RESUMEN
BACKGROUND AND OBJECTIVES: A human recombinant monoclonal anti-RhD IgG may be useful to prevent RhD allo-immunization. Roledumab is such an antibody with a glycosylation pattern optimized for biological activity. The objective of the study was to assess the safety and pharmacokinetics of roledumab in healthy RhD-negative volunteers. MATERIALS AND METHODS: A total of 46 subjects received doses of 30-3000 µg i.v. of roledumab or placebo using a double-blind escalating single-dose design; 12 of these subjects also received 300 µg i.m. of roledumab. Subjects were followed for 6 months after administration. Serum roledumab concentrations were determined using flow cytometry. RESULTS: Fourteen treatment-emergent adverse events related to treatment were reported in nine subjects, with no apparent difference in their frequency or nature after placebo or roledumab administration. No anti-roledumab antibodies were detected. AUC(last) increased from 4·4 ng/ml.day at 30 µg i.v. to 2257 ng/ml.day at 3000 g i.v. The t(½) ranged from 18 to 22 days, and the absolute bioavailability after i.m. administration was between 73% and 80%. CONCLUSION: Roledumab is safe and well tolerated in healthy RhD-negative volunteers and shows a pharmacokinetic profile similar to that of polyclonal anti-RhD immunoglobulin.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Fragmentos Fc de Inmunoglobulinas/inmunología , Receptores de IgG/inmunología , Sistema del Grupo Sanguíneo Rh-Hr/inmunología , Adulto , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Estudios Cruzados , Método Doble Ciego , Femenino , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Masculino , Persona de Mediana Edad , Receptores de IgG/genética , Receptores de IgG/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Adulto JovenRESUMEN
In the past 20 years, numerous monoclonal anti-D antibodies have been developed in order to replace the human plasma derived anti-D immunoglobulins, using different in vitro functional assays as screening methods. Some of these monoclonal antibodies have been evaluated in exploratory in vivo clinical trials, notably for their ability to mediate the clearance of D-positive red cells. A review of these reported trials is presented and the results are analyzed in the light of the newly published hypothesis conferring an important role to some Fc-FcgammaR interactions and to the glycosylation-dependent potency of the monoclonal anti-D antibodies.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Isoanticuerpos/uso terapéutico , Isoinmunización Rh/prevención & control , Animales , Anticuerpos Monoclonales/química , Citotoxicidad Celular Dependiente de Anticuerpos , Ensayos Clínicos como Asunto , Citotoxicidad Inmunológica , Femenino , Glicosilación , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/uso terapéutico , Isoanticuerpos/química , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Animales , Modelos Biológicos , Pan troglodytes , Fagocitosis , Embarazo , Procesamiento Proteico-Postraduccional , Receptores de IgG/inmunología , Globulina Inmune rho(D) , Relación Estructura-Actividad , Resultado del TratamientoRESUMEN
At present, rhesus prophylaxis concerns RhD negative pregnant women, even though 30 to 40% of them are bearing a RhD negative child. Knowing the RhD fetal genotype could change this quite irrational practice of prophylaxis (exposing many more women than needed to blood derived products) without reducing its efficacy. RhD fetal genotype determined on amniotic fluid has an excellent sensitivity. Presence of silent D genes slightly impairs its specificity which remains acceptable. However women have to be informed of possible false positives. Fetal RhD genotyping on maternal blood is more complex. Sensitivity is good from 10 GW and excellent after 15 GW. In case of a first negative result, it is recommended to control fetal RhD on a second sample drawn a few weeks later. Another new perspective for rhesus prophylaxis is the attempt to substitute polyclonal IgG anti-D into human monoclonal IgG anti-D. The main difficulty is to elaborate monoclonal antibodies with a capacity to neutralize RhD positive red blood cells equivalent to those of polyclonal anti-D. A new generation of antibodies is in process and preliminary clinical results are suggesting a possible use of these monoclonal antibodies for future rhesus prophylaxis but long-term follow-up is required to draw further conclusions.
Asunto(s)
Sangre Fetal/inmunología , Enfermedades Fetales/genética , Isoinmunización Rh/prevención & control , Sistema del Grupo Sanguíneo Rh-Hr/genética , Globulina Inmune rho(D)/uso terapéutico , Eritroblastosis Fetal/diagnóstico , Eritroblastosis Fetal/genética , Femenino , Enfermedades Fetales/diagnóstico , Genotipo , Humanos , Recién Nacido , Reacción en Cadena de la Polimerasa , Embarazo , Diagnóstico Prenatal , Globulina Inmune rho(D)/inmunología , Sensibilidad y EspecificidadRESUMEN
Sixty-four IgG Rh monoclonal antibodies (Mabs) submitted to the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens were characterised and tested in quantitative functional assays at five laboratories. The biological assays measured the ability of anti-D to mediate phagocytosis or extracellular lysis of RBC by IgG Fc receptor (Fc gamma R)-bearing effector cells. Interactions of RBC pre-sensitised with anti-D (EA-IgG) with monocytes in chemiluminescence (CL) assays were found proportional to the amount of IgG anti-D on the RBC. Using antibodies to inhibit Fc gamma RI, Fc gamma RII or Fc gamma RIII, the only receptor utilised in the monocyte CL and ADCC assays for interactions with EA-IgG1 was found to be Fc gamma RI. In these assays, enhanced interactions were promoted by EA-IgG3 and additional Fc gamma receptors may have contributed. IgG2 anti-D was not reactive in these assays and EA-IgG4 promoted weak reactions through Fc gamma RI. A macrophage ADCC assay showed that haemolysis of EA-IgG3 was greater than that of EA-IgG1, mediated mainly through Fc gamma RIII. In ADCC assays using lymphocytes (NK cells) as effector cells and papainised RBC target cells, only a minority of IgG1 anti-D Mabs were shown to be able to mediate haemolysis in the presence of monomeric IgG (AB serum or IVIg). These interactions were mediated solely through Fc gamma RIII. Haemolysis via Fc gamma RIII may depend on the presence of certain sugars on the oligosaccharide moiety of IgG. Most Mabs (IgG1, IgG2, IgG3 and IgG4) elicited intermediate, low or no haemolysis in these assays. Blocking studies indicated that low activity IgG1 and IgG4 anti-D utilised only Fc gamma RI. Other IgG1 and IgG3 Mabs appeared to promote haemolysis through Fc gamma RI and Fc gamma RIII while IgG2 was inhibited by Mabs to both Fc gamma RII and Fc gamma RIII, suggesting a variety of Fc gamma R are utilised for anti-D of low haemolytic activity. Excellent agreement between the results of the lymphocyte ADCC assays and antibody quantitation was observed between the participating laboratories.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Inmunoglobulina G/inmunología , Isoanticuerpos/inmunología , Receptores de IgG/inmunología , Anticuerpos Monoclonales/química , Citotoxicidad Celular Dependiente de Anticuerpos , Glicosilación , Hemólisis , Humanos , Inmunoglobulina G/química , Inmunoglobulinas Intravenosas/inmunología , Isoanticuerpos/química , Células Asesinas Naturales/inmunología , Mediciones Luminiscentes , Linfocitos/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Oligosacáridos/inmunología , Fagocitosis , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Receptores de IgG/clasificación , Globulina Inmune rho(D)RESUMEN
Erythropoietin (Epo) is a 166 amino acids protein containing three N-glycosylation sites (Asn-24, Asn-38, and Asn-83) and 1 O- glycosylation site (Ser-126) and involved in the regulation of the level of red blood cells. Today, only one recombinant human Epo (rHuEpo), produced in CHO cell line, is extensively used in therapy to cure severe anemia. The structure of the glycan chains of this rHuEpo slightly differ of those of the urinary human Epo (uHuEpo), considered as the natural Epo molecule. In an attempt to produce a rHuEpo as close as possible to the uHuEpo, Epo gene was expressed in a human lymphoblastoid cell line, named RPMI 1788. In order to fully characterize the Epo-RPMI, structural characterizations of the protein skeleton as well as glycan chains were undergone. As expected, the amino acid sequence of the Epo-RPMI conformed to that of uHuEpo. Surprisingly, the structure of some N-glycan chains, as mainly determined by ESI-MS, revealed some unusual characteristics. Thus, 80% of N-glycans possess a bisecting GlcNAc residue, 25% bear a second fucose residue which is present, in a large part, in a sialyl Le(x)motif, and 13% contain more than three LacNAc repeats (up to five per molecule). Despite these unusual structural characteristics, the data concerning the in vitro and in vivo biological activities were not impaired when compared to Epo-CHO and uHuEpo.
Asunto(s)
Eritropoyetina/química , Eritropoyetina/metabolismo , Linfocitos/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Línea Celular , Fraccionamiento Químico , Cricetinae , Eritropoyetina/genética , Glicopéptidos/análisis , Glicopéptidos/aislamiento & purificación , Glicosilación , Humanos , Metilación , Datos de Secuencia Molecular , Monosacáridos/análisis , Ácido N-Acetilneuramínico/metabolismo , Oligosacáridos/análisis , Farmacocinética , Proteínas Recombinantes , Antígeno Sialil Lewis X , Relación Estructura-ActividadRESUMEN
Therapeutic use of human monoclonal antibodies has so far been hampered by their poor availability. Recent developments in recombinant DNA technologies are expected to fill this gap; the variable and constant sequences of antibodies can be selected independently and then subsequently joined to express whole antibodies. We assessed here the potential of this methodology to obtain novel anti-D antibodies with improved biological characteristics. The sequences coding for heavy and light chains of two anti-Rh(D) monoclonal antibodies (one IgG1 and one IgG3) were isolated and co-expressed into murine myeloma cell P3X63.Ag8.653, either directly (parental antibodies) or after exchange of constant heavy chain sequences (class-switched antibodies). Parental antibodies produced either by transfectomas or by hybridomas behaved similarly in analysis of biochemical, binding and effector properties. Class-switched antibodies displayed altered functional properties over parental antibodies. Of particular interest, one of them showed improved phagocytosis potencies over both parental antibodies. Additionally, these results indicate that functional properties do not always simply reflect the addition of properties of constant and variable parts but that interactions between constant and variable regions may interfere.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Cambio de Clase de Inmunoglobulina , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Afinidad de Anticuerpos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Línea Celular , Clonación Molecular , Vectores Genéticos , Humanos , Fagocitosis/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Células Tumorales CultivadasRESUMEN
The mechanism whereby passive Rh (D) immunoglobulins suppress the fetomaternal alloimmunization is still unclear. New in vitro tests are needed to better characterize the functional properties of polyclonal anti-Ds. The DAF assay was developed to monitor the antibody-dependent cell-mediated cytotoxicity (ADCC) and the phagocytosis of anti-Rh (D)-sensitized RBCs by effector cells. The principle of this test is based on the oxydization of the 2,7-diaminofluorene (DAF) by the pseudoperoxidase activity of free hemoglobin. The reaction is proportional to the hemoglobin concentration. This test was performed to determine and emphasize the efficacy of different polyclonal anti-D immunoglobulin preparations to mediate lysis and phagocytosis of sensitized RBCs by human peripheral mononuclear cells. The functional properties of different human RhD monoclonal antibodies were also analyzed and compared. The test was found to be convenient to perform and allowed the avoidance of radioactive labelling of RBCs for ADCC studies. It is mainly useful for the direct quantitation of phagocytosis.
Asunto(s)
Sistema del Grupo Sanguíneo Rh-Hr/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Colorimetría , Relación Dosis-Respuesta Inmunológica , Fluorenos , Humanos , Cinética , FagocitosisRESUMEN
Eight murine anti-TNF alpha monoclonal antibodies (mAb) were produced after immunization of BALB/c mice with rhTNF alpha. Six of these mAbs were able to neutralize cytotoxic activity of TNF alpha against L929 cells. Two other mAbs had no neutralizing effect. Epitope mapping studies were performed by ELISA and by using a BIAcore system (Pharmacia). The described mAbs were allowed to define 4 different epitopes on TNF alpha. Three of them were involved in the binding of TNF alpha with its receptor (cytotoxic neutralization of TNF alpha). Another epitope was defined by non-neutralizing mAbs.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Mapeo Epitopo/métodos , Factor de Necrosis Tumoral alfa/inmunología , Animales , Técnicas Biosensibles , Pruebas Inmunológicas de Citotoxicidad , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos BALB C , Pruebas de NeutralizaciónRESUMEN
Three major yolk proteins of 140, 100, 80 KD and a faint band of 440 KD were determined by gradient gel electrophoresis in the mature eggs of Helix aspersa. Polyclonal and monoclonal antibodies were raised against mature oocyte extracts. The binding sites of these rabbit and hybridoma antibodies with the different yolk protein components were identified with a combination of WESTERN blotting, ELISA, immunofluorescence and immunogold staining. All these techniques demonstrated materials immunologically similar to vitellins in the hemolymph and in the glandular cells of the digestive gland. The data suggest that, for its vitellogenesis, the garden-snail utilizes a heterosynthetic mechanism similar to that known in oviparous animals. The vitellogenins would be produced by the digestive gland.
Asunto(s)
Proteínas del Huevo/análisis , Caracoles Helix/química , Oocitos/química , Precursores de Proteínas/análisis , Animales , Anticuerpos Monoclonales , Sistema Digestivo/química , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Hemolinfa/química , Inmunohistoquímica , Vitelogeninas/análisisRESUMEN
In a multicenter pilot study, 32 patients showing steroid-resistant acute graft-versus-host disease (GVHD) were treated by in vivo administration of anti-interleukin-2 (IL-2) receptor monoclonal antibody (MoAb B-B10). Twenty-three patients received marrow from HLA-matched related donors, four from matched unrelated donors and five from partially matched related donors. The overall grade of GVHD was II in 16 patients, III in two, and IV in five. Five milligrams of B-B10 MoAb was infused in bolus daily for 10 days and then every second day for a further 10 days in an attempt to reduce GVHD recurrence. No clinical side effects were noted during the B-B10 treatment period. A complete response (CR) acute GVHD was achieved in 21 patients (65.6%). Six patients (18.7%) showed partial improvement (PR) and 5 patients (15.6%) no response (NR). A significant factor associated with GVHD response was the delay between the onset of the GVHD and the first day of B-B10 infusion. The earlier B-B10 was introduced, the greater the probability of CR (P = .03). There was no correlation between the serum B-B10 level and GVHD response (P = .69). There was, however, a significant correlation between the clinical response and the B-B10 kinetics as a function of time: serum B-B10 levels attained a plateau level more rapidly in the CR group than in the PR/NR group. Among the 26 complete and partial evaluable responders, GVHD recurred in 10 cases (38.4%). Host anti-B-B10 MoAb immune response occurred in only one (7.1%) of the 14 patients analyzed. Fourteen of the 32 patients (43.7%) are currently alive between 2 and 14 months after GVHD treatment with B-B10 was completed.
Asunto(s)
Corticoesteroides/inmunología , Anticuerpos Monoclonales/administración & dosificación , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Receptores de Interleucina-2/inmunología , Adolescente , Adulto , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Formación de Anticuerpos , Niño , Preescolar , Evaluación de Medicamentos , Resistencia a Medicamentos/inmunología , Femenino , Francia , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/mortalidad , Humanos , Lactante , Masculino , Estudios Multicéntricos como Asunto , Proyectos Piloto , Receptores de Interleucina-2/sangreRESUMEN
A monoclonal antibody against the IL2-Receptor (B-B10) has been developed which inhibits activated T cells. Large batches of B-B10 have been purified from ascites and were checked for the absence of endotoxins, murine DNA and virus before being released for clinical use. Thirty-two patients with corticosteroid-resistant acute Graft versus Host Disease have been treated with B-B10. No side effects were observed and 68% of the patients showed full response to treatment.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Enfermedad Injerto contra Huésped/terapia , Receptores de Interleucina-2/inmunología , Animales , Células Cultivadas , Niño , Humanos , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Estudios Multicéntricos como Asunto , Proyectos Piloto , Conejos , Linfocitos T/efectos de los fármacosRESUMEN
Rabbit articular chondrocytes in confluent monolayer cultures were treated with D-Penicillamine (D-Pen) during 3 or 5 days. The [35S]-sulfate incorporation in neosynthesized proteoglycans was not modified by D-Pen doses ranging from 50 to 800 micrograms/ml. After treatment during 5 days with D-Pen concentrations of 50 or 400 micrograms/ml, the chemical characteristics of proteoglycans from medium and cell-layer were determined. The aggregation capacity of proteoglycans from medium, the monomer molecular size, the glycosaminoglycan chain length and the relative rates of the different glycosaminoglycans (chondroitins, chondroitin 6-sulfate, chondroitin 4-sulfate, hyaluronic acid) remained unchanged. These results suggest that D-Pen does not alter some of the cartilage mechanical properties due to the presence of proteoglycans.
Asunto(s)
Cartílago Articular/efectos de los fármacos , Penicilamina/farmacología , Proteoglicanos/biosíntesis , Animales , Cartílago Articular/metabolismo , Células Cultivadas , Cromatografía en Gel , Matriz Extracelular/metabolismo , Glicosaminoglicanos/biosíntesis , Glicosaminoglicanos/aislamiento & purificación , Conformación Molecular , Peso Molecular , Proteoglicanos/aislamiento & purificación , ConejosRESUMEN
In rabbit articular chondrocytes, phorbol myristate acetate (PMA), 1,2-dioctanoyl-sn-glycerol (DG) and calcium ionophore (A23187), reduced the proteoglycan synthesis, in a dose-dependent manner. The combined treatment by PMA and A23187 resulted in an enhanced inhibition of proteoglycan production, indicating a synergistic effect. In presence of PMA or A23187, the release of prostaglandin E2 (PGE2) was dramatically increased. The addition of indomethacin and BW755c to chondrocytes stimulated by PMA or A23187, suppressed the liberation of PGE2, but did not stop the decrease of proteoglycan synthesis.
Asunto(s)
Calcimicina/farmacología , Cartílago Articular/metabolismo , Prostaglandinas E/metabolismo , Proteoglicanos/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , 4,5-dihidro-1-(3-(trifluorometil)fenil)-1H-pirazol-3-amina , Animales , Ácidos Araquidónicos/fisiología , Diglicéridos/farmacología , Dinoprostona , Sinergismo Farmacológico , Indometacina/farmacología , Pirazoles/farmacología , ConejosRESUMEN
Arthritic-rendered rabbits were treated in vivo with 50 mg/kg D-penicillamine (D-Pen) daily during 4 months. Glycosaminoglycan (GAG) synthesis by synovial fibroblast cultures from D-Pen treated and untreated normal or arthritic animals was studied. Cells from arthritic-rendered animals synthesized hyaluronic acid (HA) at the same rate as cells isolated from control rabbits. When D-Pen was administered to arthritic-rendered rabbits, it significantly inhibited GAG production by fibroblasts. The hyaluronate synthetase activity determined on synovial fibroblast homogenates, however, was not modified whatever the treatment undergone by the rabbits. Moreover, synovial fibroblasts from arthritic rabbits treated or not with D-Pen generally synthesized HA with a high molecular weight similar to that produced by D-Pen treated or untreated control animals.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Artritis/tratamiento farmacológico , Glicosaminoglicanos/biosíntesis , Glicosiltransferasas , Proteínas de la Membrana , Penicilamina/uso terapéutico , Membrana Sinovial/efectos de los fármacos , Transferasas , Proteínas de Xenopus , Animales , Artritis Experimental/metabolismo , Células Cultivadas , Cromatografía de Afinidad , Cromatografía DEAE-Celulosa , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Glucuronosiltransferasa/análisis , Hialuronano Sintasas , Masculino , Peso Molecular , Pronasa/metabolismo , Conejos , Membrana Sinovial/citología , Membrana Sinovial/metabolismoRESUMEN
In order to get further insight into the mechanism of D-penicillamine action on synovial tissue collagen synthesis, fibroblasts derived from drug-treated arthritic rabbits were cultured and labelled with radioactive proline. No evident correlation was found between the amount of newly synthesized collagen and the previous treatment of animals. In contrast, the prolyl-hydroxylase activity was reduced in cells from rabbits receiving D-penicillamine. This finding suggests that culture conditions may influence the collagen-synthesizing potentiality of the synovial fibroblasts without changing the level of enzyme activity. Therefore, the prolyl-hydroxylase activity could be considered here as a more reliable reflection of the in vivo situation. The ratio of type III to type I procollagens, as estimated by DEAE-cellulose chromatography, showed a rise in cultures from D-penicillamine-treated rabbits as compared to controls. This result indicates that long-term administration of the drug may alter the collagen composition of synovial tissue matrix in rheumatoid arthritis. The question remains, however, whether this alteration contributes to the beneficial effect of the drug.
Asunto(s)
Artritis/metabolismo , Colágeno/biosíntesis , Penicilamina/farmacología , Membrana Sinovial/metabolismo , Animales , Artritis/tratamiento farmacológico , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Técnicas In Vitro , Masculino , Procolágeno/biosíntesis , Procolágeno-Prolina Dioxigenasa/metabolismo , Conejos , Membrana Sinovial/efectos de los fármacosRESUMEN
Collagen production was investigated in cultured rabbit synovial fibroblasts exposed in vitro to D-penicillamine (D-Pen). The results show that these cells are rather insensitive to the drug since only a slight increase of the collagen amount secreted was observed for 48-h exposure to concentrations of 200-400 micrograms/ml. However, fibroblasts derived from the synovium of arthritic rabbits proved to be more susceptible to D-Pen, responding by a marked increase of collagen secretion even for concentrations of 50 micrograms/ml. This finding suggests that synovial fibroblasts of arthritic patients, probably stimulated by the inflammation process, could be target cells for the D-Pen action. The activities of 4-prolyl-hydroxylase (4-PH) and galactosylglucosyl-transferase (GGT) were assayed in the same cultures. A correlation has been found between the 4-PH activity and the collagen amount produced. In contrast, no alteration in the level of GGT on exposure to D-Pen was detected. Finally, D-Pen was shown to reduce in vitro the production of collagen-inhibiting factors by phytohaemagglutinin-stimulated mononuclear cells. This effect was associated with an inhibition of the release of monocyte cell factor (MCF/interleukin-1), suggesting that D-Pen could indirectly affect synovial collagen synthesis by interfering with interleukin-1 secretion.
Asunto(s)
Colágeno/biosíntesis , Interleucina-1/biosíntesis , Penicilamina/farmacología , Membrana Sinovial/efectos de los fármacos , Animales , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Conejos , Membrana Sinovial/metabolismoRESUMEN
A partially purified monocyte factor, with Interleukin-1 properties (MCF/IL-1), enhances the proteoglycan synthesis of human neonatal articular chondrocytes in culture, and changes the repartition of these macromolecules between medium and cell layer. The size of the proteoglycan monomers, the length of the glycosaminoglycan chains and the respective levels of chondroitin-6-sulfate, chondroitin-4-sulfate and non sulfated chondroitin remain unchanged under MCF/IL-1 exposures. The addition of indomethacin reduces the stimulation effect by 60-70% only, suggesting that the MCF/IL-1 action is partially dependent on prostaglandins but seems also related to mechanisms distinct from the cyclooxygenase pathway.
Asunto(s)
Cartílago Articular/metabolismo , Interleucina-1/farmacología , Monocitos/fisiología , Proteoglicanos/biosíntesis , Cartílago Articular/citología , Células Cultivadas , Glicosaminoglicanos/análisis , Humanos , Indometacina/farmacología , Peso Molecular , Monocitos/análisis , Monocinas , Prostaglandinas/fisiología , Proteínas/farmacología , Proteoglicanos/análisisRESUMEN
Treatment of cultured human synovial cells with a mononuclear cell factor (MCF) enhanced their ability to synthesize glycosaminoglycans (GAG), but GAG repartition between extracellular, pericellular and intracellular compartments was found to be the same as in control. Hyaluronic acid (HA) production, which represents 80-90% of all secreted GAG, was stimulated 2 1/2-3-fold, but the HA molecular weight was not modified. The MCF increased the hyaluronate synthetase activity of synovial cells in similar proportions. Actinomycin D inhibited the increase in hyaluronate synthetase activity produced by MCF, indicating that this increase involves new synthesis of mRNA. Stimulation of both HA synthesis and hyaluronate synthetase activity by MCF was suppressed by 10(-4)-10(-5) M indomethacin (an inhibitor of cyclo-oxygenase), suggesting that MCF effect is prostaglandin-dependent.
Asunto(s)
Glicosiltransferasas , Ácido Hialurónico/biosíntesis , Proteínas de la Membrana , Proteínas/farmacología , Membrana Sinovial/metabolismo , Transferasas , Proteínas de Xenopus , Anciano , Células Cultivadas , Dactinomicina/farmacología , Femenino , Glucosamina/metabolismo , Glucuronosiltransferasa/metabolismo , Humanos , Hialuronano Sintasas , Indometacina/farmacología , Masculino , Persona de Mediana Edad , Peso Molecular , Monocinas , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/enzimologíaRESUMEN
Human PHA-stimulated mononuclear cells produce a factor which inhibits synovial cell collagen and non-collagen protein synthesis, whereas it enhances hyaluronic acid (HA) production. Indomethacin (10(-4)-10(-6) M), a cyclo-oxygenase inhibitor, suppresses this effect, suggesting that the mechanism is prostaglandin-mediated. The active material, of apparent molecular weight 12 000-20 000, also displays the properties of the mononuclear cell factor (MCF) previously described by others, since its stimulates collagenase and PGE2 release by the cultured synovial cells. Furthermore, it co-purifies with interleukin 1 (IL 1) as shown by lymphocyte-activating factor activity. This strongly suggests that IL 1 could be responsible for some (or all) the effects observed on MCF-exposed synovial cells. From these data, we deduce the possibility that mononuclear cells may participate in limiting synovial collagen deposition in rheumatoid arthritis.