RESUMEN
The Guillain-Barré Syndrome (GBS) is an acute inflammatory polyneuroradiculopathy, which is considered to be caused by autoimmune processes. A number of single antigenic structures has been suggested to be targeted by the immune system, but a conclusive etiological concept has not been evolved yet. We compared reactions of sera from GBS patients (N=28) and from both two pathological control groups, 25 Multiple sclerosis (MS) patients and 32 patients with other non-inflammatory peripheral polyneuropathies (ONP) and from sex- and age-matched healthy controls (N=30). Porcine peripheral nerve proteins were used as antigens in a Western blot procedure. The blots were analysed by densitometry, and a multivariate statistical comparison of the antibody repertoires was carried out. Antibody patterns of GBS patients differed significantly (p<0.001) from each of the control groups. Discriminant analysis indicated that the discrimination resulted from pattern differences of specific regions of the blots containing proteins with estimated molecular weights of 58-64 and 28-29 kDa. We conclude that statistical analysis of antibody patterns may be helpful both in clinical diagnosis and in further research concerning the pathogenesis of GBS.
Asunto(s)
Autoanticuerpos/análisis , Síndrome de Guillain-Barré/inmunología , Síndrome de Guillain-Barré/patología , Adulto , Animales , Autoanticuerpos/sangre , Western Blotting , Femenino , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Análisis Multivariante , Nervio Ciático/inmunología , PorcinosRESUMEN
The screening of sera for autoantibodies with Western blots reveals complex repertoires. the compostion of such repertoires depends on genetic control of autoantibody-producing cells, the individual's history of exposure to its own and to foreign antigens, and also on the presence of autoimmune diseases. Our method shows how staining patterns of Western blots can be recoded as binary or grey-value vectors. Vectors are transferred to artificial neural networks for learning. Artificial neural networks are able to recognize group-specific antibody binding patterns. Staining patterns can be attributed to diagnostic groups. This may support diagnostic procedures.
Asunto(s)
Algoritmos , Autoanticuerpos/análisis , Western Blotting , Redes Neurales de la Computación , Autoanticuerpos/clasificación , HumanosRESUMEN
Using a new quantitative immunoblot technique (MegaBlot and artificial neural network) sera of rats with lens-induced uveitis (LIU) were tested against Western blots (WB) of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separations of protein fractions from rat lenses. The blots were scanned using digital image analysis and evaluated by multivariate analysis of discriminance and an artificial neural network. Five groups of LIU rats (male Wistar rats, n = 28) were investigated: no treatment, single doses of methylprednisolone (MPR; 7.5 mg/kg body weight, intravenous), allopurinol (AL; 50 mg/kg body weight, intravenous), a combination of both drugs (AL and MPR), and repeated application of AL (ALFR; 50 mg/kg body weight intravenous every 2 weeks during the immunization period and a daily dose of approximately 25 mg/kg body weight, orally). ALFR, MPR, and MPRAL show statistically significant immunological effects in LIU treatment. Classification of WBs using both discriminance analysis or neural network resulted in 100% correct assignment; and 82% (discriminance analysis) and 86% (neural network) of WBs were correctly assigned using only unknown blots for classification (not studied before). The MegaBlot technique is able to detect therapeutically induced changes in autoantibody repertoires allowing therapeutic control and a prognostic view of treatment.
Asunto(s)
Alopurinol/administración & dosificación , Antimetabolitos/administración & dosificación , Autoanticuerpos/análisis , Western Blotting , Procesamiento de Imagen Asistido por Computador , Redes Neurales de la Computación , Uveítis/metabolismo , Animales , Cristalinas/análisis , Masculino , Ratas , Ratas Wistar , Uveítis/sangre , Uveítis/inducido químicamenteRESUMEN
PURPOSE: The aim of this study was to evaluate the immunomodulating activity of allopurinol using a model of lens-induced uveitis (LIU) and to compare these effects to those of steroids. METHODS: We tested the sera of both LIU and control rats against western blots (WB) of SDS-PAGE separations of protein fractions from normal and LIU rat lenses. These blots were scanned using digital image analysis. A newly developed technique was used to compare the complex autoantibody (AAB) repertoires. Five groups of LIU rats were investigated: no treatment; single doses of methylprednisolone (MPR; 7.5 mg/kg body wt.i.v.); allopurinol (AL; 50 mg/kg body wt. i.v.); a combination of both drugs (AL and MPR); repeated application of AL (ALFR; 50 mg/kg body wt.i.v. every 2 weeks during the immunization period and a daily dose of approx. 25 mg/kg body wt. orally). RESULTS: Immunization induced complex antibody repertoires against lens proteins. Antibody repertoires of LIU rats were identical, regardless of whether the proteins were obtained from control, uveitis eyes, or corresponding healthy eyes of the same individual. AL showed a dose-dependent immunological effect in LIU treatment. Given as a single dose, AL revealed no significant change in the AAB repertoire; however, ALFR showed very clear modification of the AAB repertoires compared to both controls and rats receiving steroids. CONCLUSIONS: These results suggest dose-dependent effects of allopurinol in LIU treatment. Repeated application during the immunization period induced a strong immunomodulating effect of AL that was not observed after single doses.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Alopurinol/farmacología , Antimetabolitos/farmacología , Uveítis/tratamiento farmacológico , Adyuvantes Inmunológicos/administración & dosificación , Alopurinol/administración & dosificación , Animales , Antimetabolitos/administración & dosificación , Autoanticuerpos/análisis , Autoanticuerpos/efectos de los fármacos , Autoantígenos , Western Blotting , Cristalinas , Relación Dosis-Respuesta a Droga , Vías de Administración de Medicamentos , Quimioterapia Combinada , Electroforesis en Gel de Poliacrilamida , Glucocorticoides/administración & dosificación , Glucocorticoides/farmacología , Masculino , Metilprednisolona/administración & dosificación , Metilprednisolona/farmacología , Ratas , Ratas Wistar , Uveítis/inducido químicamenteRESUMEN
A procedure for the quantification and comparison of complex (auto-) antibody repertoires of many individuals is described. It is based on multivariate statistical analyses of densitometric data of individual staining patterns, in this case Western blots. Implementation of algorithms can be based on standard software and hardware components. This procedure allows (i) the quantitative assessment of the reproducibility and reliability of any electrophoretic procedure, such as for the production of the immunological substrate, (ii) the statistical comparison of any group of staining patterns, e.g., derived from patients with autoimmune diseases or normal controls, (iii) the identification of the bands that contribute most to the differences between such groups, and (iv) the determination whether an unknown individual sample belongs to a known group. A statistical analysis of autoantibody repertoires has important possible applications: it detects spontaneous or therapeutically induced changes of repertoires, it identifies regions of interest, it supports the isolation of relevant antigens, and it will also be helpful in the diagnosis of autoimmune diseases.
Asunto(s)
Autoanticuerpos/análisis , Western Blotting , Electroforesis en Gel de Poliacrilamida , Enfermedades Musculares/inmunología , Algoritmos , Análisis Discriminante , Humanos , Análisis Multivariante , Músculo Esquelético/inmunologíaRESUMEN
Flow cytometry (FC) is an useful tool for the analysis of subpopulations in complex cell suspensions. When applying this method to the cerebrospinal fluid (CSF), some characteristic properties of this cell type must be taken into consideration: there are only few cells which decay rapidly in their native medium and during centrifugation. One aim of the immunostaining procedure preceding flow cytometric analysis must be to minimize cell loss in order to get an undistorted picture of 'true' CSF cell populations. Consequently, morphological flow cytometric plots of high resolution are an indispensable precondition for reliable determination of subpopulations defined by monoclonal antibody (Mab) binding. We describe a standardized protocol for the flow cytometric examination of CSF cells which minimizes undesired cell loss. By the use of a 'quality control' the extent of cell loss could be monitored. Examples of morphological flow cytometric plots are given. The subsequent determination of Mab binding subpopulations is critical when fluorescence intensities of antigen positive and negative cells are non-disjunct. A statistical test was developed for these cases often seen when cell surface determinants are expressed at low levels only.
Asunto(s)
Líquido Cefalorraquídeo/citología , Citometría de Flujo/métodos , Anticuerpos Monoclonales , Antígenos CD/análisis , Centrifugación , Femenino , Fluoresceína-5-Isotiocianato , Humanos , Técnicas Inmunológicas , Recuento de Leucocitos , Linfocitos/inmunología , Masculino , Control de Calidad , Coloración y Etiquetado , Factores de TiempoRESUMEN
Sera from 69 healthy blood donors were screened for autoantibodies using Western blots of human muscle fractions to assess whether prevalence and repertoires of autoantibodies against muscle tissue would change with age. Each serum contained immunoglobulins binding to a broad spectrum of antigens. Their molecular masses ranged from 20 to more than 200 kDa. Although each staining pattern appeared to be a singular combination of detected bands and staining intensities, the patterns were not different in men and women. When sera were grouped according to age (decades between 10 and over 70 years) age-dependent changes were not found. Individual repertoires of natural autoantibodies against muscle tissue are complex when examined by Western blotting. Mature normal repertoires of low-affinity autoantibodies are found at the age of 10 years which is the lower cut-off in this study. The repertoires remain stable throughout a normal lifespan.
Asunto(s)
Autoanticuerpos/sangre , Músculos/inmunología , Adolescente , Adulto , Factores de Edad , Anciano , Autoantígenos/aislamiento & purificación , Western Blotting , Niño , Femenino , Humanos , Inmunidad Innata , Masculino , Persona de Mediana Edad , Proteínas Musculares/inmunología , Caracteres SexualesRESUMEN
In a study of central autonomic dynamics in early multiple sclerosis, we measured the temporal oscillations of the momentary heart rate (heart rate dynamics). 11 young patients suffering from relapsing remitting definite multiple sclerosis in relapse-free and early stages of illness and 11 healthy controls were examined under vagotonic and sympathicotonic conditions. The temporal structure of the heart rate dynamics was operationalized phase-space analytically through the estimation of the largest Lyapunov exponent. Positive Lyapunov exponents were found in all participants under all conditions indicating deterministically chaotic heart rate oscillations. The variance analysis of these exponents detected no significant effect of sympathetic or vagal activity (experimental condition) but a significant group difference (p < .02). The multiple sclerosis patients were characterized by significantly lower Lyapunov exponents than the healthy controls. This finding suggests a more stable and thus less adaptive central-autonomic organization in early multiple sclerosis.
Asunto(s)
Sistema Nervioso Autónomo/fisiología , Sistema Nervioso Autónomo/fisiopatología , Frecuencia Cardíaca , Esclerosis Múltiple/fisiopatología , Adolescente , Adulto , Electrocardiografía , Femenino , Humanos , Matemática , Modelos Cardiovasculares , Análisis Multivariante , Valores de ReferenciaRESUMEN
If weakness of the eye muscles remains the only symptom of myasthenia gravis (MG) for more than 2 years, the condition is operationally defined as ocular MG (OMG). A number of clinical, genetic, and immunological differences between this variant and generalized MG (GMG) have been described. We analyzed repertoires of autoantibodies against proteins of skeletal and extraocular muscle in sex- and age-matched groups of patients with either GMG or OMG (n = 10 in each group). All GMG sera detected a group of three proteins larger than 200 kDa which were not detected by any of the OMG sera. Two components with apparent molecular weights of 50 and 60 kDa were stained by seven of the ten OMG sera but by none of the GMG group. These antigens are probably soluble, cytoplasmatic proteins of the eye muscle. OMG sera, furthermore, detected a protein of about 45 kDa in the pellet fraction of eye muscle but failed to do so after adsorption with skeletal muscle fractions. We conclude that OMG and GMG sera contain autoantibodies of different specificities. Our findings further support immunological heterogeneity in MG.
Asunto(s)
Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Proteínas del Ojo/inmunología , Proteínas Musculares/inmunología , Miastenia Gravis/inmunología , Músculos Oculomotores/inmunología , Adulto , Anciano , Especificidad de Anticuerpos , Estudios de Cohortes , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Immunoblotting , Masculino , Persona de Mediana Edad , Miastenia Gravis/clasificaciónRESUMEN
Latency in nervous tissue caused by herpes simplex virus 1 (HSV-1) and by varicella-zoster virus (VZV) is an intriguing feature of herpes-virus' neurotropism. HSV-1 and VZV latency are the causes of ophthalmic zoster and recurrent HSV infections in the distributions of the trigeminal branches. HSV-1 neuronal latency may play a role in the etiopathogenesis of HSV encephalitis. We attempted to determine the prevalence and age distribution of VZV and HSV latency. We applied nested polymerase chain reaction (PCR) assays to detect HSV-1 and VZV genome in trigeminal ganglia and olfactory bulbs which were obtained from 109 human corpses at forensic postmortems. HSV-1 latency was found in 72.5% of trigeminal ganglia and in 15.5% of olfactory bulbs. VZV latency was 63.3% in trigeminal ganglia and 1% in olfactory bulbs. Simultaneous latency of VZV and HSV genome occurs in 48.8% of trigeminal ganglia. The age-group specific prevalence of HSV neuronal latency increases from 18.2% in 0-20 years to reach finally 100% in persons older than 60 years. Age specific prevalences of VZV peaked for a first time with 82% between 21-30 years, fell to 50% for 40-50 years, and rose to 89% for 71-80 years. If the latent trigeminal ganglion HSV-1 genome were the source of endogenously acquired encephalitis, the peak incidence of HSV encephalitis in older subjects correlates with our findings. Increased VZV latency prevalence in nervous tissue of younger people without subsequent disease indicates sufficient immune surveillance.
Asunto(s)
Envejecimiento/fisiología , ADN Viral/análisis , Genoma Viral , Herpesvirus Humano 3/aislamiento & purificación , Bulbo Olfatorio/microbiología , Simplexvirus/aislamiento & purificación , Ganglio del Trigémino/microbiología , Adulto , Anciano , Secuencia de Bases , Niño , ADN/genética , ADN/aislamiento & purificación , ADN Viral/genética , Electroforesis en Gel de Agar , Exones , Femenino , Genes p53 , Herpesvirus Humano 3/genética , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Bulbo Olfatorio/crecimiento & desarrollo , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , Simplexvirus/genética , Ganglio del Trigémino/crecimiento & desarrolloAsunto(s)
Infecciones Bacterianas/inmunología , Activación de Complemento/fisiología , Interleucina-1/fisiología , Meningitis/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Corticoesteroides/administración & dosificación , Adulto , Infecciones Bacterianas/tratamiento farmacológico , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/inmunología , Humanos , Meningitis/tratamiento farmacológicoRESUMEN
We report the case of a 69-year old male caucasian patient who developed a lateral neck tumor while under immunosuppression with azathioprine. The tumor was diagnosed finally as an abscess caused by Salmonella enteritidis after isolation of the agent from blood, tumor biopsy and feces. This extremely rare manifestation of an infection by Salmonella enteritidis is considered as a complication of immunosuppressive therapy.
Asunto(s)
Absceso/microbiología , Azatioprina/efectos adversos , Miastenia Gravis/tratamiento farmacológico , Infecciones Oportunistas/microbiología , Infecciones por Salmonella/microbiología , Salmonella enteritidis/aislamiento & purificación , Timoma/tratamiento farmacológico , Neoplasias del Timo/tratamiento farmacológico , Tiroiditis Supurativa/microbiología , Anciano , Azatioprina/administración & dosificación , Humanos , Persona de Mediana Edad , Factores de RiesgoRESUMEN
Sera of patients with Graves' orbitopathy (GOP) often contain antibodies against retroorbital tissue components. The presence of such autoantibodies has been considered to indicate an autoimmune pathogenesis of the disease. However, their specificity has not been conclusively tested, because studies demonstrating autoantibodies used controls with no lesion in the extraocular eye muscles. Although ocular myositis (OM) is clinically distinct from GOP, damage to the retroorbital muscles is a common histopathological finding in both conditions. Using an immunoblot technique, reactions of sera from patients suffering from either disease were compared. Sera from both groups contained antibodies against a variety of antigenic determinants recognized by both sera. These autoantibodies may have been induced secondary to tissue damage and should be considered to be nonspecific. Because the role of tissue damage was not accounted for in previous studies, evidence concerning antigens supposedly specific for GOP should be reevaluated. The reaction patterns of OM and GOP sera were slightly different. These differences were specific enough to suggest that sera from patients with GOP contain antibodies against eye muscle components that are not present in the sera of patients with idiopathic OM. These findings support the assumption that GOP is an autoimmune disease. However, the major autoimmune targets remain to be identified and their pathogenic relevance is still unclear.
Asunto(s)
Autoanticuerpos/sangre , Enfermedad de Graves/diagnóstico , Miositis/diagnóstico , Adulto , Anciano , Autoanticuerpos/inmunología , Densitometría , Electroforesis en Gel de Poliacrilamida , Epítopos/inmunología , Femenino , Enfermedad de Graves/inmunología , Humanos , Immunoblotting , Masculino , Persona de Mediana Edad , Músculos/inmunología , Miositis/inmunología , Músculos Oculomotores/inmunología , Valor Predictivo de las PruebasRESUMEN
In vivo activated T cells could be isolated from cerebrospinal fluid (CSF) of a patient suffering from chronic meningitis of unclear origin. Although the patient's skin reactivity to purified protein derivative (PPD) was negative, and peripheral T cells did not proliferate to this antigen in vitro, the majority of T cell clones from CSF specifically recognized PPD on either autologous or allogeneic HLA class II compatible macrophages. Remarkably, peripheral blood mononuclear cells potently suppressed the PPD-specific proliferative responses of healthy donors. The selective enrichment of oligoclonal IgG in the CSF but not in the patient's serum further indicated T and B cell responses lacking systemic feedback control. Analyses of a persisting immune stimulation in the CSF provide a potent diagnostic tool and may explain neurological complications as observed in a number of autoimmune diseases and chronic infections.
Asunto(s)
Linfocitos B/inmunología , Líquido Cefalorraquídeo/inmunología , Linfocitos T/inmunología , Tuberculina/inmunología , Tuberculosis Meníngea/inmunología , Adulto , Células Presentadoras de Antígenos/inmunología , Antígenos de Diferenciación de Linfocitos T/análisis , Células Clonales , Relación Dosis-Respuesta Inmunológica , Femenino , Antígenos HLA-D/inmunología , Humanos , Activación de Linfocitos , Linfocitos/ultraestructura , Microscopía Electrónica , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Thirty-six biopsy specimens of human biceps and vastus lateralis muscles were examined by histometric analysis and determination of enzyme activities (phosphorylase, triosephosphate dehydrogenase, 3-hydroxacyl-CoA-dehydrogenase, lactate dehydrogenase, hexose isomerase, citrate synthetase, 6-phosphogluconate dehydrogenase). The series included 13 specimens from patients suffering from a benign form of muscular dystrophy (limb girdle and Becker type of muscular dystrophy) and 12 specimens from patients with an acute (n = 5) or chronic (n = 7) form of myositis. Muscle fibres were atrophic in myositis and hypertrophic (with an increased variation of fibre diameters) in muscular dystrophies, as has been shown previously. When myositis samples were compared with either normal or dystrophic muscles, a highly significant lowering of glycolytic enzyme activity was found in chronic myositis, while the activity of 6-phosphogluconate dehydrogenase was elevated to highly significant levels. Measurements of the latter enzyme's activity might be of additional value in differentiating chronic forms of myositis from benign muscular dystrophies.
Asunto(s)
Músculos/enzimología , Miositis/diagnóstico , Fosfogluconato Deshidrogenasa/metabolismo , Adolescente , Adulto , Anciano , Biopsia , Citrato (si)-Sintasa/metabolismo , Diagnóstico Diferencial , Femenino , Glucólisis , Humanos , Masculino , Persona de Mediana Edad , Músculos/patología , Distrofias Musculares/diagnóstico , Distrofias Musculares/enzimología , Distrofias Musculares/patología , Miositis/enzimología , Miositis/patología , Oxidorreductasas/metabolismoRESUMEN
Sera of 45 patients with myasthenia gravis (MG) and of 30 healthy controls were screened for antibodies against muscle antigens in an immunoblotting solid-phase assay using a preparation of human amputated muscle as the substrate. In each group, the sera showed several bands on the blots. The findings are thought to indicate that antibodies against muscle components are normally present in human sera. Sera from patients or controls could be distinguished by differences in the band staining patterns. It is suggested that antibodies which are not directed against the acetylcholine receptor may nonetheless play an important role in the pathogenesis and clinical course of myasthenia gravis.