretoRESUMO
With the aim of proposing an alternative model to animal experimentation, we investigated cytokine production in response to antigens in an in vitro system. This is a co-culture system of healthy human leukocytes and enterocyte-like Caco-2 cells. The antigens tested, EgA31, EgTrp, and FABP1, are candidates for vaccines in infections caused by Echinococcus spp. in the gut. All three have previously been described in the protoscolex stage and belong to protein families which confer protective immunity against several helminths. In this study, we evaluate the Th1/Th2 profile (Th1: IL-12, IFN-gamma; Th2: IL-6, IL-10) in response to protoscoleces, EgA31 and the mixture of EgA31, EgTrp and FABP1. No cytokine production was detected in response to protoscoleces. Neither IFN-gamma nor IL-6, but a significant IL-10 and IL-12 concentration was detected in response to both types of antigens. These findings suggest that EgA31 and the mixture EgA31/EgTrp/FABP1 generated an immunogenic response associated with a mixed Th1/Th2 cytokine.
Assuntos
Antígenos de Helmintos/imunologia , Proteínas de Transporte/imunologia , Citocinas/metabolismo , Echinococcus/imunologia , Proteínas de Peixes , Leucócitos/imunologia , Animais , Células CACO-2 , Técnicas de Cultura de Células , Linhagem Celular , Técnicas de Cocultura , Proteínas de Ligação a Ácido Graxo , Humanos , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Interleucina-6/metabolismoRESUMO
The purpose of this study is to develop a reliable in vitro human model to test new immunotherapeutic approaches for squamous cell carcinoma that develop on mucosal surfaces. The organotypic (raft) culture permits cells to proliferate and differentiate at an air-liquid interface on a dermal equivalent support. Normal keratinocytes stratify and fully differentiate in a manner similar to the normal squamous epithelial tissues, while human papillomavirus-immortalized and established squamous carcinoma cell lines exhibit dysplastic morphologies similar to (pre)neoplastic lesions seen in vivo. We have demonstrated the ability of these organotypic cultures to be manipulated by altering the epithelial stratification with cytokines (interferon-gamma and tumor necrosis factor-alpha) and by integrating activated lymphocytes or dendritic cells into the in vitro formed epithelial sheet. This model may provide a useful tool to investigate the factors contributing to the presence and function of immunocompetent cells within a neoplastic epithelium that develops on a mucosal surface.
Assuntos
Imunoterapia/métodos , Queratinócitos/imunologia , Papillomaviridae/imunologia , Vacinas Anticâncer/isolamento & purificação , Vacinas Anticâncer/farmacologia , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/prevenção & controle , Linhagem Celular Transformada , Transformação Celular Viral , Células Dendríticas/imunologia , Células Dendríticas/patologia , Epitélio/imunologia , Epitélio/patologia , Humanos , Imunidade nas Mucosas , Técnicas In Vitro , Interferon gama/farmacologia , Queratinócitos/virologia , Modelos Biológicos , Mucosa/imunologia , Mucosa/patologia , Papillomaviridae/genética , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/prevenção & controle , Lesões Pré-Cancerosas/imunologia , Lesões Pré-Cancerosas/patologia , Lesões Pré-Cancerosas/prevenção & controle , Proteínas Recombinantes , Fator de Necrose Tumoral alfa/farmacologia , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/prevenção & controleRESUMO
The phosphoramidate triester prodrugs of anti-human HIV 2', 3'-dideoxynucleoside analogs (ddN) represent a convenient approach to bypass the first phosphorylation to ddN 5'-monophosphate (ddNMP), resulting in an improved formation of ddN 5'-triphosphate and, hence, higher antiviral efficacy. Although phosphoramidate derivatization markedly increases the anti-HIV activity of 2',3'-didehydro-2', 3'-dideoxythymidine (d4T) in both wild-type and thymidine kinase-deficient CEM cells, the concept is far less successful for the 3'-azido-2',3'-dideoxythymidine (AZT) triesters. We now investigated the metabolism of triester prodrugs of d4T and AZT using pure enzymes or different biological media. The efficiency of the first activation step, mediated by carboxylesterases, consists of the formation of the amino acyl ddNMP metabolite. The efficiency of this step was shown to be dependent on the amino acid, alkyl ester, and ddN moiety. Triesters that showed no conversion to the amino acyl ddNMP accumulated as the phenyl-containing intermediate and had poor, if any, anti-HIV activity. In contrast to the relative stability of the triesters in human serum, carboxylesterase-mediated cleavage of the prodrugs was found to be remarkably high in mouse serum. The subsequent conversion of the amino acyl ddNMP metabolite to ddNMP or ddN was highest in rat liver cytosolic enzyme preparations. Although L-alaninyl-d4TMP was efficiently converted to d4TMP, the main metabolite formed from L-alaninyl-AZTMP was the free nucleoside (AZT), thus explaining why d4T prodrugs, but not AZT prodrugs, retain anti-HIV activity in HIV-infected thymidine kinase-deficient cell cultures. The rat liver phosphoramidase responsible for the formation of ddNMP was shown to be distinct from creatine kinase, alkaline phosphatase, and phosphodiesterase.
Assuntos
Fármacos Anti-HIV/metabolismo , Fígado/metabolismo , Pró-Fármacos/metabolismo , Inibidores da Transcriptase Reversa/metabolismo , Estavudina/metabolismo , Zidovudina/metabolismo , Aminoácidos/metabolismo , Animais , Biotransformação , Didesoxinucleotídeos , Desenho de Fármacos , Esterases/metabolismo , Ésteres/metabolismo , HIV/efeitos dos fármacos , Humanos , Fígado/enzimologia , Masculino , Camundongos , Nucleosídeos/metabolismo , Ratos , Ratos Wistar , Estavudina/análogos & derivados , Nucleotídeos de Timina/metabolismo , Células Tumorais Cultivadas , Zidovudina/análogos & derivadosRESUMO
Antiviral activity was characterized from the culture broth of the Basidiomycete Macrocystidia cucumis (Pers. ex Fr.) Heim. When the stationary phase was reached (21 d), the culture broth was shown through an ELISA assay to contain antiviral activity against herpes simplex virus type 1 (HSV-1), as assessed in baby hamster kidney cells (BHK-21). Once the presence of the anti-HSV-1 activity in the culture broth was demonstrated, we proceeded with the purification and isolation of the active principle using a semi-preparative HPLC technique. The activity was associated with a purine nucleoside designated McA. This compound displayed no cytotoxicity at antivirally effective concentrations and proved to be a novel nucleoside analogue.