RESUMEN
BACKGROUND: Cancer, in particular mucinous adenocarcinoma, is associated with venous thromboembolism (VTE). Tissue factor (TF), initiator of coagulation, plays a central role in the paradigm that clotting and tumor growth form a vicious circle, in which hypercoagulability facilitates the aggressive biology of cancer and vice versa. Expression of TF in tumors is associated with poor differentiation and poor prognosis. PATIENT/METHODS: We investigated the association between clinically manifest VTE and procoagulant properties of circulating microparticles (MP) isolated from blood of unselected pancreatic and breast adenocarcinoma patients' consecutive subjects, who presented with ultrasound or CT-scan confirmed VTE, and healthy subjects. RESULTS: Patients with disseminated breast and pancreatic cancer had significantly increased levels of MP-associated TF activity compared with healthy controls, subjects with idiopathic acute VTE and non-metastatic cancer patients. Patients with both high MP-associated TF-activity and MP-associated epithelial mucin (MUC1) had a lower survival rate at 3-9 months follow-up than those with low TF-activity and no MUC1 expression: the likelihood of survival was 0.42 (95% CI: 0.19- 0.94) for an individual with these two predictor variables present, after adjustment for other factors (age cohort, type of cancer, VTE) in the Cox proportional hazards model. CONCLUSIONS: Our results suggest an important role for MP-associated TF and MUC1 in the pathogenesis of thrombosis in disseminated mucinous adenocarcinoma patients. Future studies should reveal the mechanism underlying the observed associations.
Asunto(s)
Adenocarcinoma Mucinoso/sangre , Neoplasias de la Mama/sangre , Vesículas Citoplasmáticas/metabolismo , Neoplasias Pancreáticas/sangre , Tromboembolia/etiología , Tromboplastina/metabolismo , Trombosis de la Vena/etiología , Adenocarcinoma Mucinoso/complicaciones , Adenocarcinoma Mucinoso/mortalidad , Adenocarcinoma Mucinoso/patología , Adulto , Anciano , Antígenos de Neoplasias/sangre , Coagulación Sanguínea , Neoplasias de la Mama/complicaciones , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Estudios de Casos y Controles , Diferenciación Celular , Femenino , Estudios de Seguimiento , Humanos , Estimación de Kaplan-Meier , Funciones de Verosimilitud , Masculino , Persona de Mediana Edad , Mucina-1 , Mucinas/sangre , Invasividad Neoplásica , Estadificación de Neoplasias , Neoplasias Pancreáticas/complicaciones , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Valor Predictivo de las Pruebas , Pronóstico , Modelos de Riesgos Proporcionales , Medición de Riesgo , Tromboembolia/sangre , Tromboembolia/mortalidad , Factores de Tiempo , Trombosis de la Vena/sangre , Trombosis de la Vena/mortalidadRESUMEN
Ep-CAM is a new type of cell adhesion molecule (CAM) which does not structurally resemble the members of the four major families (cadherins, integrins, selectins, and CAMs of the immunoglobulin superfamily) and mediates Ca(2+)-independent, homophilic adhesions. The extracellular domain of Ep-CAM consists of a cysteine-rich region, containing two type II epidermal growth factor (EGF)-like repeats, followed by a cysteine-poor region. We generated mutated Ep-CAM forms with various deletions in the extracellular domain. These deletion mutants, together with monoclonal antibodies recognizing different epitopes in the extracellular domain, were used to investigate the role of the EGF-like repeats in the formation of intercellular contacts mediated by Ep-CAM molecules. We established that both EGF-like repeats are required for the formation of Ep-CAM-mediated homophilic adhesions, including the accumulation of Ep-CAM molecules at the cell-cell boundaries, and the anchorage of the Ep-CAM adhesion complex to F-actin via alpha-actinin. Deletion of either EGF-like repeat was sufficient to inhibit the adhesion properties of the molecule. The first EGF-like repeat of Ep-CAM is required for reciprocal interactions between Ep-CAM molecules on adjacent cells, as was demonstrated with blocking antibodies. The second EGF-like repeat was mainly required for lateral interactions between Ep-CAM molecules. Lateral interactions between Ep-CAM molecules result in the formation of tetramers, which might be the first and necessary step in the formation of Ep-CAM-mediated intercellular contacts.
Asunto(s)
Antígenos de Neoplasias/fisiología , Moléculas de Adhesión Celular/fisiología , Factor de Crecimiento Epidérmico/genética , Adhesión Celular/genética , Línea Celular , Molécula de Adhesión Celular Epitelial , Células Epiteliales/citología , Células Epiteliales/fisiología , Regulación de la Expresión Génica , Humanos , Mutación , Secuencias Repetidas en Tándem/genéticaRESUMEN
Reflection contrast microscopy (RCM) is a light microscopic method to image cells at high definition and enhanced sensitivity compared to conventional bright-field microscopy. RCM images have very high contrast, which makes them easily applicable for digital image analysis. Because ultrathin sections are mostly used in this method, RCM also functions by bridging light with electron microscopy: the combination of ultrastructural with histochemical studies. RCM can also replace electron microscopy for rapid and simple screening of large quantities of samples for immunocytochemical staining. Special attention is paid to small biological objects, which have to be processed for RCM. If you encounter the limits of brightfield microscopy, in resolution, sensitivity or handling of the specimen, RCM will be a feasible option. Reflection contrast microscopy methods use only slightly adjusted electron microscopy methods for specimen preparation. Therefore, many familiar techniques for ultrathin specimen preparation can be applied. It is essential that only refractive index differences exist in those areas that are of interest and that the further specimen is as optically homogenic as possible, with a refractive index as close to that of glass as possible. Therefore, plastic embedding is recommended.
Asunto(s)
Técnicas de Preparación Histocitológica , Microscopía/métodos , Animales , Colorantes Fluorescentes/metabolismo , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Glomérulos Renales/patología , Glomérulos Renales/ultraestructura , Laminina/análisis , Ratones , Microscopía/instrumentación , Microscopía Electrónica/métodosRESUMEN
The bcl-2 oncogene is expressed in lymphoid and myeloid cells as well as in neurons and several types of epithelial cells and inhibits programmed cell death (apoptosis). Deregulation by the t(14;18) translocation in lymphoid malignancies induces inappropriate cell survival and serves as one of the steps toward a fully malignant behavior. Using pre- and postembedding immunoelectron microscopy in normal and neoplastic lymphocytes, we demonstrate bcl-2 immunoreactivity to the mitochondrial outer circumference and the nuclear envelope and to a lesser degree to the cell membrane. Mitochondrial staining was patchy, reminiscent of mitochondrial contact zones. Additionally, there was a suggestion of association with nuclear pores. In these regions, transmembrane transport is mediated. This may suggest that bcl-2 exerts its function in this process.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Linfocitos/química , Proteínas Proto-Oncogénicas/análisis , 3,3'-Diaminobencidina/análisis , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 18 , Humanos , Membranas Intracelulares/química , Mitocondrias/química , Membrana Nuclear/química , Proteínas Proto-Oncogénicas c-bcl-2 , Translocación GenéticaRESUMEN
To clarify where and how beta-amyloid begins to deposit in senile plaques, we examined the ultrastructural localization of amyloid beta protein (Abeta) in diffuse plaques of brains with hereditary cerebral hemorrhage with amyloidosis-Dutch type. Alzheimer disease (AD), and from nondemented aged subjects. Serial ultrathin sections of osmium-plastic blocks were immunogold-labeled for Abetax-42 (Abeta42), and sections on grids were observed under the electron microscope (EM) after observing the exact localization of the diffuse plaques in sections on glass slides by the reflection contrast microscope. Abeta42 deposition, which was decollated with gold particles, appeared in 3 forms in all subjects under the EM: 1) Scattered small bundles of amyloid fibrils between cell processes, frequently seen in the densely stained area of diffuse plaques. 2) Scattered small foci of nonfibrillar materials between cell processes as a relatively minor form. 3) Abeta42 on a part of the cell surface plasma membrane of normal appearing cell processes, a major form in weakly immunostained areas. The last form was not associated with degenerative neurites or reactive glia. Abeta42 deposition on the cell surface plasma membrane appears to be an initial event in diffuse plaques, and then it develops into amorphous/fibrillar amyloid between cell processes.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Amiloidosis/complicaciones , Encéfalo/metabolismo , Hemorragia Cerebral/genética , Hemorragia Cerebral/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Amiloidosis/patología , Encéfalo/patología , Membrana Celular/metabolismo , Cerebelo/patología , Hemorragia Cerebral/complicaciones , Hemorragia Cerebral/patología , Lóbulo Frontal/patología , Humanos , Microscopía Electrónica , Persona de Mediana Edad , Placa Amiloide/metabolismo , Placa Amiloide/patología , Valores de ReferenciaRESUMEN
Allogeneic islet transplantation in Type I diabetic patients is considerably hampered by the variable outcome of islet isolation and purification. After collagenase digestion of the pancreas, islet isolation is traditionally performed under hypothermic conditions in physiological solutions such as Hanks and RPMI. The University of Wisconsin solution (UWS) has been shown superior for hypothermic preservation of the pancreas. We, therefore, compared the UWS and RPMI for canine islet isolation and subsequent purification in either a conventional hyperosmotic density gradient of dextran in Hanks, or a novel normosmotic density gradient of Percoll in UWS. The isolation solution did not affect islet yield before purification (51% of the native islet mass). Loss of amylase (30%) and swelling of the acinar cells were observed in RPMI. In contrast, no loss of amylase and slight shrinkage of the acinar cells were observed in the UWS. Cell swelling affected the density separation and viability of the cells. Dextran density separation resulted in a 15% purity and 41% recovery of the islets isolated in RPMI, as compared to a 93% purity and 52% recovery of islets isolated in UWS. Percoll density separation improved the purity (99%) and recovery (74%) of islets isolated in UWS. Islets isolated in UWS demonstrated a superior basal and glucose stimulated insulin release during perifusion. Electron microscopy demonstrated a well-preserved islet ultrastructure after isolation in both solutions--except for slightly swollen mitochondria after isolation in RPMI. Autotransplantation of islets in pancreatectomised dogs was successful both after isolation in UWS and RPMI. We conclude that prevention of cell swelling during isolation and purification in the UWS resulted in an improved yield of viable and consistent virtually pure islets. Prevention of cell swelling during islet isolation should facilitate the analysis and control of other factors affecting outcome in man.
Asunto(s)
Islotes Pancreáticos/citología , Conservación de Tejido/métodos , Animales , Medios de Cultivo , Perros , Islotes Pancreáticos/ultraestructura , Trasplante de Islotes Pancreáticos , Soluciones , Trasplante AutólogoRESUMEN
The outcome of islet isolation is considered uncertain because of the large variability of islet and insulin yield, but comparison of the isolated and native islet population has not been attempted. We therefore addressed the efficacy of collagenase digestion, and density gradient purification of islets from the splenic dog pancreas (n = 31) by morphometry of the islet volume and size distribution, and by extraction of insulin and amylase, in samples from the pancreas, the digest, and gradient fractions. In contrast to a approximately 90% recovery of pancreatic insulin and amylase after digestion, islet yield amounted to 50% of the islet content of the pancreas. After density separation, islets were mainly found in the purified fractions, while half of the recovered insulin was located in the acinar fraction of the gradients-indicating a substantial proportion of islets entrapped in acinar fragments. The islet and insulin content of the pancreas correlated well with islet and insulin yield after digestion (r = 0.7, p < .0001). The insulin content of digest suspensions did neither correlate with islet nor insulin recovery in the purified fraction of the gradients (r = 0.4) as opposed to the islet content of digest suspensions, which correlated with both (r = 0.7, p < .0001). After density separation near 100% purity was obtained, and no loss of insulin from isolated islets was demonstrated by extraction and microscopy. Size distributions of native and isolated islets demonstrated no fragmentation. We conclude that the variability of isolation outcome may be attributed to a large extent to the variability of the native endocrine pancreas. Isolation efficacy was best documented by morphometry, because insulin extraction did not discriminate between free and entrapped islets. However, assessment by both morphometry and extraction allowed the quantitation of entrapped islets, and demonstrated preservation of beta-cell granulation. Similar studies should facilitate the analysis of other factors affecting islet isolation in man.
Asunto(s)
Separación Celular/métodos , Islotes Pancreáticos/citología , Páncreas/citología , Animales , Separación Celular/instrumentación , Centrifugación por Gradiente de Densidad/instrumentación , Centrifugación por Gradiente de Densidad/métodos , Colagenasas , Perros , Femenino , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Cinética , Perfusión , Factores de TiempoRESUMEN
The authors recently showed variable subcellular immunoreactivity of the Bcl-2 and Bax proteins after fixation of cell monolayers with acetone, methanol, or paraformaldehyde (PF) followed by methanol (PF/methanol). Here, the authors demonstrate by reflection contrast microscopy and transmission electron microscopy that acetone or methanol fixation result in complete loss of integrity of intracellular structures in contrast with PF or glutaraldehyde fixation. Scanning electron microscopy revealed poor preservation of plasma membrane integrity after fixation in acetone or methanol. Fixation with PF before methanol reduced damage to intracellular and plasma membranes. In addition, Western blot analysis demonstrated loss of Bcl-2 and Bax protein during acetone or methanol fixation, whereas PF fixation before methanol permeabilization markedly reduced this loss. For studies on the intracellular localization of soluble or unknown types of antigen, the authors discourage the use of acetone and methanol as single fixatives.
Asunto(s)
Estructuras Celulares/efectos de los fármacos , Fijación del Tejido/normas , Acetona/farmacología , Western Blotting , Estructuras Celulares/ultraestructura , Formaldehído/farmacología , Humanos , Metanol/farmacología , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Microscopía de Contraste de Fase , Polímeros/farmacología , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Fijación del Tejido/métodos , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2Asunto(s)
Islotes Pancreáticos/embriología , Embarazo en Diabéticas/fisiopatología , Animales , Femenino , Enfermedades Fetales/etiología , Retardo del Crecimiento Fetal/etiología , Humanos , Hiperglucemia/etiología , Hiperinsulinismo/etiología , Hiperplasia , Insulina/sangre , Islotes Pancreáticos/patología , Islotes Pancreáticos/fisiopatología , Intercambio Materno-Fetal , Embarazo , RatasAsunto(s)
Separación Celular/métodos , Islotes Pancreáticos/citología , Soluciones Preservantes de Órganos , Adenosina , Alopurinol , Amilasas/metabolismo , Animales , Peso Corporal , Centrifugación por Gradiente de Densidad/métodos , Colagenasas , Perros , Glutatión , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Páncreas/anatomía & histología , Páncreas/citología , Páncreas/enzimología , Rafinosa , Conservación de Tejido/métodosRESUMEN
Limbal transplants in humans show a high rate of rejection even under local and systemic immunotherapy. In order to test immunomodulatory treatments a new limbal transplant model in the rat was developed using enhanced green fluorescent protein (E-GFP) as marker for follow-up. Sixty E-GFP-positive limbal transplants from Sprague-Dawley TgN(act-EGFP)Osb4 rats were transplanted onto 18 wild-type inbred Sprague-Dawley (isografts) rats, six wild-type litter mate Sprague-Dawley (sibling) rats, 18 Fischer 344 (allografts) rats, and 18 Fischer 344 rats depleted from monocytes and macrophages by subconjunctival treatment with clodronate liposomes. All rats were monitored three times a week with fluorescence microscopy, until fluorescence had disappeared. At postoperative days 6, 9, 12, and 15, three rats of all groups were killed for immunohistochemical analysis of infiltrating cells. Using a modified digital fluorescence microscope, we were able to monitor transplant behavior over time without disturbance of the ocular surface. The average days of rejection were 14 days in the isograft group, the sibling group, and the untreated allograft group. However, the average day of rejection in the allogeneic macrophage-depleted group was 27 days. Marked infiltration of macrophages and lymphocytes was seen in the untreated isografts and allografts. In the clodronate liposome-treated allografts infiltration was minor. A successful new limbal transplant model is described. The transplant can be accurately followed up in vivo by E-GFP labeling of the donor tissue without disturbing the corneal surface. Although E-GFP itself proved to be immunogenic, local clodronate liposome injections significantly increased graft survival. So the model seems to be useful for testing immunosuppressive or modulatory agents in limbal transplantation studies.
Asunto(s)
Trasplante de Córnea/métodos , Supervivencia de Injerto/inmunología , Proteínas Fluorescentes Verdes/análisis , Limbo de la Córnea/cirugía , Sustancias Luminiscentes/análisis , Animales , Antígenos/inmunología , Biomarcadores/análisis , Ácido Clodrónico/administración & dosificación , Trasplante de Córnea/inmunología , Femenino , Rechazo de Injerto/inmunología , Inmunohistoquímica/métodos , Limbo de la Córnea/inmunología , Liposomas , Linfocitos/inmunología , Macrófagos/inmunología , Microscopía Fluorescente/métodos , Modelos Animales , Ratas , Ratas Endogámicas F344 , Ratas Sprague-DawleyRESUMEN
One simple post-embedding method for combined light- and electron microscopy is presented. Different types of antigens in normal rat and mouse kidneys as well as in tissues from cases of experimental induced nephritis were stained after Lowicryl K4M embedding by an immunogold (silver) method. The (silver-enhanced) gold particles were visualized by light microscopy, e.g. bright-field (BFM)- and reflection contrast (RCM) microscopy, as well as by electron microscopy. The potentials of RCM visualization in this field were investigated, resulting in the successful detection of colloidal gold (15 nm) particles, or silver enhanced gold particles, on ultrathin sections. Furthermore, an increased detection sensitivity of RCM compared with BFM together with an increase in the sensitivity of the immunostaining by RCM visualization was found. The different ways to use RCM, alone or in combination with bright-field- or phase contrast microscopy for visualization of plastic sections varying in thickness, type of plastic and staining, are discussed.
Asunto(s)
Inmunohistoquímica/métodos , Microscopía/métodos , Animales , Coloides , Enfermedad Injerto contra Huésped/metabolismo , Riñón/metabolismo , Riñón/ultraestructura , Ratones , Microscopía Electrónica , Microtomía , Ratas , Plata , Coloración y Etiquetado/métodosRESUMEN
Insulin concentrations in amniotic fluid were correlated with fetal age and with fetal weight in order to test the hypothesis that fetal insulin regulates fetal growth. No significant correlation could be detected. Insulin levels in amniotic fluid in diabetics, however, were significantly higher than in nondiabetics. In the same samples of amniotic fluid C-peptide proved to be very unstable during long term storage.
Asunto(s)
Líquido Amniótico/metabolismo , Feto/fisiología , Insulina/metabolismo , Péptido C/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Edad Gestacional , Crecimiento , Humanos , Recién Nacido , Recién Nacido Pequeño para la Edad Gestacional , Embarazo , Embarazo en Diabéticas/metabolismoRESUMEN
Intrauterine growth retardation (IUGR) was induced in rats by uterine artery ligation according to Wigglesworth. Control and growth-retarded animals were studied at days 21 and 22 of fetal life and 2 h after spontaneous birth. Fetuses with IUGR, when compared to controls, showed significantly lower glycemia and insulinemia levels. Newborn rats with IUGR had reduced glycemia levels but identical insulin values in comparison to controls. Dysmature and control animals had significantly different absolute weights of endocrine pancreas, but they had equal amounts of endocrine pancreatic tissue in proportion to their total body weight. Within the endocrine pancreas the same number of islet cells was counted in dysmature and control animals, but the animals with IUGR ended up with a reduced percentage of insulin-containing granulated B cells after birth.
Asunto(s)
Animales Recién Nacidos/anatomía & histología , Retardo del Crecimiento Fetal/complicaciones , Islotes Pancreáticos/embriología , Animales , Arterias/fisiología , Glucemia/metabolismo , Femenino , Retardo del Crecimiento Fetal/etiología , Insulina/sangre , Islotes Pancreáticos/citología , Islotes Pancreáticos/fisiología , Ligadura , Tamaño de los Órganos , Embarazo , Ratas , Ratas Endogámicas , Útero/irrigación sanguíneaRESUMEN
Reflection contrast microscopy (RCM) of ultrathin sections was recently introduced as a sensitive technique for visualization with enhanced definition in immunogold histochemistry. Experience of using RCM as a major tool in immunocytochemical research in different fields is summarized, e.g. oncology, nephrology and embryology. The sensitive visualization of immunocytochemical labels, gold particles or peroxidase-diaminobenzidine deposits in or on ultrathin sections, by RCM instead of electron microscopy is demonstrated. RCM of ultrathin sections is an adequate light microscopical alternative for immunoelectron microscopy, since an overview of both label and tissue is obtained with a high image definition and high contrast of label. In the studies presented, RCM is shown to provide a better gradation in staining intensity and staining pattern than other light microscopical methods. Moreover, a precise localization of multiple labels is obtained with this method. Besides the applications shown, ultrathin section visualization by RCM is very useful for correlative light- and electron microscopical studies of fine structures. Commercially available fluorescence microscopes can be adapted for proper RCM functioning; an adaptation scheme and list of microscopes tested is provided.
Asunto(s)
Inmunohistoquímica/métodos , Microscopía de Contraste de Fase/métodos , Microtomía/métodos , Animales , Anticuerpos Monoclonales , Antígenos/análisis , Perros , Riñón/química , Riñón/ultraestructura , Ratones , Páncreas/química , Páncreas/ultraestructura , Ratas , Aglutininas del Germen de TrigoRESUMEN
Proximal tubular cells (PTC) were isolated from porcine kidney by collagenase treatment, subsequently purified on a discontinuous density gradient and finally cultured. Porcine PTC (PPTC) in primary culture expressed keratin, characteristics of epithelia and brush border specific glycoproteins (FX1A). In addition, vimentin was present. All cells were negative for the endothelial marker pal-E. Less than 0.1% expressed the Tamm-Horsfall protein, characteristic of the distal tubule, while less than 0.3% of all cells in culture expressed desmin, characteristic of connective tissue (i.e. fibroblasts) and mesangial cells. Ultrastructural analysis revealed microvilli, tight junctions and abundant mitochondrial and lysosomes, all characteristics of proximal tubular cells. Freshly isolated PPTC were validated as in vitro model to detect nephrotoxicity by studying the effect of mercuric chloride, cis-platin, p-aminophenol and the halogenated alkenes 1,2 dichlorovinyl-l-cysteine, S-(1,1-difluoro-2,2-dichloroethyl)-L-cysteine (DCDFE-cys) and the glutathione conjugate of DCDFE on viability and mitochondrial membrane potential. The cells responded, time- and dose-dependently, to the nephrotoxic compounds with a decrease in mitochondrial membrane potential and loss of viability. The sensitivity of the porcine cells in detecting toxic effects corresponded favorably with in vitro systems derived from other animals.
Asunto(s)
Alquenos/toxicidad , Aminofenoles/toxicidad , Cisplatino/toxicidad , Túbulos Renales Proximales/efectos de los fármacos , Cloruro de Mercurio/toxicidad , Animales , Separación Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Inmunohistoquímica , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/fisiología , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/ultraestructura , Potenciales de la Membrana/efectos de los fármacos , Microscopía Electrónica , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Mitocondrias/ultraestructura , PorcinosRESUMEN
Alterations in glomerular function and structure were studied in protein-overload nephrosis in the rat induced by intraperitoneal administration of bovine serum albumin (BSA). Fractional clearance (C/GFR) studies using inulin and tracer proteins of different molecular size and charge revealed in proteinuric rats 1) unchanged glomerular filtration rate and renal plasma flow; 2) a 34-fold increase in C/GFR of rat serum albumin, reaching values similar to BSA; 3) a 2-fold increase in C/GFR for anionic horse radish peroxidase (HRP), but normal values for neutral and cationic HRP, and 4) an 11- and 3-fold increase for heterologous IgG and IgM, respectively. Glomerular epithelial cells showed degenerative changes, but the distribution of anionic sites in the glomerular basement membrane was found to be unaltered, as determined by polyethyleneimine binding studies. In summary, an elevation of serum albumin concentration resulted in an increased transcapillary albumin transport. This was found to lead to degenerative changes of glomerular epithelial cells with development of large pore defects, which were completely reversible.
Asunto(s)
Proteinuria/metabolismo , Animales , Capilares/patología , Permeabilidad Capilar , Femenino , Peroxidasa de Rábano Silvestre/metabolismo , Inmunoglobulina G/metabolismo , Inmunoglobulina M/metabolismo , Glomérulos Renales/irrigación sanguínea , Glomérulos Renales/patología , Tasa de Depuración Metabólica , Proteinuria/etiología , Proteinuria/patología , Ratas , Ratas Endogámicas , Albúmina Sérica/metabolismo , Albúmina Sérica Bovina/metabolismoRESUMEN
The recently developed postembedding immunoelectron microscopic technique using low temperature embedding in Lowicryl K4M has been applied for the localization and relative quantitation of nephritogenic antibodies in rats with nephrotoxic serum nephritis. Nephrotoxic serum nephritis with different degrees of severity was induced by intravenous administration of increasing doses of rabbit anti-rat glomerular basement membrane antiserum to male Wistar rats. Twenty-four hours after administration, renal tissue was processed for immunoelectron microscopy using Lowicryl embedding after fixation in paraformaldehyde. Thin sections were stained with colloidal gold-labeled antibodies to rabbit IgG. By morphometric analysis the amount of gold label per nanometer of glomerular basement membrane was counted and a strong correlation was found with the amount of administered antiserum. These data show that postembedding immunoelectron microscopy using low temperature embedding in Lowicryl K4M and immunogold staining can be applied to study the localization and relative quantitation of tissue-bound immunoglobulins.
Asunto(s)
Anticuerpos/análisis , Glomerulonefritis/inmunología , Glomérulos Renales/inmunología , Animales , Membrana Basal/inmunología , Técnica del Anticuerpo Fluorescente , Histocitoquímica , Inmunoglobulina G/análisis , Masculino , Microscopía Electrónica , Ratas , Ratas EndogámicasRESUMEN
The present study describes the development of membranous glomerulopathy (MGP) with high proteinuria in DZB rats exposed to mercuric chloride (HgCl2). IgG1 and IgG2a antibodies, eluted from glomeruli with subepithelial immune deposits, bind to the interface of the GBM and epithelial cells. High reactivity to GBM was demonstrated by ELISA and Western blotting, which could be absorbed for 30% by laminin or laminin-associated extracellular matrix components. No reactivity was found with type IV collagen, fibronectin, heparan sulfate proteoglycans, or tubular brush border antigens. Absorption to GBM removed the reactivity to renal antigens. Passively transferred eluted antibodies bind in a predominantly linear pattern along the GBM, causing focal ultrastructural transformations of the podocytes. These results suggest that this type of HgCl2-induced MGP, associated with epithelial cell injury and proteinuria, is caused by autoantibodies to basement membrane components which are located at the epithelial cell-basement membrane interface and may be involved in cell-matrix binding.
Asunto(s)
Autoanticuerpos/inmunología , Glomerulonefritis Membranosa/inmunología , Animales , Especificidad de Anticuerpos , Western Blotting , Colágeno/inmunología , Complemento C3/biosíntesis , Complemento C9/biosíntesis , Complejo de Ataque a Membrana del Sistema Complemento/biosíntesis , Reacciones Cruzadas , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Fibronectinas/inmunología , Glomerulonefritis Membranosa/inducido químicamente , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/inmunología , Inmunoglobulina E/biosíntesis , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Inmunohistoquímica , Glomérulos Renales/inmunología , Laminina/inmunología , Cloruro de Mercurio , Microscopía Electrónica , Proteinuria/inducido químicamente , Proteoglicanos/inmunología , Ratas , Ratas Endogámicas , Factores de TiempoRESUMEN
BACKGROUND: In murine chronic graft-versus-host disease, an experimental model for lupus nephritis, autoantibodies against renal tubular epithelium can be found. Part of these antibodies are directed against a constituent of renal tubular epithelium, the enzyme Dipeptidyl peptidase IV (DPP IV). DPP IV is present on the cell membrane of glomerular epithelial and endothelial cells, and plays an important role in cell-extracellular matrix interactions. In mice and rats, administration of heterologous anti-DPP IV antibodies can induce proteinuria and podocyte effacement. EXPERIMENTAL DESIGN: In this study, the glomerular DPP IV enzyme activity was investigated in the course of graft-versus-host disease in (C57BL/10 x DBA/2) F1 hybrids both by light- and electron microscopy using a DPP IV-specific substratum. RESULTS: Light microscopical examination revealed an overall reduction of DPP IV activity and an altered distribution pattern in glomeruli as early as 4 weeks after induction of graft-versus-host disease. Immunofluorescence studies using anti-DPP IV antibodies showed actual redistribution, excluding antibody-mediated enzyme inactivation. Enzyme electron microscopy revealed an irregular deposition of reaction product characterized by a patchy, "moth eaten" appearance of the endothelial and epithelial membranes. This process occurred simultaneously with the development of albuminuria and preceded the effacement of epithelial foot processes. In control mice, DPP IV showed a continuous distribution along endothelial and podocyte membranes. CONCLUSIONS: In view of these findings we postulate that impairment of the function of DPP IV as a non integrin adhesion molecule may be one of the causative factors underlying the structural and functional lesions observed in this model for lupus nephritis.