RESUMEN
A 53-year-old man with a known history of alcohol abuse was admitted to hospital after a minor collapse. He had a laceration to the forehead and three rib fractures. Laboratory blood-analysis showed raised non-cholestatic liver-enzyme levels suggesting alcohol-abuse. On history taking the patient was shown to have been suffering from personality changes and multiple hallucinogenic episodes for the previous two years. He had been seen and evaluated by a neurologist to that effect. The patient's family had accepted the situation and thought of it as dementia, probably caused by alcohol abuse. He had been treated for atrial flutter and was taking acenocoumerol, atorvastatin, quinapril and metoprolol 50 mg twice daily as medication. During admission the patient appeared to be suffering from a delirium with complex visual and auditory hallucinations, for which he was given haloperidol. Revision of medication use led to the stopping of metoprolol, which had been started two years earlier. Within 24 hours the delirium had disappeared completely. There was spontaneous fall in the liver enzymes. At his last follow-up, the patient had had no psychiatric symptoms for 6 months. The relationship between stopping metoprolol and the disappearance of the psychosis appeared to be a causal one and this is supported by the limited literature available on this subject.
Asunto(s)
Antagonistas Adrenérgicos beta/efectos adversos , Delirio/inducido químicamente , Metoprolol/efectos adversos , Antagonistas Adrenérgicos beta/uso terapéutico , Alcoholismo/complicaciones , Humanos , Hígado/enzimología , Masculino , Metoprolol/uso terapéutico , Persona de Mediana EdadRESUMEN
We evaluated inhibitor formation in a group of patients with mild haemophilia A caused by an Arg593 to Cys mutation. A remarkably high cumulative inhibitor incidence of 14% over 22 years was observed. Three of 49 patients developed transient, low-titre inhibitors, which remained below 2.0 BU mL(-1). Four patients with an Arg593 to Cys mutation developed high-titre inhibitors (>5.0 BU mL(-1)). Three of these patients have been described previously. In this study, we characterized inhibitory antibodies in a fourth patient with high-titre inhibitors. Epitope mapping studies revealed that antibodies were predominantly directed to the A2 domain of factor VIII. We addressed the role of human leucocyte antigen (HLA) class II alleles in inhibitor development in patients with an Arg593 to Cys mutation by HLA genotyping. In the group of inhibitor patients raised frequencies of HLA-DRB1*01 and HLA-DQB1*05 were observed that did not reached statistical significance. Our data suggest that inhibitor development in mild haemophilia A patients with an Arg593 to Cys mutation is not linked to HLA class II profile.
Asunto(s)
Anticuerpos/análisis , Factor VIII/genética , Genes MHC Clase II/genética , Hemofilia A/genética , Mutación/genética , Mapeo Epitopo , Factor VIII/antagonistas & inhibidores , Factor VIII/inmunología , Genotipo , HumanosRESUMEN
A well-known complication of factor VIII replacement therapy in patients with hemophilia A is the development of inhibitory antibodies. Several studies have demonstrated the presence of a binding site for factor VIII inhibitors in the A3 domain. Six different human monoclonal single-chain variable domain antibody fragments (scFv) directed toward the A3-C1 domains of factor VIII have been isolated, using phage display technology. Sequence analysis revealed that the V(H) domains of 2 scFv were encoded by germline gene segments from the V(H)1 gene family and 4 by germline gene segments belonging to the V(H)3 gene family. Epitope mapping of the scFv was performed, using a series of hybrid factor VIII/factor V light chain fragments. This analysis revealed that 5 of 6 scFv were directed against a region encompassing amino acid sequence Q1778-D1840 in the A3 domain, a previously identified binding site for factor VIII inhibitors. Only 2 of 5 scFv directed against amino acid sequence Q1778-D1840 inhibited the procoagulant activity of factor VIII. Our results define the properties of human antibodies directed against region Q1778-D1840 in the A3 domain. Binding of one, noninhibitory scFv was independent of the region Q1778-D1840, suggesting the presence of an additional binding site for anti-factor VIII antibodies in the A3-C1 domains of factor VIII.
Asunto(s)
Epítopos/inmunología , Factor VIII/inmunología , Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes de Inmunoglobulinas , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Isoanticuerpos/inmunología , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Epítopos/química , Factor VIII/química , Humanos , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Isoanticuerpos/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Biblioteca de Péptidos , Estructura Terciaria de Proteína , Proteínas Recombinantes/inmunología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de SuperficieRESUMEN
We describe a previously healthy woman who at the age of 44 years developed a factor VIII inhibitor, that over the years increased to a maximum level of 3600 Bethesda units (BU) mL(-1) in 1978. The epitope specificity of the factor VIII inhibitor was investigated and antibodies directed against the A2 and C2 domains of factor VIII were detected. The majority of these antibodies were of subclass IgG4. Over the years, the inhibitor titre gradually decreased and in 1989, the inhibitor could no longer be detected. Shortly after, the patient developed autoimmune haemolytic anaemia. A possible link between the disappearance of factor VIII inhibitors and the development of other autoantibodies may be explained by concomitant development of anti-idiotypic antibodies that neutralize the activity of factor VIII inhibitors. We were unable to detect anti-idiotypic antibodies, which could explain the decline in factor VIII inhibitor titre in this patient.
Asunto(s)
Anemia Hemolítica Autoinmune/inmunología , Autoanticuerpos/inmunología , Factor VIII/inmunología , Anciano , Anemia Hemolítica Autoinmune/sangre , Animales , Autoanticuerpos/sangre , Femenino , Humanos , Ratones , Persona de Mediana EdadRESUMEN
Hemophilia A is a X-linked bleeding disorder that is caused by the functional absence of blood coagulation factor VIII. The bleeding tendency in hemophilia A patients can be corrected by the administration of plasma-derived or recombinant factor VIII concentrates. A serious complication in hemophilia care is the development of factor VIII neutralizing antibodies (inhibitors) that arise as a consequence of factor VIII replacement therapy. The majority of factor VIII inhibitors are directed toward epitopes located within the A2, A3, and C2 domains of factor VIII. In this article, we summarize current knowledge on the epitope specificity of factor VIII inhibitors. In addition, we will discuss recent information on the molecular characteristics of human anti-factor VIII antibodies generated by phage display technology.
Asunto(s)
Factor VIII/inmunología , Biblioteca de Péptidos , Secuencia de Bases , Epítopos/química , Hemofilia A/complicaciones , Hemofilia A/tratamiento farmacológico , Hemofilia A/inmunología , Humanos , Fragmentos de Inmunoglobulinas/química , Isoanticuerpos/química , Datos de Secuencia MolecularRESUMEN
One of the major binding sites for factor VIII inhibitors is located within the A2 domain. In this study, phage display technology was used to isolate 2 human monoclonal antibodies, termed VK34 and VK41, directed toward the heavy chain of factor VIII. The V(H) domain of a single-chain variable domain antibody fragment (scFv) VK34 is encoded by germline gene segment DP-10. Epitope-mapping studies revealed that scFv VK34 is directed against amino acid residues Arg(484)-Ile(508), a previously identified binding site for factor VIII inhibitors in the A2 domain. ScFv VK34 inhibited factor VIII activity with a titer of 280 BU/mg. The V(H) domain of VK41 was encoded by germline gene segment DP-47. A phage corresponding to VK41 competed with a monoclonal antibody for binding to amino acid residues Asp(712)-Ala(736) in the acidic region adjacent to the A2 domain. Reactivity of VK41 with a factor VIII variant in which we replaced amino acid residues Asp(712)-Ala(736) for the corresponding region of heparin cofactor II was strongly reduced. In addition, substitution of Tyr(718719723) for Phe abrogated binding of VK41 to factor VIII. ScFv VK41 did not inhibit factor VIII activity. This study not only defines the primary structure of human anti-factor VIII antibodies reactive with the A2 domain, it also describes an antibody with an epitope not previously identified in the antibody repertoire of hemophilia patients with an inhibitor. (Blood. 2000;96:540-545)
Asunto(s)
Anticuerpos Monoclonales/genética , Factor VIII/inmunología , Biblioteca de Péptidos , Secuencia de Aminoácidos , Anticuerpos Monoclonales/química , Especificidad de Anticuerpos , Bacteriófagos/genética , Sitios de Unión de Anticuerpos , Mapeo Epitopo , Escherichia coli , Factor VIII/química , Hemofilia A/inmunología , Humanos , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/genética , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Anticuerpos de Cadena ÚnicaRESUMEN
A serious complication in hemophilia care is the development of factor VIII (FVIII) neutralizing antibodies (inhibitors). The authors used V gene phage display technology to define human anti-FVIII antibodies at the molecular level. The IgG4-specific, variable, heavy-chain gene repertoire of a patient with acquired hemophilia was combined with a nonimmune, variable, light-chain gene repertoire for display as single-chain variable domain antibody fragments (scFv) on filamentous phage. ScFv were selected by 4 rounds of panning on immobilized FVIII light chain. Sequence analysis revealed that isolated scFv were characterized by V(H) domains encoded by germline genes DP-10, DP-14, and DP-88, all belonging to the V(H)1 gene family. All clones displayed extensive hypermutation and were characterized by unusually long CDR3 sequences of 20 to 23 amino acids. Immunoprecipitation revealed that all scFv examined bound to the C2 domain of FVIII. Furthermore, isolated scFv competed with an inhibitory murine monoclonal antibody for binding to the C2 domain. Even though scFv bound FVIII with high affinity, they did not inhibit FVIII activity. Interestingly, the addition of scFv diminished the inhibitory potential of patient-derived antibodies with C2 domain specificity. These results suggest that the epitope of a significant portion of anti-C2 domain antibodies overlaps with that of the scFv isolated in this study. (Blood. 2000;95:558-563)
Asunto(s)
Autoanticuerpos/genética , Factor VIII/inmunología , Genes de Inmunoglobulinas , Hemofilia A/inmunología , Inmunoglobulina G/genética , Cadenas Pesadas de Inmunoglobulina/genética , Adulto , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Autoanticuerpos/inmunología , Sitios de Unión , Factor VIII/química , Femenino , Hemofilia A/sangre , Hemofilia A/genética , Humanos , Inmunoglobulina G/inmunología , Región Variable de Inmunoglobulina/genética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Alineación de SecuenciaRESUMEN
IFN-gamma-inducible protein-10 (IP-10) is a chemokine, which plays an important role in mediating inflammation by attracting activated T cells, and it has been demonstrated in inflammatory skin diseases and cutaneous T cell lymphomas. Keratinocytes can abundantly produce IP-10 mRNA after IFN-gamma treatment. In this study we explored possibilities to downregulate IP-10 expression using human cultured keratinocytes as a model system. Decreased IP-10 mRNA levels were found using specific inhibitors of protein kinase (PK)-C (H-7 and Calphostin C). Moreover, depletion of PK-C by pretreatment of the cells with phorbol myristate (PMA) also down-regulated IP-10 mRNA expression. In addition, elevated cAMP levels were shown to inhibit IP-10 mRNA expression as could be concluded from experiments with forskolin and W-7, substances which, directly or indirectly, raise the intracellular cAMP level. With Genistein, an inhibitor of tyrosine kinase, the IFN-gamma-induced IP-10 mRNA expression was also found to be diminished. These data suggest that inhibitors of the IP-10 mRNA expression in cultured keratinocytes may be potentially of clinical relevance to suppress inflammatory processes in the skin.
Asunto(s)
Quimiocinas CXC/genética , AMP Cíclico/metabolismo , Queratinocitos/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , ARN Mensajero/metabolismo , Células Cultivadas , Quimiocina CXCL10 , AMP Cíclico/fisiología , Humanos , Masculino , Proteína Quinasa C/fisiología , Proteínas Tirosina Quinasas/fisiologíaRESUMEN
Recent studies suggest that certain missense mutations associated with mild to moderate haemophilia A predispose to inhibitor development. In this study, we present a longitudinal analysis of the epitope specificity of an inhibitor that developed in a mild haemophiliac with an Arg593-->Cys mutation. Immunoprecipitation studies revealed the presence of antibodies directed towards the light chain and A2 domain of factor VIII. Limited reactivity was observed with metabolically labelled C2 domain. Almost complete inhibitor neutralization was achieved upon addition of A2 domain. Binding of the inhibitor was not affected by the presence of the Arg593-->Cys substitution in the recombinant A2 fragment. Evaluation of the epitope specificity of anti-factor VIII antibodies in plasma samples obtained at different time-points of inhibitor development revealed initial development of a low titre inhibitor directed towards the A2 domain and factor VIII light chain. A second period of factor VIII replacement therapy resulted in a dramatic rise in factor VIII inhibitor titre, which maintained their original epitope specificity. Based on the results of this and previous studies (Fijnvandraat et al., 1997; Thompson et al., 1997) it is argued that inhibitor development in patients with the Arg593-->Cys mutation may proceed via a similar mechanism.
Asunto(s)
Anticuerpos/inmunología , Factor VIII/inmunología , Hemofilia A/genética , Hemofilia A/inmunología , Mutación Missense , Anciano , Anticuerpos/sangre , Mapeo Epitopo , Epítopos/inmunología , Factor VIII/uso terapéutico , Hemofilia A/sangre , Hemofilia A/tratamiento farmacológico , HumanosRESUMEN
The development of inhibitory antibodies to factor VIII in patients affected by a mild form of hemophilia A (factor VIII > 0.05 IU/mL) is considered a rare event. In this study, we evaluated the relationship between genotype and anti-factor VIII antibody formation in a patient with mild hemophilia A. Mutation analysis showed that a missense mutation in the factor VIII gene leading to replacement of Arg593 by Cys in the A2 domain of factor VIII was associated with hemophilia A in this patient. The anti-factor VIII antibodies present in the patient's plasma were characterized using metabolically labeled factor VIII fragments expressed in insect cells. The anti-factor VIII antibodies, composed of subclasses IgG2 and IgG4, reacted with both the fragment corresponding to the factor VIII heavy chain and the A2 domain. The Arg593 --> Cys substitution was introduced into the cDNA encoding the A2 domain of factor VIII and the resulting construct was expressed in insect cells. Strikingly, the metabolically labeled A2 domain carrying the Arg593 --> Cys mutation was not recognized by the anti-factor VIII antibodies present in the plasma of the patient. These data indicate that the anti-factor VIII antibodies are exclusively directed against exogenous factor VIII. This strongly suggests that the Arg593 --> Cys substitution results in recognition of wild-type factor VIII as nonself and is thereby related to the formation of anti-factor VIII antibodies after factor VIII replacement therapy in this particular patient.
Asunto(s)
Factor VIII/genética , Factor VIII/inmunología , Hemofilia A/genética , Isoanticuerpos/biosíntesis , Mutación Puntual , Adulto , Autoantígenos/química , Autoantígenos/genética , Enfermedades en Gemelos , Epítopos/química , Epítopos/genética , Factor VIII/química , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Isoantígenos/química , Masculino , Gemelos MonocigóticosRESUMEN
Hospital resolution of mass casualty incidents can have difficulties involving "command and control" and information management, ineffective use of triage classes, and missed diagnostic procedures, leading to lower quality of care. A computer system has been developed to supply continuously updated group and patient data. The system uses barcoded identifiers to represent patients, injuries, facilities, and locations, in order to minimize errors and make exchange of data possible. The system communicates with the permanent hospital information system. This article reports the use of this technology during several experiments and real incidents. Computer registration based on bar codes, despite the greater number of items entered, still showed 25% fewer inaccuracies when compared with handwritten medical charts. Extensive training was shown to be unnecessary. Paramedical personnel judged the automated procedures to be an improvement during the admission of 143 evacuated patients.