RESUMEN
OBJECTIVE: Trigonella foenum-graecum L. (fenugreek) is widely used as a leafy vegetable and spice in China and North Africa. Recent studies have reported that fenugreek can reduce fatigue; however, its antifatigue mechanism remains unclear. Therefore, this study aimed to investigate the potential antifatigue effects of fenugreek extract (FE) on mitophagy and the underlying mechanisms. MATERIALS AND METHODS: We evaluated the potential effects of FE tablet on an exhaustive exercise-induced fatigue (EEF) rat model. Oxidative stress indicators and fatigue biomarkers in the serum and skeletal muscle were detected. Mitophagy and mitochondrial morphology were observed using transmission electron microscopy. The expression levels of mitochondrial autophagy-related proteins were detected using western blot and immunofluorescence. RESULTS: Compared with the model group, FE enhanced the activities of the antioxidant enzymes superoxide dismutase and glutathione peroxidase as well as total antioxidant capacity; however, it decreased the level of malondialdehyde in the serum and skeletal muscle after a 7-day treatment. Moreover, certain indicators of mitochondrial function, such as reactive oxygen species levels, ATP levels, cellular and mitochondrial Ca2+ levels, and ATPase activity, were significantly improved in the FE group compared with the model group. Finally, we found that mitophagy was induced by exhaustive exercise and inhibited by FE. Regarding mitochondrial autophagy-related proteins, the expression levels of LC3B, FUNDC1, PGAM5, PARKIN, and PINK1 in the skeletal muscle tissue were increased in the EEF group compared with the control group. After administration of FE and a positive control drug, a significant reversal in the expression of the above-mentioned proteins was noted. CONCLUSIONS: Our findings demonstrate that FE exerted antifatigue effects in the EEF rat model by regulating the mitophagy-related FUNDC1/LC3B signaling pathway rather than the PINK1/PARKIN signaling pathway.
Asunto(s)
Trigonella , Ratas , Animales , Trigonella/metabolismo , Antioxidantes/farmacología , Mitofagia , Ratas Wistar , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Músculo Esquelético/metabolismo , Fatiga , Comprimidos , Proteínas Quinasas , Proteínas Relacionadas con la Autofagia , Ubiquitina-Proteína Ligasas , Proteínas de la Membrana , Proteínas MitocondrialesRESUMEN
OBJECTIVE: To determine the clinical spectrum of disease in humans with mutations in the CD95 (Fas/ APO-1) receptor and to obtain mechanistic insight into the different clinical phenotypes observed. METHODS: Clinical information for each of the index cases, first-degree relatives, and any family members reported to have Canale-Smith syndrome (or another autoimmune disease) was gathered by direct interview, chart review, and verification of data by the physician or pathologist concerned. Apoptosis of activated T or B lymphocytes was induced by agonistic anti-CD95 antibodies and quantified by a cell death assay (propidium iodide staining in the subdiploid peak) or cell viability assay (alamar blue or 3H-thymidine incorporation). RESULTS: Evaluation of an additional 8 probands with novel heterozygous CD95 mutations revealed hypergammaglobulinemia and immune-mediated cytopenias in all patients, as well as urticarial rash, oral ulceration, lymphopenia, and peripheral neuropathy in some individuals. One patient (P4) had systemic lupus erythematosus (SLE) characterized by a World Health Organization class V lupus nephropathy, a recurrent, reversible multifocal central nervous system disorder, high-titer antiphospholipid autoantibodies, and autoimmune cytopenias. In the P4 pedigree, the father had reduced T and B cell apoptosis associated with a CD95 mutation, whereas an independent B cell apoptotic defect was demonstrated in maternal family members who did not have a CD95 mutation. Three cases of B cell lymphoma occurred in carriers of the CD95 mutation. CONCLUSIONS: CD95 mutations are associated with loss of regulation of B lymphocytes, which predisposes to systemic autoimmunity including SLE. The P4 family provides a model of the complex genetic and functional interactions that are required for the development of a lupus-like syndrome.
Asunto(s)
Lupus Eritematoso Sistémico/genética , Receptor fas/genética , Apoptosis , Enfermedades Autoinmunes/genética , Linfocitos B/citología , Preescolar , Salud de la Familia , Femenino , Humanos , Enfermedades Linfáticas/genética , Enfermedades Linfáticas/inmunología , Masculino , Mutación , Linaje , Síndrome , Linfocitos T/citologíaRESUMEN
Heterozygous mutations of the receptor CD95 (Fas/Apo-1) are associated with defective lymphocyte apoptosis and a clinical disease characterized by lymphadenopathy, splenomegaly, and systemic autoimmunity. From our cohort of 11 families, we studied eight patients to define the mechanisms responsible for defective CD95-mediated apoptosis. Mutations in and around the death domain of CD95 had a dominant-negative effect that was explained by interference with the recruitment of the signal adapter protein, FADD, to the death domain. The intracellular domain (ICD) mutations were associated with a highly penetrant Canale-Smith syndrome (CSS) phenotype and an autosomal dominant inheritance pattern. In contrast, mutations affecting the CD95 extracellular domain (ECD) resulted in failure of extracellular expression of the mutant protein or impaired binding to CD95 ligand. They did not have a dominant-negative effect. In each of the families with an ECD mutation, only a single individual was affected. These observations were consistent with differing mechanisms of action and modes of inheritance of ICD and ECD mutations, suggesting that individuals with an ECD mutation may require additional defect(s) for expression of CSS.
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Apoptosis/genética , Enfermedades Autoinmunes/genética , Enfermedades Linfáticas/genética , Mutación , Receptor fas/genética , Enfermedades Autoinmunes/patología , Preescolar , Femenino , Regulación de la Expresión Génica , Heterocigoto , Humanos , Enfermedades Linfáticas/patología , Masculino , EmbarazoRESUMEN
Mutations in the Fas receptor or its ligand (FasL) lead to lupus-like systemic autoimmune diseases in mice and in some humans. To determine whether a significant number of patients with systemic lupus erythematosus (SLE) have impaired FasL function, we compared T cell effector function by superantigen-activated CD4+ T cell lines or by anti-CD3- and IL-2-generated cytotoxic T cells. No differences were observed between SLE and normal control superantigen-derived CD4+ T cells in either the ability of these cells to up-regulate Fas expression or to induce apoptosis of the Fas-sensitive target B cells. When anti-CD3/IL-2-activated T cells were examined, SLE T cells had a modest reduction (-8%) in T cell cytotoxicity compared with normal controls, but the reduction was similar to the rheumatoid arthritis disease controls. A modest reduction in cytotoxicity was evident in both the Fas and perforin/granzyme pathways as determined by testing Fas-positive and -negative targets as well as by selective blockade of the perforin/granzyme pathway with concanamycin. These results indicate that no specific defects in FasL function are evident in the majority of SLE patients under the in vitro conditions tested. The proportional reduction in FasL and perforin/granzyme function in SLE and rheumatoid arthritis patients following anti-CD3/IL-2 stimulation most likely reflects subtle differences in activation in patient-derived vs normal control T cells.
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Linfocitos T CD4-Positivos/inmunología , Lupus Eritematoso Sistémico/inmunología , Glicoproteínas de Membrana/inmunología , Animales , Línea Celular , Proteína Ligando Fas , Humanos , Activación de Linfocitos , RatonesRESUMEN
BACKGROUND: The Canale-Smith syndrome is a childhood disorder characterized by lymphadenopathy and autoimmunity. The similarity between this syndrome and that in mice with the lymphoproliferation (lpr) phenotype or the generalized-lymphoproliferative-disease (gld) phenotype led us to investigate whether it too is caused by mutations of the Fas gene (lpr mice) or the Fas ligand (gld mice), which regulate apoptosis in lymphocytes. METHODS: We studied four patients with the syndrome and their families. T-lymphocyte phenotypes were analyzed, and the susceptibility of activated T cells to Fas-mediated apoptosis in vitro was determined. Mutations of Fas were sought by nucleotide-sequence analysis. RESULTS: Patients with the Canale-Smith syndrome had increased numbers of circulating double-negative T cells (>20 percent) and profoundly impaired apoptosis of activated T cells incubated with an anti-Fas antibody. Three novel Fas mutations were identified, all of which were heterozygous and predicted to impair signal transduction by Fas. Autoimmune manifestations of the disease, such as hemolytic anemia and thrombocytopenia, persisted into adolescence. Two patients followed into adulthood had intermittent lymphadenopathy, which diminished over time. Neoplasms developed in both, and one died of hepatocellular carcinoma at the age of 43. CONCLUSIONS: Patients with the Canale-Smith syndrome have mutations in Fas, which implicates this gene in the accumulation of lymphocytes and the autoimmunity characteristic of the syndrome.
Asunto(s)
Enfermedades Autoinmunes/genética , Mutación del Sistema de Lectura , Enfermedades Linfáticas/genética , Mutación Puntual , Linfocitos T , Receptor fas/genética , Adulto , Apoptosis , Enfermedades Autoinmunes/inmunología , Niño , Femenino , Humanos , Ligandos , Enfermedades Linfáticas/inmunología , Masculino , Datos de Secuencia Molecular , Linaje , Síndrome , Linfocitos T/inmunologíaRESUMEN
This study was undertaken to determine the role of antibodies against both recombinant Ro (r-Ro) and La (r-La) proteins and polypeptides derived from the recombinant La protein in predicting fetal and neonatal outcome in children at risk to develop neonatal lupus erythematosus (NLE). All sera were obtained in the perinatal period and quantitative ELISA assays were used. We collected 41 maternal sera within 2 months of delivery of a child with NLE (21 with congenital heart disease block (CHB) and 20 with dermatologic NLE) and 19 sera from anti-Ro and/or anti-La antibody-positive mothers with systemic lupus erythematosus (SLE) who delivered a child without NLE. All sera were tested for anti-r-La and anti-r-Ro antibodies by ELISA, and most sera were tested for antibodies directed against La polypeptides by immunoblot. We found significantly higher anti-r-La antibody levels in the sera from mothers of children with NLE compared with sera from mothers of unaffected children (0.67 +/- 0.43 versus 0.14 +/- 0.30; P < 0.0001). There was a statistically significant difference in the mean anti-r-La levels between the sera of mothers of children with CHB compared with dermatologic NLE (0.51 +/- 0.45 versus 0.83 +/- 0.37 respectively; P = 0.0091). When we examined antibodies directed against the recombinant 52-kD Ro protein, there was a statistically significant elevation of titres in the sera of mothers of NLE children (0.77 +/- 0.35) compared with non-NLE mothers (0.29 +/- 0.39; P < 0.0001). There was no difference in the r-Ro levels between mothers of children with dermatologic NLE compared with CHB (0.82 +/- 0.37 versus 0.71 +/- 0.74; P = 0.32). When we examined polypeptides derived from the recombinant La protein, the mean number of polypeptides recognized by sera from mothers of children with NLE was significantly higher than the mean number of polypeptides recognized by sera from mothers of unaffected children (5.1 +/- 0.54 versus 2.3 +/- 0.54 respectively; P < 0.001). More importantly, when we examined the individual polypeptides, we found that only sera from mothers of children with NLE and not from mothers of unaffected children recognized a polypeptide designated DD (30% versus 0%, respectively). These studies indicate that the autoantibody response to the Ro/La particle can differentiate sera from mothers of children with NLE and sera from mothers of unaffected children. Furthermore, there was a difference in the anti-La autoantibody response between mothers of children with CHB and dermatologic NLE.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Autoanticuerpos/inmunología , Autoantígenos/inmunología , Enfermedades del Recién Nacido/inmunología , Lupus Eritematoso Sistémico/congénito , ARN Citoplasmático Pequeño , Ribonucleoproteínas/inmunología , Western Blotting , Mapeo Epitopo , Femenino , Humanos , Recién Nacido , Lupus Eritematoso Sistémico/inmunología , Intercambio Materno-Fetal , Péptidos/inmunología , Embarazo , Proteínas Recombinantes , Antígeno SS-BRESUMEN
Fas-deficient lpr and gld mice develop lymphadenopathy due to the accumulation of T cells with an unusual double negative (DN) (CD4-CD8-) phenotype. Previous studies have shown that these abnormal cells are capable of inducing redirected lysis of certain Fc receptor-positive target cells. Since the Fas ligand (FasL) has recently been shown to be partly responsible for T cell-mediated cytotoxicity, lymph node cells from lpr and gld mice were examined for the expression of FasL mRNA. Northern blot analysis revealed that lymph node cells obtained from lpr and gld mice had a striking increase in the level of expression of FasL mRNA predominantly due to expression in the DN T cells. Furthermore, lpr, but not gld lymph node cells killed the B cell line, A20, in a Fas-dependent manner. These findings indicate that Fas mutations result in a massive up-regulation of FasL which, most likely, results from repetitive exposure to (self) antigen. This phenomenon could explain the lpr-induced wasting syndrome observed when lpr bone marrow-derived cells are adoptively transferred to wild-type recipients.
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Caquexia/fisiopatología , Enfermedad Injerto contra Huésped/fisiopatología , Glicoproteínas de Membrana/metabolismo , Factores de Edad , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Enfermedades Autoinmunes/fisiopatología , Secuencia de Bases , Citotoxicidad Inmunológica , Cartilla de ADN/química , Proteína Ligando Fas , Expresión Génica , Enfermedades Linfáticas/fisiopatología , Ratones , Ratones Endogámicos C3H , Ratones Mutantes , Datos de Secuencia Molecular , ARN Mensajero/genética , Subgrupos de Linfocitos T/metabolismo , Receptor fasRESUMEN
PURPOSE: To attempt transcorneal laser sclerostomy with the erbium laser and to control the flow of aqueous through this sclerostomy with a suture ligature. METHODS: A contact erbium laser was used to create sclerostomies through small corneal incisions in both eyes of eight rabbits. Prior to surgery, a Merocel sponge soaked in mitomycin-C (0. 4 mg/cc) was applied to the conjunctiva at the operative site in one eye of each rabbit for 5 min. In the perfused human autopsy eye, following the creation of transcorneal erbium laser sclerostomy, a ligature suture was placed at the limbus around the sclerostomy opening to limit fluid flow. RESULTS: Using the erbium laser probe and the transcorneal approach, patent sclerostomies were created in all eyes. Intraocular pressures were significantly lower in mitomycin-C-treated eyes up to four months postoperatively (p = 0. 05). Eyes not treated with mitomycin-C demonstrated failure of filtering blebs by 1 month postoperatively (mean bleb survival = 9. 3 days). All mitomycin-C-treated eyes showed evidence of bleb formation up to 4 months postoperatively. Histologic examination of transcorneal sclerostomies in rabbit eyes showed patent sclerostomies at 1 day postoperatively with minimal adjacent thermal damage. In perfused human autopsy eyes, intraocular pressure was maintained near preoperative levels following placement of a ligature suture around the sclerostomy and decreased with release of the suture. CONCLUSIONS: These results demonstrate that functional filtering blebs can be created via small transcorneal incisions using the erbium laser. Transconjunctival mitomycin-C produces lower postoperative intraocular pressures and prolongs bleb survival. Aqueous flow through the sclerostomy was controlled with a suture ligature.
RESUMEN
Antifibrosis agents have improved the success of glaucoma filtration surgery, although undesired side effects are not readily reversible and may present a major limitation in the use of these agents. Our purpose was to study the efficacy of cytosine arabinoside (Ara-C) as an adjunctive antimetabolite in glaucoma surgery in the rabbit, and reversal of toxicity due to this agent with the competitive inhibitor 2'-deoxycytidine. Posterior lip sclerectomy was performed in rabbit eyes treated with 15 mg subconjunctival Ara-C daily for 7 d then every other day for 7 d. Mean intraocular pressure was lower in eyes treated with Ara-C compared with controls at all time points following filtration surgery. On the 10th postoperative day, the mean intraocular pressure of control eyes (25.0 +/- 1.9 mm Hg) had returned to baseline levels, whereas the intraocular pressure of eyes treated with Ara-C was significantly lower (16.0 +/- 1.7 mm Hg) (P < 0.001). Bleb survival was also prolonged in the Ara-C-treated eyes. The major ocular side effect of Ara-C was corneal toxicity, with epithelial defects in 40% of eyes after 8 daily injections of 15 mg Ara-C. Reversal of toxicity was enhanced with 2'-deoxycytidine, with complete resolution of epithelial toxicity after 6.5 +/- 1.7 d following daily topical 10% 2'-deoxycytidine compared with 12.7 +/- 0.58 for control (P < 0.002). These results demonstrate that postoperative subconjunctival injection of Ara-C results in improved bleb function after filtration surgery in the rabbit. Recovery from corneal epithelial toxicity due to Ara-C is markedly enhanced with the competitive inhibitor 2'-deoxycytidine.
Asunto(s)
Córnea/efectos de los fármacos , Enfermedades de la Córnea/prevención & control , Citarabina/toxicidad , Citarabina/uso terapéutico , Desoxicitidina/farmacología , Cirugía Filtrante , Animales , Quimioterapia Adyuvante , Enfermedades de la Córnea/inducido químicamente , Citarabina/antagonistas & inhibidores , Epitelio/efectos de los fármacos , Glaucoma/tratamiento farmacológico , Glaucoma/cirugía , Inyecciones , Presión Intraocular , Conejos , EscleroplastiaRESUMEN
The heparinized-coated tube is widely used for arterial bypass, mainly in patients with aneurysms or trauma of the thoracic aorta. Its application in venous bypass was less frequent. We report a 26-year-old woman who sustained blunt abdominal injury which resulted in complete severance of the suprahepatic hepatic vein and multiple partial lacerations of the inferior vena cava. For hemostasis, total hepatectomy with interruption of the inferior vena cava was performed. During the anhepatic phase, end-to-end portocaval anastomosis and a bypass shunt using 9 mm Gott tube from the right femoral vein to the right atrium were set up for veno-venous bypass. Orthotopic liver transplantation was performed later. Gott tube was placed for 18 hours with good venous return. We concluded that heparinized-coated tube is good for veno-venous bypass with the following advantages: 1) simple operative procedure, 2) enough flexibility to be kept out of the operative field, 3) no anticoagulant is necessary, consequently less bleeding resulted.
Asunto(s)
Circulación Extracorporea/instrumentación , Vena Femoral/cirugía , Trasplante de Hígado/métodos , Hígado/lesiones , Heridas no Penetrantes/cirugía , Adulto , Circulación Extracorporea/métodos , Femenino , Atrios Cardíacos/cirugía , Heparina , Humanos , Intubación/instrumentación , Derivación Portocava Quirúrgica/métodosRESUMEN
Fas is a cell surface protein of the tumor necrosis factor receptor, nerve growth factor receptor, CD40 family, and is involved in the control of lymphocyte apoptosis. A mutation in the Fas gene in MRL/lpr mice results in massive lymphoproliferation (lpr) and accelerated autoimmunity. To further study the nature of this defect, Fas mRNA expression was evaluated by reverse transcriptase polymerase chain reaction as well as by Northern blotting. These studies revealed that the wild-type Fas message was produced at approximately 10-fold lower levels in the lpr compared with the ++ substrain of MRL mice. In addition to the wild-type transcript, lpr mice also synthesized chimeric transcripts containing an insertion of the early retrotransposon (ETn). Molecular cloning and nucleotide sequencing of a Fas-ETn chimeric cDNA suggested that the striking reduction in wild-type Fas mRNA levels and the presence of aberrant transcripts in MRL/lpr mice are most likely explained by the insertion of the ETn retrotransposon into an intron of the Fas gene and induction of alternative splicing involving the 5' ETn long terminal repeat.
Asunto(s)
Antígenos de Superficie/genética , Enfermedades Autoinmunes/genética , Elementos Transponibles de ADN , Mutación , ARN Mensajero/biosíntesis , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , ADN , Tejido Linfoide/metabolismo , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Homología de Secuencia de Ácido Nucleico , Receptor fasRESUMEN
The ribosomal P proteins are necessary for GTPase activity during protein synthesis. In addition to antibodies to the P proteins, sera from lupus patients contain anti-rRNA activity. To determine whether lupus antiribosomal sera recognize the region of 28S rRNA recently proposed to form part of the ribosomal GTPase center, an rRNA fragment corresponding to nucleotides (nt) 1922-2020 was transcribed in vitro and tested for antigenicity. 18 of 24 (75%) lupus sera containing anti-P antibodies, but only 2 of 24 (8%) lupus sera without anti-P, immunoprecipitated this rRNA fragment (p less than 0.001). The binding was specific, since no significant differences were observed between anti-P positive and negative lupus sera in binding to the RNA fragment transcribed in the antisense orientation or to a control region of rRNA. The majority of sera tested protected a rRNA fragment of approximately 68 nucleotides. To evaluate the fine specificity of the anti-28S antibodies, deletions and site-directed mutations were made in the RNA fragment. The anti-28S antisera required nt 1944-1955 for recognition and were remarkably sensitive to destabilizing as well as nondestabilizing mutations in the stems of the RNA fragments. Detection of antiprotein and anti-RNA antibodies directed against a functionally related domain in the ribosome, together with the remarkable specificity of anti-28S antibodies, strongly suggests a direct role for this region of the ribosome in initiating and/or maintaining antiribosomal autoantibody production.
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Autoanticuerpos/inmunología , Lupus Eritematoso Sistémico/inmunología , ARN Ribosómico 28S/inmunología , Proteínas Ribosómicas/inmunología , Especificidad de Anticuerpos , Secuencia de Bases , Clonación Molecular , Análisis Mutacional de ADN , GTP Fosfohidrolasas/inmunología , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Fosfoproteínas/inmunología , Pruebas de Precipitina , Homología de Secuencia de Ácido Nucleico , Relación Estructura-ActividadRESUMEN
Nucleotide sequencing of HeLa genomic DNA amplified by the polymerase chain reaction revealed 696-bp and 841-bp introns within the gene encoding the proteins SmB and SmB', respectively. Since splice consensus sequences were observed flanking the 145-bp insertion detected in the cDNA encoding SmB, we conclude that SmB and SmB' are derived from alternative splicing of a single gene transcript.
Asunto(s)
Autoantígenos/genética , Ribonucleoproteínas Nucleares Pequeñas/genética , Secuencia de Bases , Genes , Células HeLa , Humanos , Intrones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Nucleares snRNPRESUMEN
SmB and SmB' are the major antigenic proteins contained within small nuclear RNP particles that are recognized by both human SLE and MRL mouse anti-Sm sera. We amplified cDNA obtained from HeLa cells by using the polymerase chain reaction and identified two clones, U2 and L13, that encode SmB and SmB', respectively. The nucleotide sequences of these two clones were identical except for the insertion of a 145-bp sequence in U2 that contained an early in frame termination codon and a potential 3' consensus splice site. The predicted amino acid sequences of HeLa SmB and B' proteins were therefore identical except for the COOH terminal 2 (U2) and 11 (L13) amino acids. U2, L13, and four subclones of U2 (F-B, B-R, F-X, and X-B) were ligated to pATH vectors and expressed as trpE fusion proteins. Epitope mapping with 12 human SLE and 12 MRL/lpr mouse anti-SmB/B' sera revealed that antibodies directed against the X-B peptide accounted for most (65.5 +/- 15.4 and 63.2 +/- 25.3%), B-R intermediate levels (51.5 +/- 30.8 and 18 +/- 19.6%), and F-X none of the anti-SmB activity in human and mouse sera, respectively. Ten human and two mouse sera contained antibodies that cross-reacted with epitopes located within the proline-rich, COOH-terminal, 27-residue peptide encoded by B-R and the NH2-proximal F-B peptide. These observations suggest that a) the polymerase chain reaction is a powerful ancillary method to synthesize autoantigens, b) SmB and B' in HeLa cells are derived from alternative splicing of a common RNA transcript, and c) both SLE and MRL anti-SmB/B' sera recognize multiple epitopes (some shared and some unique) on these proteins.
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Autoantígenos/inmunología , Ribonucleoproteínas/inmunología , Secuencia de Aminoácidos , Autoantígenos/genética , Secuencia de Bases , Western Blotting , Clonación Molecular , Epítopos , Células HeLa , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/inmunología , Ribonucleoproteínas Nucleares PequeñasRESUMEN
Autoantibodies to a polymerase III transcription factor, La (SS-B), are frequently detected in the serum of patients with Sjogren's syndrome and systemic lupus erythematosus. To define the humoral immune response to this protein, we analyzed the patterns of antibody recognition toward 13 recombinant La peptides by immunoblotting and determined the heterogeneity of antibodies reactive with the immunodominant epitopes. The smallest epitopes that were strongly antigenic and recognized by greater than 70% of sera tested (immunodominant) were encoded by the subclones BgX and XA located in the 5' and 3' halves of the La cDNA, respectively. Conformation of the immunodominant La peptides played a major role in antibody recognition. Although greater diversity in antibody binding to carboxyl-terminal La peptides was observed, the overall pattern of peptide recognition by anti-La antibodies was similar in different diseases. The antibody responses to the immunodominant peptides were strongly correlated (r = 0.68, P less than 0.001). One- and two-dimensional isoelectric focusing of affinity purified IgG anti-La peptide antibodies revealed restricted heterogeneity and oligoclonal bands (kappa light chains). These observations suggest that anti-La antibodies are induced and/or maintained by the self antigen and that their diversity is constrained either by mechanisms related to tolerance or by affinity maturation of the humoral immune response.
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Autoanticuerpos/análisis , Autoantígenos/inmunología , Lupus Eritematoso Sistémico/inmunología , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes/inmunología , Ribonucleoproteínas , Síndrome de Sjögren/inmunología , Animales , Autoantígenos/genética , ADN/análisis , Perros , Ensayo de Inmunoadsorción Enzimática , Epítopos/análisis , Humanos , Inmunoglobulina G/análisis , Antígeno SS-BRESUMEN
Antibody binding to total protein extracted from a mammalian source (HeLa cells) and from a prokaryotic source (Escherichia coli) was compared in sera from patients with systemic lupus erythematosus (SLE) and sera from normal subjects. When the average numbers of peptides or proteins recognized by IgG antibodies were compared on immunoblots, SLE sera bound to a significantly greater number of proteins from the HeLa cell extract than did sera from normal individuals (P less than 0.001). In contrast, SLE sera actually bound to fewer E coli proteins than did the sera obtained from normal controls, although the difference was not statistically significant. There was no correlation between the number of E coli proteins and HeLa proteins recognized by individual SLE sera, and there was no trend toward reactivity with a larger number of antigens in sera containing higher levels of IgG. IgG from SLE sera did not bind to 6 purified eukaryotic protein standards (selected solely on the basis of differences in size and charge) either in their denatured state or in their native state. These findings indicate that the high levels of IgG antibodies against selected eukaryotic intracellular proteins in patients with SLE cannot be explained by a random polyclonal B cell activation.
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Autoanticuerpos/inmunología , Linfocitos B/fisiología , Membranas Intracelulares/inmunología , Lupus Eritematoso Sistémico/inmunología , Activación de Linfocitos , Proteínas de la Membrana/inmunología , Adolescente , Adulto , Anticuerpos Antiidiotipos/análisis , Autoanticuerpos/análisis , Escherichia coli/análisis , Células HeLa/análisis , Células HeLa/inmunología , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina G/inmunología , Membranas Intracelulares/análisis , Lupus Eritematoso Sistémico/sangre , Proteínas de la Membrana/análisis , Persona de Mediana Edad , Distribución Aleatoria , Valores de ReferenciaRESUMEN
Improved methods for high resolution composite gel electrophoresis under dissociating conditions and electrophoretic transfer of immunoglobulins (Mr 150,000-1,800,000) to nitrocellulose have been developed. Using these techniques and highly specific antisera to detect either light or heavy chains on nitrocellulose transfers, the immunoglobulin and clonal composition of washed cryoglobulins (3 micrograms each) could be determined within two days. The results were confirmed by isoelectric focusing and immunoblotting of selected samples. In addition, the method was used for detection of an idiotypic determinant and rheumatoid factor activity within components of the cryoglobulins.
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Anticuerpos/análisis , Crioglobulinemia/inmunología , Idiotipos de Inmunoglobulinas/análisis , Isotipos de Inmunoglobulinas/análisis , Crioglobulinas/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Focalización IsoeléctricaRESUMEN
A 22-amino acid synthetic peptide (P peptide) containing the conserved, shared autoantigenic determinants of the ribosomal P proteins was conjugated to rabbit serum albumin and used to analyze anti-P heterogeneity in 17 lupus sera. Anti-P peptide antibodies demonstrated moderate restriction in isotype (IgM and IgG, but not IgA), subclass (predominantly IgG1 and IgG2), light chain type (predominantly kappa) and spectrotype. In one serum, almost exclusive use of IgG2 and the kappa light chain was observed. These findings indicate that there is a nonrandom selection of heavy and light chain constant region genes as well as limited variable region diversity in the anti-P peptide response.
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Autoanticuerpos/análisis , Inmunoglobulina G/análisis , Lupus Eritematoso Sistémico/inmunología , Fosfoproteínas/inmunología , Proteínas Ribosómicas/inmunología , ADN/inmunología , Humanos , Inmunoglobulina G/clasificación , Cadenas Ligeras de Inmunoglobulina/análisisRESUMEN
The spectrotypes of IgA and IgM rheumatoid factors (RF) were analysed in whole serum as well as immunoglobulin fractions and purified RF from patients with one of three autoimmune disorders. As predicted from the pI ranges of normal human serum IgM and IgA in agarose, IgM RF had near neutral pIs, whereas IgA RF showed more acidic (lower) pI values. Serum IgA and IgM RF from patients with rheumatoid arthritis or the sicca syndrome showed considerable charge heterogeneity whereas IgM RF from mixed cryoglobulinaemia or rheumatoid vasculitis patients showed monoclonal and oligoclonal banding patterns respectively. Clonotypic analysis was best achieved with isolated light and heavy chains from highly purified RF. The IgA RF spectrotypes from unmodified paired serum and saliva IgA were clearly different whereas after desialation, similar pI values and, in one case, similar spectrotypes were observed. These observations are compatible with the hypothesis that serum and saliva IgA RF are derived from similar clonal precursors. The methods used in these studies may also be of use in the analysis of IgA and IgM antibody diversity in a number of other situations.
Asunto(s)
Diversidad de Anticuerpos , Enfermedades Autoinmunes/inmunología , Inmunoglobulina A/análisis , Inmunoglobulina M/análisis , Factor Reumatoide/inmunología , Artritis Reumatoide/inmunología , Crioglobulinemia/inmunología , Humanos , Inmunoglobulina A Secretora/análisis , Focalización Isoeléctrica , Saliva/inmunología , Síndrome de Sjögren/inmunologíaRESUMEN
A composite agarose-polyacrylamide gel containing urea and sodium dodecyl sulfate reliably resolved unreduced human immunoglobulins according to their molecular weight. Intact immunoglobulins and a number of other macromolecules were readily transferred to nitrocellulose paper by either capillary or electrophoretic blotting, although the latter technique was more effective. Conventional antigen probing as well as immobilized antibody studies can be performed on the nitrocellulose transfers.