RESUMEN
An isoquinoline derivative, 5-methyl-7,8-dimethoxy-1-phenylpyrazolo[5,4-c]isoquinoline (compound 1), was identified as a novel inhibitor of LPS-induced TNF-alpha production by cell-based screening. Compound 1 suppressed LPS-induced TNF-alpha production in RAW264.7 cells and murine peritoneal macrophages in a dose-dependent manner similar to SB203580, known as a specific inhibitor of p38 MAPK. It also inhibited an LPS-induced increase in serum TNF-alpha in a mouse endotoxic shock model with an ED(50) of approximately 10 mg/kg. Compound 1 had little effect on the incorporation of [3H]-leucine into the cells, while it suppressed LPS-induced TNF-alpha mRNA levels in RAW264.7 cells. The results indicate that suppression of TNF-alpha production was not a result of nonspecific inhibition of de novo translation but was based on the decreased TNF-alpha mRNA levels. The in vitro kinase assay revealed that compound 1 did not strongly inhibit p38 MAPK activity, its potency being much lower than that of SB203580, suggesting that the TNF-alpha-suppressive action of compound 1 cannot be attributed to the inhibition of p38 MAPK. Furthermore, in contrast to SB203580, it significantly inhibited the growth of RAW264.7 and THP-1 cells in a cytostatic manner. Compound 1 is likely to have antiinflammatory and antiproliferative effects by acting on some molecule other than p38 MAPK that contributes to both LPS-induced TNF-alpha production and the cell growth of monocyte/macrophages.
Asunto(s)
División Celular/efectos de los fármacos , Isoquinolinas/farmacología , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Monocitos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Transcripción Activador 2 , Animales , Recuento de Células , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Imidazoles/farmacología , Isoquinolinas/química , Leucina/efectos de los fármacos , Leucina/metabolismo , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Monocitos/citología , Monocitos/metabolismo , Piridinas/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/metabolismo , Tritio , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genética , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Monoclonal antibodies (MoAbs) against N-domain of carcinoembryonic antigen (CEA), C249, K348, K1338, and K1444, that inhibit CEA-mediated cell adhesion, did not crossreact with nonspecific cross-reacting antigen (NCA). To determine amino acid sequences involved in the adhesion, epitopes of the MoAbs were mapped with recombinant NCAs carrying CEA-NCA chimeric N-domain. The data showed that the epitopes of C249, K1338, K1444 are located within the regions 1-32, 1-32, and 33-59 of CEA, respectively, and that two discrete regions 1-32 and 60-93 may be related to the epitope of K348. Comparison of amino acid sequences between CEA and NCA suggested that four residues (21, 27-29), eight residues (21, 27-29, 66, 78, 79, 89), and three residues (43, 44, 46) are important for recognition by C249 (or K1338), K348, and K1444, respectively. These residues seem to participate in the cell adhesion mediated by CEA.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígeno Carcinoembrionario/química , Mapeo Epitopo , Secuencia de Aminoácidos , Reacciones Antígeno-Anticuerpo , Antígeno Carcinoembrionario/inmunología , Adhesión Celular/inmunología , Datos de Secuencia Molecular , Estructura Terciaria de ProteínaRESUMEN
An anti-human tumor necrosis factor-alpha (TNF-alpha) monoclonal antibody, designated as 3B10, inhibits the biological activity of human TNF-alpha. In the present study, we constructed humanized version of the antibody by grafting its complementarity-determining regions (CDRs) onto a human antibody, HBS-1. Using a molecular model of mouse 3B10, framework residues affecting the CDR conformation were identified. Thus, these residues were also introduced into the framework together with the CDRs in a stepwise manner, depending on the degree of the possible importance of the residues. As a result, one humanized version (h3B10-9) which possesses nine mouse framework residues showed the same binding activity as that of the chimeric version. This humanized anti-TNF-alpha antibody is expected to be less immunogenic and thus more suitable for possible clinical use.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Secuencia de Aminoácidos , Animales , Células COS/metabolismo , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/metabolismo , Ratones , Datos de Secuencia Molecular , Pruebas de Neutralización , Reacción en Cadena de la Polimerasa , Conformación Proteica , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Transfección , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
A mouse anti-human tumor necrosis factor-alpha (TNF-alpha) monoclonal antibody (MoAb), designated as 3B10, has previously been produced and characterized by our laboratory. We report here the construction and the expression of mouse-human chimeric antibody derived from the MoAb. cDNAs encoding variable regions of heavy and light chains were prepared from 3B10 cells by polymerase chain reaction, and introduced to mammalian expression vectors containing cDNA for human gamma1 and kappa constant regions, respectively. Cotransfection of the vectors into CHO cells resulted in production of antibody reacting with human TNF-alpha. In SDS-PAGE analysis, the chimeric antibody, c3B10, migrated at 170 kDa under a nonreducing condition, whereas two bands with 58 and 28 kDa appeared following treatment with 2-mercaptoethanol. Both c3B10 and mouse 3B10 neutralized the cytotoxic activity of human TNF-alpha to the same level, indicating that c3B10 holds the binding activity of its original MoAb. These findings suggest that the introduced genes for chimeric heavy and light chains are transcribed and translated to produce the chimeric heavy and light chain peptides, and that the peptides are assembled to form native IgG molecule. The chimeric anti-TNF-alpha antibody described in this study is expected to be less immunogenic and thus more suitable for possible clinical use.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Factor de Necrosis Tumoral alfa/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Células CHO , Cricetinae , Electroforesis en Gel de Poliacrilamida , Genes de Inmunoglobulinas , Vectores Genéticos , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Ratones , Pruebas de Neutralización , Proteínas Recombinantes de Fusión/inmunología , Transfección , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In order to investigate the role of L-selectin (CD62L) in delayed-type hypersensitivity (DTH) reaction, effect of fucoidin, a potent inhibitor of CD62L function, was examined in a model of mouse contact hypersensitivity reaction. Intravenous injection of fucoidin to sensitized mice just before hapten challenge resulted in a significant and dose-dependent reduction of the ear swelling in contact hypersensitivity. The ear swelling caused by the hapten challenge was also inhibited when fucoidin was administered at the sensitization phase. Histological analyses of the ear sections revealed that the fucoidin-induced suppression of contact hypersensitivity reflected a marked inhibition of the ear edema and the leukocyte infiltration. The activity of fucoidin was specific in that its related saccharides exerted little effect on the reaction. These results suggest that CD62L may play an important role in both afferent and efferent phases of cutaneous DTH reaction. Since DTH response is one of the most significant features of several chronic inflammatory diseases, our data also show that blocking of CD62L function may be beneficial for the treatment of these diseases in humans.
Asunto(s)
Dermatitis por Contacto/fisiopatología , Selectina L/fisiología , Polisacáridos/farmacología , Animales , Modelos Animales de Enfermedad , Femenino , Leucocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB CRESUMEN
Polyclonal antibody against human natriuretic peptide receptor B (NPR-B) was produced using as immunogen a soluble chimeric protein consisting of the extracellular domain of the receptor fused with Fc portion of human IgG. The antibody was purified with protein A column, and then subjected to an adsorption of anti-Fc antibody using IgG column. The purified antibody recognized human NPR-B but not the related receptor NPR-A. The antibody inhibited C-type natriuretic peptide (CNP)-mediated intracellular cGMP accumulation in a dose-dependent manner. With regard to specific activity for the neutralization, the antibody purified with IgG column was significantly stronger than that before the adsorption step, indicating that the purification of the antibody with IgG column was extremely effective to remove the contaminating anti-Fc antibody from anti-NPR-B antibody. Western blot analysis using the purified antibody revealed that while the native NPR-B exists as an oligomer, the truncated NPR-B lacking most of its cytoplasmic domain is a monomer. This finding suggests that the cytoplasmic region may be involved in the oligomerization of the receptor. The results in this study demonstrate that soluble IgG fusion protein is very effective and useful for generating specific antibodies to the proteins expressed on cell surface.
Asunto(s)
Anticuerpos/inmunología , Anticuerpos/farmacología , Guanilato Ciclasa/inmunología , Receptores del Factor Natriurético Atrial/inmunología , Animales , Anticuerpos/aislamiento & purificación , Formación de Anticuerpos , Especificidad de Anticuerpos , Western Blotting , Células CHO , Cricetinae , GMP Cíclico/biosíntesis , Guanilato Ciclasa/análisis , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Conejos , Receptores del Factor Natriurético Atrial/análisis , Proteínas Recombinantes de Fusión/inmunología , Solubilidad , Transfección , Vacunas Sintéticas/farmacologíaRESUMEN
Monoclonal antibodies (mAbs) against human natriuretic peptide receptor-A (NPR-A) or NPR-B were produced using NPR-expressing Chinese hamster ovary (CHO) cells and soluble chimeric NPRs consisting of the extracellular domain of each receptor fused to Fc region of human IgG. Three anti-NPR-A mAbs, designated as A144, A397 and A416, bound to human NPR-A but not to NPR-B, while an anti-NPR-B mAb B136 reacted with human NPR-B but not with NPR-A. Competition analysis with the anti-NPR-A mAbs revealed that two mAbs, A144 and A416, recognize an identical or the adjacent site of the receptor and that A397 is directed against another epitope. No anti-NPR-A mAb affected binding of atrial natriuretic peptide (ANP) to NPR-A, while the anti-NPR-B mAb B136 inhibited binding of C-type natriuretic peptide (CNP) to NPR-B. Inhibition of the ligand-binding by B136 is specific in that the mAb showed no effect on the binding of ANP to NPR-A. B136 also blocked CNP-mediated intracellular cGMP accumulation in NPR-B-expressing cells. These results suggest that the region recognized by B136 may be related to the ligand-binding region of NPR-B. NPR-A- and NPR-B-expressing cells were selectively detected by immunostaining using the mAbs. These findings demonstrate that the mAbs will be useful to elucidate the role of the natriuretic peptides and their receptors in normal and disease states in humans [correction of human].
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Guanilato Ciclasa/inmunología , Receptores del Factor Natriurético Atrial/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Factor Natriurético Atrial/metabolismo , Secuencia de Bases , Western Blotting , Células CHO , Clonación Molecular , Cricetinae , ADN Complementario , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Expresión Génica , Humanos , Inmunohistoquímica , Datos de Secuencia MolecularRESUMEN
A monoclonal antibody (mAb) against human tumor necrosis factor-alpha (TNF-alpha), designated 3B10, neutralizes biological activity of TNF-alpha, while another anti-TNF-alpha mAb 10F10 does not. In Western blot analysis, both mAbs bound to SDS-denatured TNF-alpha, indicating that the epitopes recognized by the mAbs are sequential but not conformational. To map precisely the epitopes of the mAbs, 76 overlapping octapeptides corresponding to an entire sequence of TNF-alpha were synthesized and their abilities to react with the mAbs were examined by enzyme-linked immunosorbent assay (ELISA). 3B10 bound to only one peptide at position 81-88 of TNF-alpha, SRIAVSYQ, whereas 10F10 was reactive with three overlapping peptides, ANALLANG (33-40), ALLANGVE (35-42), and LANGVELR (37-44). These results demonstrate that the 81-88 and 37-40 regions are important for the recognition of TNF-alpha by 3B10 and by 10F10, respectively. In solid-phase ELISA, 3B10 inhibited the binding of TNF-alpha to soluble TNF receptors, sTNF-RI and sTNF-RII. In contrast, 10F10 exerted little effect on the binding. TNF-alpha was detected by sandwich-type ELISA where 3B10 alone was used for both capture and detection, suggesting that 3B10 did not interfere with the trimer formation of TNF-alpha. The results obtained in this study suggest that the 81-88 region of TNF-alpha may participate in the receptor binding and that 3B10 neutralizes the activities of TNF-alpha by blocking the region.