RESUMO
To reduce the risk of transmission of hepatitis A virus, an Octapharma produced factor VIII (fVIII) concentrate treated with solvent detergent (FVIII-SD) was further pasteurized after purification. This product, Octavi SDPlus (FVIII-SDP), was marketed in Europe in 1993 to 1995. Inhibitors appeared from September to October, 1995, in 12 of 109 previously treated German hemophilia A patients. A study of similarly treated Belgian patients, who also developed inhibitors, had shown antibodies to the fVIII light chain (domains A3-C1-C2) only. In the present study, the epitope specificity of 8 German inhibitor plasmas was also found to be restricted to the light chain. In radioimmunoprecipitation assays to localize the light chain epitope(s), antibody binding to heavy chain (domains A1-A2-B) was 11-148 fold lower than to the C2 domain, and binding to recombinant A3-C1 was barely detectable. These results were supported by >95% neutralization of a high responder inhibitor titer by the C2 domain.
Assuntos
Fator VIII/imunologia , Fator VIII/uso terapêutico , Hemofilia A/tratamento farmacológico , Hemofilia A/imunologia , Epitopos Imunodominantes/imunologia , Adolescente , Adulto , Anticorpos/imunologia , Especificidade de Anticorpos , Criança , Alemanha , Humanos , Radioimunoensaio , VírusRESUMO
Factor VIII (fVIII) is a protein cofactor essential for blood coagulation, and it binds in the factor Xase complex to factors IXa, X, and phospholipid. In about 30% of severe hemophilia A patients, treatment with fVIII leads to production of anti-fVIII antibodies. Anti-fVIII autoantibodies also rarely appear in normal individuals. Those antibodies that inactivate fVIII (inhibitors) prevent optimal fVIII therapy. Inhibitor epitopes were previously localized to the fVIII A2, A3, and C2 domains and to an acidic amino acid region between A1 and A2. Such anti-fVIII antibodies interfere with fVIII binding to components of the factor Xase complex and prevent blood coagulation. When total anti-fVIII titers were determined for each fVIII domain in 43 inhibitor plasmas by immunoprecipitation (IP) and inhibitor neutralization assays, the anti-light chain (LCh) antibody titer was highest, anti-A2 was intermediate, and anti-A1 and anti-B were low. The relative immunogenicity of the fVIII domains in hemophilic and autoantibody inhibitor patients was similar.
Assuntos
Autoanticorpos/sangue , Fator VIII/química , Fator VIII/imunologia , Hemofilia A/sangue , Anticorpos Monoclonais , Coagulação Sanguínea , Fator VIII/antagonistas & inibidores , Hemofilia A/imunologia , Humanos , Substâncias Macromoleculares , Testes de Neutralização , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologiaRESUMO
Interleukin-6 (IL-6) is a pleiotropic cytokine having several functions, including the regulation of immunologic and inflammatory responses. It is produced by many cell types, including lymphocytes, macrophages, and fibroblasts, and is believed to play a major role in pulmonary fibrosis, a condition resulting from expansion of the fibroblast compartment and the accumulation of extracellular matrices secreted primarily by fibroblasts. Production of IL-6 by lung fibroblasts has been well documented; however, it was not known whether all murine lung fibroblasts secreted IL-6 or only subsets thereof. Previous studies in our laboratory have shown that murine lung fibroblasts can be divided into subpopulations based on Thy 1 expression. These subpopulations, Thy 1+ and Thy 1-, differ in morphology, expression of surface markers, and function. IL-6 mRNA was detected in both Thy 1+ and Thy 1- murine fibroblasts and clones using reverse transcriptase polymerase chain reaction (RT-PCR). Interestingly, semi-quantitative RT-PCR and Northern blot analysis demonstrated that IL-6 mRNA was down-regulated in confluent fibroblast cultures versus cultures in log phase growth. Also, IL-6 activity was detected in the supernatants of murine lung fibroblast lines and clones using an IL-6-dependent hybridoma assay. Hybridoma proliferation was inhibited by the addition of a neutralizing anti-mouse IL-6 antibody, indicating that the activity was indeed due to IL-6. The lung fibroblasts expressed IL-6 receptors on their surface as determined by flow cytometry using a rat anti-mouse IL-6 receptor antibody (15A7).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Fibroblastos/metabolismo , Interleucina-6/biossíntese , Pulmão/metabolismo , Antígenos Thy-1/análise , Animais , Divisão Celular , Fibroblastos/química , Fibroblastos/citologia , Interleucina-6/metabolismo , Pulmão/citologia , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Testes de Neutralização , RNA Mensageiro/biossíntese , Receptores de Interleucina/análise , Receptores de Interleucina-6RESUMO
The treatment of hemophilia A patients has improved in this decade by production of recombinant factor VIII (FVIII) in mammalian cells, which has significantly reduced contamination by infectious agents. A remaining serious problem in 25-30% of patients with severe hemophilia A is the appearance of antibodies that inactivate FVIII. The present therapy for this condition is frequent treatment with high doses of FVIII to induce tolerance, which is defined as a negative Bethesda assay. Serial plasma samples from 50 evaluable patients in a large study of 72 previously untreated patients were tested to determine whether tolerized patients have actually lost all their anti-FVIII by using a 10 fold more sensitive immunoprecipitation (IP) method of measuring all anti-FVIII antibodies. Six of the 22 patients with inhibitors were given tolerance therapy, and 3 of them were only partially tolerized as determined by IP assay. Seven patients with 1-11 BU/mL lost their inhibitor spontaneously, while 5 non-inhibitor patients with low level immune responses similarly became antibody negative. In a smaller study, a tolerized patient with 0 BU/mL had remaining non-inhibitory antibody levels high enough to reduce the FVIII half-life significantly. Plasmas from 2 patients who were not tolerized, were tested by the IP assay for the A2 and/or C2 domain specificity of the anti-FVIII over time. The antibodies detected were directed against both the heavy and light chains of FVIII, and they increased and decreased at the same rate before and during tolerance therapy.
Assuntos
Fator VIII/imunologia , Hemofilia A/tratamento farmacológico , Tolerância Imunológica , Isoanticorpos/sangue , Especificidade de Anticorpos , Fator VIII/administração & dosagem , Hemofilia A/complicações , Hemofilia A/imunologia , Humanos , Epitopos Imunodominantes , Testes de PrecipitinaRESUMO
Factor VIII (fVIII) functions as a cofactor of factor IXa in the intrinsic pathway of blood coagulation. Its absence or abnormality causes the bleeding disorder hemophilia A. About 23% of hemophiliacs who receive therapeutic fVIII infusions develop antibodies that inhibit its activity. We previously showed by inhibitor neutralization assays that the fVIII A2 and C2 domain polypeptides contain common inhibitor epitopes. Often hemophilic inhibitor plasmas were partially neutralized by C2 and more completely neutralized by fVIII light chain (A3-C1-C2), suggesting the presence of an additional major inhibitor epitope(s) within the A3-C1 domains. In immunoprecipitation assays, 17 of 18 inhibitor IgGs bound to recombinant 35S-A3-C1. Amino acids 1811-1818 of the A3 domain comprise a binding site for factors IX and IXa. Three inhibitor IgGs prevented binding of factor IXa to fVIII light chain, and the binding of each IgG to light chain was competed by A3 peptide 1804-1819. The generation of factor Xa by the fVIIIa/fIXa complex in a chromogenic assay was prevented by these inhibitors. Therefore, we propose that another important mechanism of fVIII inactivation by human inhibitors is the prevention of fVIIIa/fIXa association.
Assuntos
Anticorpos Bloqueadores/imunologia , Fator IX/metabolismo , Fator VIII/metabolismo , Anticorpos Bloqueadores/farmacologia , Sítios de Ligação/imunologia , Epitopos/imunologia , Fator IX/imunologia , Fator VIII/imunologia , Humanos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
We have determined that murine lung fibroblasts are divisible into two major subpopulations based on expression of Thy 1. Twenty-four to fifty-three percent of freshly isolated lung cells displayed Thy 1 and were separated using FACS into Thy 1+ and Thy 1- fractions for morphologic examination. Analysis by electron microscopy revealed that both the Thy 1+ and Thy 1- fractions contained fibroblasts. Freshly isolated lung cells cultured for 2 wk consisted of greater than 95% fibroblasts, with 28 to 49% displaying Thy 1. These cells were sorted by FACS into Thy 1+ and Thy 1- lines that maintained a stable phenotype over many weeks and that were used as a source to obtain stable fibroblast clones. Adherent pulmonary fibroblasts are not phagocytic and lack the markers of macrophages, dendritic cells, B lymphocytes, and T lymphocytes (with the exception of Thy 1). Interestingly, the Thy 1- fibroblasts spread more and contained a more extensive microfilament and microtubule network than did the spindly and often lipid-containing Thy 1+ population. Both populations of fibroblasts synthesized collagen. Class I MHC expression was very low on Thy 1+ and Thy 1- fibroblasts, but high levels were displayed after gamma-IFN treatment. Most exciting was the unexpected finding that only the Thy 1- lines and clones displayed class II MHC (Ia) in response to treatment with gamma-IFN. Moreover, only the Thy 1- fraction (gamma-IFN-treated) presented antigen to T lymphocyte clones, an observation that suggests that this subset of cells may be involved primarily in promoting chronic lung inflammation, which is associated with developing fibrosis. Thus, two populations of pulmonary fibroblasts exist, defined by the expression of Thy 1, distinguishing morphology, inducibility for Ia expression, and antigen-presenting function. It should now be possible, using these characteristics, to ascertain the role of pulmonary fibroblast subpopulations in developing fibrosis.
Assuntos
Antígenos de Superfície/análise , Genes MHC da Classe II , Pulmão/citologia , Animais , Linhagem Celular , Células Cultivadas , Colágeno/biossíntese , Fibroblastos/citologia , Fibroblastos/imunologia , Fibroblastos/fisiologia , Citometria de Fluxo , Imunofluorescência , Antígenos de Histocompatibilidade Classe II/análise , Interferon gama/farmacologia , Pulmão/imunologia , Pulmão/fisiologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos , Fagocitose/efeitos dos fármacos , Proteínas Recombinantes , Linfócitos T/imunologia , Antígenos Thy-1RESUMO
The purpose of this investigation was to ascertain whether the alpha 6 integrin subunit was present on normal murine lung cells and fibroblasts, and if so, to determine the identity of the beta subunit coordinately expressed with alpha 6 and whether or not these integrin subunits could be regulated by cytokines. Previously, our laboratory isolated populations of Thy 1+ and Thy 1- fibroblasts from normal murine lung tissue. These cells differed in surface marker expression and in response to, and production of, pro-inflammatory cytokines. Research defining the properties of these two populations has led to the hypothesis that unique groups of fibroblasts exist within the murine lung. Though alpha 6 beta 1 is known to be expressed by platelets, lymphocytes, and epithelial cells, its presence and regulation on lung fibroblast subsets has not been explored. We now report the following findings: 1) the laminin receptor, alpha 6 beta 1, is present on 20-30% of freshly isolated normal murine lung cells in all three murine strains tested; 2) established Thy 1+ and Thy 1- murine lung fibroblast subsets and clones constitutively express alpha 6 beta 1 at varied levels; and 3) alpha 6 beta 1 expression on fibroblast lines and clones can be upregulated by treatment with IFN-gamma or TNF-alpha. Since these T cell and macrophage derived cytokines are known to be present during an inflammatory response, upregulation of alpha 6 beta 1 expression may facilitate recruitment and retention of lung fibroblasts in regions undergoing repair.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Integrinas/metabolismo , Interferon gama/farmacologia , Pulmão/metabolismo , Receptores de Laminina/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Antígenos de Superfície/análise , Sequência de Bases , Citocinas/farmacologia , Fibroblastos , Expressão Gênica/efeitos dos fármacos , Técnicas In Vitro , Pulmão/citologia , Glicoproteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , RNA Mensageiro/genética , Proteínas Recombinantes/farmacologia , Antígenos Thy-1RESUMO
Pulmonary fibrosis resulting from diverse etiologies is characterized by proliferation of fibroblasts and excessive accumulation of interstitial collagen. Whether fibrosis is associated with selective expansion of fibroblast subpopulations differing in amounts or types of collagens synthesized is unknown. We have previously isolated lines and clones of normal murine lung fibroblasts based on the presence of the Thy 1 surface antigen. These subpopulations differ in morphology, growth characteristics, and display of class II major histocompatibility complex antigens (R.P. Phipps, D.P. Penney, P. Keng, H. Quill, A. Paxhia, S. Derdak, and M. E. Felch. Am. J. Respir. Cell Mol. Biol. 1: 65-74, 1989). We evaluated the amounts and types of collagen and fibronectin synthesized by Thy 1+ (Fib2-T-3+) and Thy 1- (Fib2-T-4-) lung fibroblast lines and clones. Thy 1+ fibroblast line synthesized two- to threefold more collagen and noncollagen protein than the Thy 1- line. In contrast, both the Thy 1+ and Thy 1- lines synthesized similar amounts of fibronectin. Thy 1+ and Thy 1- lines and clones expressed mRNA for alpha 1(I)-and alpha 1(III)-procollagen and synthesized both types (predominantly type I and lesser amounts of type III) of collagen, protein, and mRNA. The fibroblast clones varied significantly in total collagen and fibronectin production, with one Thy 1- clone (D3) synthesizing the largest amount of collagen but relatively little fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Antígenos de Superfície/análise , Colágeno/biossíntese , Fibronectinas/biossíntese , Pulmão/metabolismo , Glicoproteínas de Membrana/análise , Animais , Colágeno/genética , Fibroblastos/imunologia , Fibroblastos/metabolismo , Pulmão/citologia , Fenótipo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Antígenos Thy-1RESUMO
The finding that human factor VIII (fVIII) inhibitor antibodies with C2 domain epitopes interfere with the binding of fVIII to phosphatidylserine (PS) suggested that this is the mechanism by which they inactivate fVIII. We constructed a recombinant C2 domain polypeptide and demonstrated that it bound to all six human inhibitors with fVIII light chain specificity. Thus, some antibodies within the polyclonal anti-light chain population require only amino acids within C2 for binding. Recombinant C2 also partially or completely neutralized the inhibitor titer of these plasmas, demonstrating that anti-C2 antibodies inhibit fVIII activity. Immunoblotting of a series of C2 deletion polypeptides, expressed in Escherichia coli, with inhibitor plasmas showed that the epitopes for human inhibitors consist of a common core of amino acid residues 2248 through 2312 with differing extensions for individual inhibitors. The epitope of inhibitory monoclonal antibody (MoAb) ESH8 was localized to residues 2248 through 2285. Three human antibodies and anti-C2 MoAb NMC-VIII/5 bound to a synthetic peptide consisting of amino acids 2303 through 2332, a PS-binding site, but MoAb ESH8 did not. These antibodies also inhibited the binding of fVIII to synthetic phospholipid membranes of PS and phosphatidylcholine, confirming that the blocked epitopes contribute to membrane binding as well as binding to PS. In contrast, MoAb ESH8 did not inhibit binding. As the maximal function of activated fVIII in the intrinsic factor Xase complex requires its binding to a phospholipid membrane, we propose that fVIII inhibition by anti-C2 antibodies is related to the overlap of their epitopes with the PS-binding site. MoAb ESH8 did not inhibit fVIII binding to PS-containing membranes, suggesting the existence of a second mechanism of fVIII inhibition by anti-C2 antibodies.