RESUMEN
The antigenic domains of histone 5 (H5), a highly conserved variant of histone 1 (H1), were studied in relation to their reactivity with autoantibodies found in the sera of patients with systemic lupus erythematosus (SLE) and drug-induced lupus (DIL). While some H5 antibodies cross-react with H1, adsorption and immunoblotting studies have identified H5-specific antibodies as well. After proteolytic cleavage of H5 peptides, the reactivity of sera from these patients was tested by Western immunoblotting. All SLE (9/9) and DIL (7/7) sera bound an antigenic determinant in the carboxyl (C) terminus of H5 while none of the sera bound to the amino (N) terminus or the central hydrophobic domain. Although the reactivity of DIL sera with the purified H5 peptides was weaker than that of SLE sera, the antigenic domains bound by both groups of sera were the same. These observations demonstrate that the H5 domains reacting with DIL sera are restricted to the carboxyl terminus and are therefore no less restricted than those reacting with SLE sera. Further, the potential epitopes in the carboxyl terminus of H5 do not have a high degree of sequence identity with known mammalian peptides.
Asunto(s)
Autoanticuerpos/sangre , Histonas/inmunología , Lupus Eritematoso Sistémico/inmunología , Secuencia de Aminoácidos , Animales , Pollos , Reacciones Cruzadas , Epítopos/inmunología , Humanos , Hidralazina/efectos adversos , Immunoblotting , Lupus Eritematoso Sistémico/inducido químicamente , Datos de Secuencia Molecular , Péptidos/inmunología , Procainamida/efectos adversosRESUMEN
Calf thymus histone 1 (H1) was cleaved by chemical and enzymatic methods and the resulting polypeptides were fractionated by high-performance cation-exchange. Up to 1 mg of H1 polypeptides were loaded onto a 50 x 5 mm I.D. cation-exchange column and fractionated to greater than 95% purity in less than 30 min. This is the first report on the separation of H1 polypeptides by a strong cation-exchange matrix. In addition, the high-performance cation-exchange chromatography protocol represents a significant decrease in fractionation time when compared to conventional ion-exchange and gel filtration chromatography. The utility of this procedure is shown when the H1 peptides purified by the protocol were used to define antigenic domains of H1 band by procainamide-induced lupus and idiopathic systemic lupus erythematosus. The majority of the sera tested by enzyme-linked immunoassay (ELISA) reacted to the C-terminal peptides of H1 indicating this to be the major antigenic domain of H1.
Asunto(s)
Cromatografía Liquida/métodos , Histonas/aislamiento & purificación , Animales , Bromosuccinimida/farmacología , Bovinos , Quimotripsina/farmacología , Ensayo de Inmunoadsorción Enzimática , Histonas/análisis , Histonas/efectos de los fármacos , Histonas/inmunología , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/inmunología , Procainamida , Cloruro de Sodio , Trombina/farmacología , Timo/análisisRESUMEN
HL-60 is a human promyelocytic cell line which was found to be capable of differentiating toward a macrophage-like or granulocyte-like phenotype. Histochemical analysis demonstrated that incubation of cells in the presence of phorbol myristate acetate (PMA) or 1,25-dihydroxyvitamin D3 induced varying degrees of monocytic differentiation, while incubation in the presence of retinoic acid (RA) or dimethyl sulfoxide (DMSO) induced granulocytic differentiation. The differentiation induced by PMA, RA, and to a lesser extent DMSO, was accompanied by the induction of plasminogen activator inhibitor expression. mRNA analysis of control and PMA-induced cultures revealed the induction of a 2-kb message in treated cells which hybridized with a PAI-2-specific oligonucleotide probe. This is consistent with the literature concerning the expression of PAI by macrophages and granulocytes. No hybridization was detected with a PAI-1 specific probe. Expression of PAI by cells of hematopoietic origin appears to be associated with differentiation or stimulation of committed cells. Furthermore, PAI-2 expression by HL-60 cells is not restricted to one specific hematopoietic lineage. Since other cells of hematopoietic origin such as platelets express PAI-1, future studies using pluripotential cell lines could provide information on the initial events of lineage commitment and gene expression.
Asunto(s)
Diferenciación Celular , Regulación Neoplásica de la Expresión Génica , Genes , Proteínas Gestacionales/genética , Células Tumorales Cultivadas/citología , Línea Celular , Genes/efectos de los fármacos , Humanos , Leucemia Promielocítica Aguda , Inactivadores Plasminogénicos , Proteínas Gestacionales/biosíntesis , Proteínas Gestacionales/aislamiento & purificación , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
Rabbit antibodies have been raised against rat liver nuclear envelopes. An enzyme-linked immunosorbent assay (ELISA) demonstrated high titer antiserum specific for the nuclear envelope preparation. Immunocytochemical studies showed that the antiserum stained the nuclear envelopes, but not intra-nuclear components of HEp-2 (human malignant epithelial) cells. When electrophoretically separated peptides were tested by immunoblotting techniques, the rabbit antiserum specifically stained proteins with molecular masses of 26 and 28 kD. These peptides had similar mobilities to purified histone 1 (H1). Indeed purified calf thymus H1 recognized the antiserum. The antigens are not loosely bound to the nuclear envelope, as they could not be extracted with low salt. Therefore, we have established that the 26 and 28 kD nuclear envelope peptides are not contaminants of the nuclear envelope preparation and that they express determinants that are immunologically cross-reactive with purified H1, but not with intra-nuclear H1.
Asunto(s)
Histonas/análisis , Hígado/ultraestructura , Membrana Nuclear/análisis , Animales , Antígenos/inmunología , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Sueros Inmunes/inmunología , Masculino , Peso Molecular , Membrana Nuclear/inmunología , Ratas , Ratas EndogámicasRESUMEN
Antibodies from 5 patients with systemic sclerosis reacted with an antigen localized to the metaphase chromatin, the cleavage furrow and the midbody of anaphase and telophase HEp-2 cells. The titer of antimidbody antibodies ranged from 1:160 to 1:1280. Four patients had systemic sclerosis and one had idiopathic Raynaud's phenomenon. In situ biochemical characterization of the antigen revealed that it was resistant to DNase I, micrococcal nuclease and RNase A, but was sensitive to trypsin treatment. The antigen remained insoluble in 400 mM acetic acid but was extracted from the cells with 400 mM hydrochloric acid. The antibody was not seen in sera from 2500 normal female blood donors, 120 patients with systemic lupus, 60 patients with rheumatoid arthritis, 15 patients with linear scleroderma or 25 patients with Raynaud's disease.
Asunto(s)
Antígenos/inmunología , Células/inmunología , Cromatina/inmunología , Esclerodermia Sistémica/sangre , Línea Celular , Células/ultraestructura , Humanos , Metafase , Esclerodermia Sistémica/inmunologíaRESUMEN
The sera of patients with systemic lupus erythematosus (SLE) and drug-induced lupus (DIL) were used to study the antigenic regions of histone 1 (H1) that bind antibodies in these sera. ELISA and immunoblotting techniques using enzymatically and chemically derived peptides of H1 showed that the major antigenic domain is in the carboxyl (C) terminus. None of the 24 SLE or 11 DIL sera bound to the central hydrophobic polypeptide by ELISA. The reactivity of DIL sera with the purified H1 peptides was similar to that observed with SLE sera. This observation suggests a common immune pathway for DIL and SLE.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Histonas/inmunología , Lupus Eritematoso Sistémico/inmunología , Aminoácidos/análisis , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Lupus Eritematoso Sistémico/inducido químicamente , Péptidos/análisis , ProcainamidaRESUMEN
An indirect immunoenzyme (IIE) kit to detect autoantibodies in the sera of patients with systemic rheumatic diseases has been evaluated and compared to a conventional indirect immunofluorescence (IIF) assay. Both IIF and IIE were performed on a human epithelial cell line (HEp-2) using sera categorized on the basis of their autoantibody specificity. The correlation coefficient between the two assays was greater than 0.77 for all autoantibodies except antimitochondrial antibodies, which had higher end-point titers with the IIE kit. The inter- and intratest variability of IIE and IIF was comparable, differing by no more than one tube dilution. The IIE test had less background staining, allowing for better resolution and easier interpretation of staining patterns. IIE assay in the form of a commercially available kit is a reliable alternative to IIF.
Asunto(s)
Autoanticuerpos/análisis , Enfermedades Autoinmunes/inmunología , Enfermedades Reumáticas/inmunología , Línea Celular , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Juego de Reactivos para DiagnósticoRESUMEN
The sera of patients with systemic lupus erythematosus (SLE) and drug-induced lupus (DIL) were used to study the antigenic epitopes on nuclear histones that bind antibodies in these sera. ELISA and immunoblotting techniques showed that antibodies from both patient groups bound all classes of intact histone: H1 greater than H2B greater than H2A greater than H3 greater than H4. The different classes of histone were enzymatically or chemically cleaved to produce a series of peptide fragments which were then used to map the reactive epitopes by ELISA and immunoblotting. Ten of 11 DIL sera and 11 of 12 SLE sera bound the carboxy and amino terminal peptides. Only one sera of each group bound to the central hydrophobic polypeptide. The reactivity of DIL sera with fractionated histone polypeptides was similar to that observed with SLE sera. This observation suggests that the histone epitopes reacting with DIL sera are no less restricted than those reacting with SLE.