RESUMEN
AIMS: To describe the clinical and pathological features of 10 further cases of fibroblastic polyps (FP), a recently described, distinctive type of colorectal mucosal polyp. METHODS AND RESULTS: The patients were seven women and three men with ages ranging from 44 to 63 years. The lesions ranged in size from 2 to 4 mm. Eight of the polyps were located in the sigmoid colon. Five cases were associated with hyperplastic polyps. Histologically, FP displayed bland, plump spindle cells with oval nuclei arranged as bundles parallel to the surface or as haphazardly orientated sheets with a focal periglandular or perivascular concentric arrangement. Eight polyps represented mixed fibroblastic/hyperplastic polyps as they contained serrated (hyperplastic) crypts. Immunohistochemically, all cases were positive for vimentin and negative for desmin, smooth-muscle actin, h-caldesmon, S100 protein, c-Kit, epithelial membrane antigen, cytokeratin AE1/3, CD34, CD68, COX-2, and factor XIIIa. Ultrastructural examination supported the fibroblastic nature of the tumour cells. CONCLUSIONS: FP is a distinctive type of benign mucosal colorectal polyp characterized by its distal location, small size, frequent association with hyperplastic polyps, distinct morphological appearance and typical immunonegativity for markers of specific differentiation. FP with serrated crypts (mixed fibroblastic/hyperplastic polyp) represents a frequent variant of this lesion. Pathologists should recognize FP and discriminate it from other types of colorectal polyps.
Asunto(s)
Colon/patología , Pólipos del Colon/patología , Adulto , Colon/química , Colon/ultraestructura , Pólipos del Colon/metabolismo , Femenino , Fibroblastos/patología , Humanos , Hiperplasia , Inmunohistoquímica , Mucosa Intestinal/química , Mucosa Intestinal/patología , Mucosa Intestinal/ultraestructura , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Vimentina/análisisRESUMEN
BACKGROUND: 15-Lipoxygenase (15-LO) is a nonheme iron-containing enzyme that catalyzes the peroxidation of fatty acids. Herein, we studied the effect of 15-LO overexpression in the vascular endothelium on thymocyte apoptosis by evaluating thymuses from low-density lipoprotein receptor-deficient (LDL-RD) mice and LDL-RD/15-LO mice. Thymuses were evaluated by immunohistochemistry and by TUNEL whereas in vitro studies were carried out by employing freshly isolated thymocytes from the respective mice and evaluation of apoptosis by propidium iodide and annexin V cytometry. METHODS AND RESULTS: The apoptotic index in LDL-RD/15-LO mice was significantly higher than in the LDL-RD mice. In the thymic medulla the difference was smaller, although still significant. Freshly isolated thymus cells from LDL-RD/15-LO mice exhibited a higher rate of spontaneous cell death than controls. Incubation of thymus cells in the presence of the cell-permeable caspase-3 inhibitor DEVD-CMK resulted in a decrease in the frequency of apoptotic cells in LDL-RD/15-LO thymocytes, whereas no effect was evident in control thymocytes. The antioxidant N-acetylcysteine causes the increase in apoptosis in both groups. CONCLUSION: LDL-RD/15-LO mice exhibit increased thymocyte apoptosis both in vivo and in vitro. These findings may suggest a role for 15-LO in the natural selection of thymocytes.
Asunto(s)
Araquidonato 15-Lipooxigenasa/fisiología , Supresión Clonal/fisiología , Endotelio Vascular/metabolismo , Linfocitos T/citología , Timo/citología , Acetilcisteína/farmacología , Animales , Antioxidantes/farmacología , Apoptosis , Araquidonato 15-Lipooxigenasa/genética , Caspasa 3 , Inhibidores de Caspasas , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Inhibidores de Cisteína Proteinasa/farmacología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Oligopéptidos/farmacología , Receptores de LDL/deficiencia , Receptores de LDL/genética , Timo/irrigación sanguíneaRESUMEN
Mitochondrial DNA plays a crucial role in oxidative production of energy. Thus, defects in mitochondrial DNA can affect virtually all organ systems. The point mutation A --> G at position 3243 in the mitochondrial tRNAleu(UUR) gene is the cause of several distinct types of mitochondrial cytopathy and several clinical phenotypes, including encephalomyopathy with lactic acidosis and stroke-like episodes and maternally inherited diabetes and deafness. This mutation has been recently described also in association with kidney disease, mainly focal and segmental glomerulosclerosis. At present, little is known about the prevalence of this mitochondrial nephropathy, its clinical course and the pathogenesis of glomerular damage. We describe 2 unrelated patients, who presented with proteinuria and progressed to end-stage renal failure. Other clinical features were short stature, severe headache, hearing loss, diabetes mellitus and hypertrophic cardiomyopathy. The main histological finding was an increased number of abnormal mitochondria in tubular cells and podocytes. Analysis of mitochondrial DNA from leukocytes and urine sediment revealed heteroplasmy for the A3243G mutation in tRNAleu(UUR) gene in both patients. Recognition of the characteristic clinical and histological features of the mitochondrial A3243G mutation-associated glomerulopathy will enable correct diagnosis and better management of a disease which is likely to be underdiagnosed.
Asunto(s)
Enfermedades Renales/genética , Mitocondrias/genética , Mutación , ARN de Transferencia/genética , Adulto , Progresión de la Enfermedad , Femenino , Humanos , MasculinoRESUMEN
CoQ transfers electrons from complexes I and II of the mitochondrial respiratory chain to complex III. There are very few reports on human CoQ deficiency. The clinical presentation is usually characterized by: epilepsy, muscle weakness, ataxia, cerebellar atrophy, migraine, myogloblinuria and developmental delay. We describe a patient who presented with neonatal liver and pancreatic insufficiency, tyrosinemia and hyperammonemia and later developed sensorineural hearing loss and Leigh syndrome. Liver biopsy revealed markedly reduced complex I+III and II+III. Addition of CoQ to the liver homogenate restored the activities, suggesting CoQ depletion. Histological staining showed prominent bridging; septal fibrosis and widening of portal spaces with prominent mixed inflammatory infiltrate, associated with interface hepatitis, bile duct proliferation with numerous bile plugs. Electron microscopy revealed a large number of mitochondria, which were altered in shape and size, widened and disordered intercristal spaces. This may be the first case of Leigh syndrome with liver and pancreas insufficiency, possibly caused by CoQ responsive oxphos deficiency.
Asunto(s)
Enfermedad de Leigh/enzimología , Fallo Hepático Agudo/enzimología , Hígado/patología , Enfermedades Mitocondriales , Ubiquinona/metabolismo , Biopsia , Complejo I de Transporte de Electrón/deficiencia , Complejo II de Transporte de Electrones/deficiencia , Complejo III de Transporte de Electrones/deficiencia , Pérdida Auditiva Sensorineural/enzimología , Pérdida Auditiva Sensorineural/fisiopatología , Humanos , Hiperamonemia/enzimología , Lactante , Enfermedad de Leigh/fisiopatología , Hígado/enzimología , Hígado/ultraestructura , Fallo Hepático Agudo/patología , Masculino , Errores Innatos del Metabolismo/enzimología , Mitocondrias Hepáticas/enzimología , Fosforilación Oxidativa , Páncreas/enzimología , Páncreas/patología , Ubiquinona/deficienciaRESUMEN
We determined apoptosis in whole rat colonic tissue and in isolated colonocytes from the various rat crypt regions in preneoplastic stages up to frank neoplasia following administration of the procarcinogen, dimethylhydrazine (DMH). Apoptotic cells were determined by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-method, by evaluating sections stained with hematoxylin and eosin, and caspase-1 immunostaining. Apoptotic cells in whole colonic tissue from untreated rats were confined to the upper crypt while, in DMH-treated rats apoptotic and caspase-1 positive cells were located in the crypt proliferative regions. Numerous apoptotic and caspase-1-positive cells were found in sections from early tumors while in the delayed tumors, apoptotic-positive cells were absent and number of caspase-1-positive cells was negligible. A marked reduction in the apoptotic index along the crypt was observed in isolated transformed colonic cells, this was not the case for caspase-1-positive cells. We conclude that: (i) in colorectal tumors at progressive stage apoptosis is altered, (ii) the mechanistic alteration in apoptosis may be located between caspase-1-protease activity and the fragmentation process of DNA.
Asunto(s)
Apoptosis , Caspasa 1/análisis , Colon/citología , Neoplasias del Colon/fisiopatología , Lesiones Precancerosas/fisiopatología , 1,2-Dimetilhidrazina , Animales , Biomarcadores/análisis , Carcinógenos , Transformación Celular Neoplásica/metabolismo , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/patología , Etiquetado Corte-Fin in Situ , Masculino , Lesiones Precancerosas/inducido químicamente , Lesiones Precancerosas/patología , RatasRESUMEN
Microvillous inclusion disease (MID) is characterized by diffuse villous atrophy without inflammatory changes. While increased apoptosis has been related to mucosal flattening in celiac disease, the role of apoptosis in the pathogenesis of MID is unknown. The aim of this study was to assess the rates of apoptosis and cell proliferation in MID and to compare them with those of normal controls and celiac disease. Small intestinal biopsies from 5 infants with MID, 10 children with normal villous architecture, and 10 children with untreated celiac disease were stained with the terminal uridine deoxynucleotidyl nick end labeling (TUNEL) method to assess apoptotic activity, and with Ki-67 immunohistochemistry to assess cellular proliferation. TUNEL and Ki-67 positive enterocytes were counted in a minimum of 20 well oriented half crypts per section. The percentage of apoptotic cells per crypt (apoptotic index) in normal, MID, and celiac biopsies was 0.03 +/- 0.01%, 0.08 +/- 0.08%, and 0.16 +/- 0.3%, respectively. Significant differences were found between normal and MID, and between normal and celiac cases. The percentage of Ki-67 positive cells per crypt (proliferation index) in normal, MID, and celiac cases was 14 +/- 2.5%, 28 +/- 9.2%, and 56 +/- 14%. Significant differences were found between the 3 groups. In conclusion, (1) enterocyte apoptosis and proliferation are increased in MID; (2) apoptosis appears to be an important factor of cell loss and may be, at least in part, responsible for villous atrophy in MID; and (3) crypts in MID are hyperplastic and not hypoplastic. HUM PATHOL 31:1404-1410.
Asunto(s)
Apoptosis , Diarrea Infantil/patología , Enterocitos/patología , Intestino Delgado/patología , Microvellosidades/patología , Atrofia , ADN/análisis , Diarrea Infantil/genética , Diarrea Infantil/metabolismo , Enterocitos/metabolismo , Femenino , Humanos , Etiquetado Corte-Fin in Situ , Lactante , Recién Nacido , Intestino Delgado/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Microvellosidades/metabolismo , Índice MitóticoRESUMEN
Cationic colloidal gold (CCG), a polycationic histochemical probe, was used to analyze the distribution of glomerular basement membrane (GBM) polyanions, mainly heparan sulfate proteoglycan in spontaneous hypertensive rats (SHR) with or without salt loading and antihypertensive treatment with propranolol. The changes of mean GBM width and anionic sites distribution were assessed by electron microscopy. Plasma and urinary nitrates (NO(x)) were measured by nitrite (NO2) + nitrate (NO3), stable metabolites of NO. SHR had decreased NO production and increased GBM width (27%) compared with the control Wistar-Kyoto (WKY) rats. The chronic high dietary salt intake resulted in a significant increase in blood pressure, proteinuria, and renal function in the SHR rats. The chronic high salt dietary intake resulted in a decrease in NO in the WKY and a further reduction in NO production in the SHR. The GBM anionic sites count was similar in the SHR and WKY nonsalt-loaded groups, 13.5 +/- 0.5 and 12.8 +/- 0.4 CCG counts/microm GBM, respectively, but significantly lower in both salt-loaded SHR and WKY, 9.9 +/- 0.55 (P < .01) and 9.6 +/- 0.55 (P < .01) CCG counts/microm GBM, respectively. Antihypertensive treatment with propranolol in the salt-loaded SHR group resulted in lower blood pressure, a further decrease in NO production, but no significant changes in GBM width and anionic sites count. It is concluded that chronic high salt intake may be deleterious to the permselectivity of the GBM. A low NO production state that results from chronic salt loading in already hypertensive rats will result in more severe organ (renal) damage, most probably by the addition of the loss of GBM permselectivity to the existing pathomorphologic changes.
Asunto(s)
Hipertensión/metabolismo , Glomérulos Renales/metabolismo , Óxido Nítrico/metabolismo , Polímeros/metabolismo , Animales , Antihipertensivos/uso terapéutico , Membrana Basal/metabolismo , Membrana Basal/patología , Presión Sanguínea/efectos de los fármacos , Dieta Hiposódica , Femenino , Oro/metabolismo , Hipertensión/tratamiento farmacológico , Hipertensión/patología , Hipertensión/fisiopatología , Riñón/patología , Riñón/fisiopatología , Glomérulos Renales/patología , Masculino , Microscopía Electrónica , Nitratos/sangre , Nitritos/sangre , Polielectrolitos , Polilisina/metabolismo , Propranolol/uso terapéutico , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Distribución TisularRESUMEN
The rates of glucose transport and of glycolysis and the expression of the glucose transporters GLUT-1 through GLUT-4 were measured in T47D human breast cancer cells that underwent differentiation by retinoic acid. Glucose transport was found to be the rate-limiting step of glycolysis in control and differentiated cells. The transporters GLUT-1, GLUT-3, and GLUT-4 were present in the cell membrane and in the cytoplasm, and GLUT-2 was present solely in the cytoplasm. Differentiation led to a reduction in GLUT-1 and to an increase in cytoplasmic GLUT-2 and GLUT-3 with no change in GLUT-4. Differentiation also caused a reduction in the maximal velocity of glucose transport by approximately 40% without affecting the Michaelis-Menten constant of glucose transport. These changes did not alter the steady-state concentration of the phosphate metabolites regulating cell energetics but increased the content of phospholipid breakdown phosphodiesters. In conclusion, differentiation of human breast cancer cells appears to be associated with decreased glycolysis by a mechanism that involves a reduction in GLUT-1 and a slowdown of glucose transport.
Asunto(s)
Neoplasias de la Mama/patología , Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas del Tejido Nervioso , Tretinoina/farmacología , Algoritmos , Neoplasias de la Mama/ultraestructura , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 2 , Transportador de Glucosa de Tipo 3 , Humanos , Queratinas/biosíntesis , Cinética , Espectroscopía de Resonancia Magnética , Microscopía Electrónica , Proteínas de Transporte de Monosacáridos/biosíntesis , Proteínas de Transporte de Monosacáridos/genética , Fosfatos/metabolismo , Células Tumorales CultivadasRESUMEN
Calcifying epithelial odontogenic tumor, also known as Pindborg tumor, is a rare benign tumor with locally aggressive behavior. It is characterized by squamous epithelial cells, calcifications, and eosinophilic deposits that have been identified as amyloid. We report a case of calcifying epithelial odontogenic tumor and investigate the nature of the amyloid, using histologic, immunohistochemical, and ultrastructural studies. The amyloid was immunohistochemically negative for basement membrane components and positive for all cytokeratin stains performed (cocktail of cytokeratins 1, 5, 6, 8, 13, and 16, and cytokeratins AE1 and AE3). The amyloid stained focally in a glandular-like pattern, reminiscent of the epithelial glandlike structures of the tumor. We conclude that the amyloid is derived from filamentous degeneration of keratin filaments that originate from the tumor squamous epithelium. The keratin degeneration is part of a developmental or aging process that the tumor undergoes.
Asunto(s)
Amiloide/análisis , Queratinas/análisis , Neoplasias Maxilares/patología , Tumores Odontogénicos/patología , Calcinosis/patología , Células Epiteliales/patología , Femenino , Humanos , Inmunohistoquímica , Neoplasias Maxilares/diagnóstico por imagen , Neoplasias Maxilares/cirugía , Persona de Mediana Edad , Tumores Odontogénicos/diagnóstico por imagen , Tumores Odontogénicos/cirugía , RadiografíaRESUMEN
BACKGROUND: In previous studies we found that experimental Adriamycin (ADR) nephropathy is associated with the loss of glomerular basement membrane (GBM) anionic sites provided by heparan sulfate proteoglycans. Chronic saline loading in normal rats resulted in a similar effect on the GBM anionic sites. The L-arginine-nitric oxide synthase-nitric oxide system is involved in the pathogenesis of experimental chronic renal failure. The present study was performed to determine the combined effect of nitric oxide (NO) modulation and chronic saline loading in ADR nephropathy. The modulation of NO was done by chronic administration of L-arginine (NO donor) or N(w)-nitro-L-arginine, a known nitric oxide synthase inhibitor. METHODS: Systolic blood pressure was measured in awake rats by a tail-cuff method. Renal function was assessed by creatinine clearance, FeNa%, and daily protein excretion. The change of mean GBM widths and anionic sites distribution were assessed by electron microscopy. The localization of anionic sites was carried out by cationic colloidal gold. Plasma and urinary nitrates (NO(x)) were measured by nitrite (NO(2)) + nitrate (NO(3)), stable metabolites of NO. RESULTS: Two weeks after the ADR administration (3.5 mg/kg BW iv) the rats had severe renal failure (creatinine clearance 134 +/- 31 microl/min/100 g BW vs. initial values 670 +/- 29 microl/min/ 100 g BW, p < 0.001), high FeNa%, severe proteinuria, increased GBM width, significant reduction of GBM anionic sites and low urinary NO(x) excretion. The saline loading resulted in further reduction of GBM anionic sites count and blood pressure elevation. The inhibition of NO did not change the course of ADR nephropathy. The main finding of the present study is that chronic administration of L-arginine significantly alleviates the renal failure in the ADR (+/- saline loading) nephropathy. The L-arginine-treated rat had higher creatinine clearance, lower FeNa% and protein excretion and complete normalization of GBM anionic sites distribution. CONCLUSIONS: Sodium loading has a deleterious effect on GBM permselectivity. L-Arginine prevents the reduction of GBM anionic sites, decreases proteinuria and alleviates the renal insufficiency in ADR nephropathy.
Asunto(s)
Doxorrubicina/toxicidad , Enfermedades Renales/inducido químicamente , Enfermedades Renales/metabolismo , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/metabolismo , Óxido Nítrico/metabolismo , Animales , Aniones , Arginina/farmacología , Membrana Basal/efectos de los fármacos , Membrana Basal/metabolismo , Membrana Basal/patología , Sitios de Unión , Inhibidores Enzimáticos/farmacología , Femenino , Oro Coloide/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Enfermedades Renales/patología , Glomérulos Renales/patología , Masculino , Nitratos/metabolismo , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitritos/metabolismo , Nitroarginina/farmacología , Ratas , Ratas Wistar , Cloruro de SodioRESUMEN
Primary ciliary dyskinesia is an autosomal recessive condition characterised by chronic sinusitis, bronchiectasis, and subfertility. Situs inversus occurs in 50% of cases (Kartagener syndrome). It has an estimated incidence of 1 in 20 000 live births. The clinical phenotype is caused by defective ciliary function associated with a range of ultrastructural abnormalities including absent dynein arms, absent radial spokes, and disturbed ciliary orientation. The molecular genetic basis is unknown. A genome scan was performed in five Arabic families. Using GENEHUNTER, a maximal multipoint lod score (HLOD) of 4.4 was obtained on chromosome 19q13.3-qter at alpha (proportion of linked families) = 0.7. A 15 cM critical region is defined by recombinations at D19S572 and D19S218. These data provide significant evidence for a PCD locus on chromosome 19q and confirm locus heterogeneity.
Asunto(s)
Cromosomas Humanos Par 19 , Trastornos de la Motilidad Ciliar/genética , Adulto , Mapeo Cromosómico , Cuerpo Ciliar/ultraestructura , Trastornos de la Motilidad Ciliar/fisiopatología , Femenino , Humanos , Masculino , Repeticiones de Microsatélite , Linaje , Sinusitis/etiología , Situs Inversus/etiologíaRESUMEN
AIMS: To document the presence, morphology, immunophenotype and ultrastructure of multinucleated stromal cells within the anal mucosa and to discuss possible pathogenetic mechanisms for this occurrence. METHODS AND RESULTS: Multiple sections of normal anal mucosa from 30 abdominoperineal resection specimens were analysed by light microscopic, electron microscopic and immunohistochemical methods. Multinucleated stromal cells were found in 22 cases (73%). They contained two to five nuclei, arranged in a linear fashion or in a rosette or grape-like pattern. They stained positive for vimentin and negative for actin, desmin and oestrogen/progesterone receptors. Ultrastructural examination confirmed their fibroblastic lineage. Mast cells were frequently observed in the immediate vicinity of mono- and multinucleated cells. CONCLUSIONS: Multinucleated stromal cells are a common occurrence in the normal anal mucosa. They should not be misinterpreted as neoplastic cells. Mast cells may play a role in their morphogenesis.
Asunto(s)
Canal Anal/patología , Mucosa Intestinal/patología , Células del Estroma/ultraestructura , Anciano , Núcleo Celular/patología , Núcleo Celular/ultraestructura , Femenino , Humanos , Mucosa Intestinal/ultraestructura , Masculino , Persona de Mediana Edad , Células del Estroma/patologíaRESUMEN
This study was designed primarily to assess the localization of apoptosis cascade proteins along the rat colonic crypt and secondarily to test whether the activity and/or localization of these proteins are affected by the enrichment of the diet with the soluble fiber pectin. Expression of apoptosis cascade proteins was assessed in isolated colonocytes harvested from the luminal and basal crypt colonocyte populations. Two different dietary regimens were tested: a standard diet (diet A), and a diet enriched in pectin (diet B), a soluble fiber that undergoes fermentation in the cecum and produces high concentrations of intracolonic short-chain fatty acids. Caspase-1 expression was maximal in luminal colonocytes of rats fed diet B, as evidenced by Western blot and immunohistological analyses. Expression of the cleaved poly(ADP-ribose) polymerase product was elevated in both the luminal and basal colonocytes of the pectin-fed group, whereas in rats fed diet A, the expression was lower, especially in basal crypt colonocytes. The highest expression of the antiapoptotic protein Bcl-2 was observed in the lower compartments of the colonic crypt tissue and was maximal in the rat group fed a standard diet. The apoptotic index in colonocytes of rats fed diet B was higher than that measured in rats fed diet A. Cumulatively, our results indicate that apoptosis cascade proteins are differentially localized along the lumen-crypt axis, and their expression and activity may be controlled by dietary components. These results may, at least partially, account for the documented protective effect of butyrogenic fibers on colorectal cancer.
Asunto(s)
Apoptosis/fisiología , Caspasas/metabolismo , Colon/metabolismo , Colon/patología , Dieta , Animales , Masculino , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , RatasRESUMEN
Cell-to-cell adhesion, an important event in differentiation, is impaired during advanced stages of tumorigenesis. In this study, we examined the possible regulation of cell-adhesion proteins by the differentiation agent butyrate in LS174T and HM7 cells, two types of human colon cancer cells that differ in their ability to produce mucin and colonize the liver of experimental animals. The more aggressive, high-mucin-producing cell line (HM7), a clone selected from LS174T cells, showed a scattered and undifferentiated ultramorphological appearance and low basal alkaline phosphatase activity; the proteins beta-catenin and E-cadherin, as detected by immunostaining, were expressed in the cells' nuclei. All of these properties were significantly less pronounced in the less aggressive, low-mucin-producing LS174T cells. In both cell lines, butyrate treatment enhanced cell-to-cell interaction, alkaline phosphate activity, translocation of beta-catenin and E-cadherin from the nuclei to the membrane junctions, and transcription and translation of the 120-kDa E-cadherin isoform, but not of its 100-kDa isoform. Analysis of possible mechanisms of E-cadherin up-regulation revealed that butyrate induces the release of nuclear proteins from the E-cadherin promoter sequence, reducing transcription repression. We suggest that butyrate activates E-cadherin transcription through translocation of nuclear transcription factors bearing specific repressor activity. We surmise that abrogation of nuclear 100-kDa E-cadherin and beta-catenin expression following butyrate treatment is related to the control of E-cadherin gene transcription.
Asunto(s)
Butiratos/farmacología , Cadherinas/metabolismo , Neoplasias del Colon/metabolismo , Transactivadores , Transcripción Genética/efectos de los fármacos , Fosfatasa Alcalina/metabolismo , Western Blotting , Diferenciación Celular/efectos de los fármacos , Neoplasias del Colon/patología , Proteínas del Citoesqueleto/metabolismo , Humanos , Microscopía Electrónica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas/efectos de los fármacos , Regulación hacia Arriba , beta CateninaRESUMEN
The present study was aimed at evaluating the effect of high intracolonic butyrate concentrations, either through fermentation of a soluble fiber-enriched diet or via intracolonic butyrate instillation, on colon cancer in a chemically induced (dimethylhydrazine) rat model. The effects were tested in four groups of dimethylhydrazine-treated rats: (i) rats fed a standard diet, (ii) rats fed a diet enriched with 15% citrus pectin, a soluble fiber that ferments and produces a high concentration of intracolonic butyrate, (iii) rats fed a standard diet and intrarectally instilled with a sodium butyrate solution (50 mM), (iv) rats fed a standard diet and intrarectally instilled with sodium butyrate vehicle solution (100 mM NaCl). The apoptotic index in the distal colon of rats fed pectin was higher than in colonic tissue from rats fed a standard diet. The expression of caspase-1, a cysteine protease implicated in the regulation of programmed cell death, as detected by both Northern and Western analysis, showed the highest mRNA and protein levels in colonic tissue from rats intrarectally instilled with butyrate. Immunohistology confirmed the Western blot findings. Expression of the cleaved poly(ADP-ribose) polymerase product, a downstream nuclear substrate for caspase-3 in the apoptotic pathway, was elevated in both the pectin-fed and butyrate-instilled groups. Expression of the antiapoptotic protein Bcl-2 was significantly reduced following pectin feeding as well as butyrate instillation. The highest expression of Bcl-2 was observed in tumor tissue. A marked reduction in aberrant crypt number was observed in colonic tissue obtained from both the pectin-fed and butyrate-instilled groups relative to rats from the standard diet group. The average tumor volume per rat in both the pectin-fed and butyrate-instilled groups was significantly lower than in rats from the standard diet and the sodium butyrate vehicle-instilled groups. We conclude that high butyrate levels, either instilled or obtained following fermentation of soluble dietary fibers, inhibit early and late events in colon tumorigenesis by controlling the transcription expression and activity of key proteins involved in the apoptotic cascade.
Asunto(s)
Apoptosis , Butiratos/metabolismo , Colon/metabolismo , Neoplasias del Colon/metabolismo , 1,2-Dimetilhidrazina , Animales , Northern Blotting , Western Blotting , Peso Corporal , Butiratos/uso terapéutico , Caspasa 1/biosíntesis , Colon/patología , Neoplasias del Colon/patología , Neoplasias del Colon/prevención & control , Dieta , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Isobutiratos , Masculino , Modelos Biológicos , Mutágenos , Neoplasias/inducido químicamente , Pectinas/uso terapéutico , Poli(ADP-Ribosa) Polimerasas/metabolismo , ARN Mensajero/metabolismo , Ratas , Factores de TiempoRESUMEN
L-90 cells were selected to grow in the presence of serum lipoproteins and 90 microM lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). L-90 cells massively accumulate HMGR, a result of >10-fold amplification of the gene and 40-fold rise in mRNA, and also overexpress other enzymes of the mevalonate pathway. Western blot and promoter-luciferase analyses indicate that transcriptional regulation of sterol-responsive genes by 25-hydroxycholesterol or mevalonate is normal. Yet, none of these genes is regulated by lipoproteins, a result of severe impairment in the low density lipoprotein receptor pathway. Moreover, L-90 cells do not accelerate the degradation of HMGR or transfected HMGal chimera in response to 25-hydroxycholesterol or mevalonate. This aberrant phenotype persists when cells are grown without lovastatin for up to 37 days. The inability to regulate HMGR degradation is not due to its overproduction since in LP-90 cells, which were selected for lovastatin resistance in lipoprotein-deficient serum, HMGR is overexpressed, yet its turnover is regulated normally. Also, the rapid degradation of transfected alpha subunit of T cell receptor is markedly retarded in L-90 cells. These results show that in addition to gene amplification and overexpression of cholesterogenic enzymes, statin resistance can follow loss of regulated HMGR degradation.
Asunto(s)
Resistencia a Medicamentos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hidroximetilglutaril-CoA Reductasas/metabolismo , Lovastatina/farmacología , Animales , Células CHO , División Celular/efectos de los fármacos , Línea Celular , Cricetinae , Genes Reporteros , Hidroxicolesteroles/farmacología , Lípidos/biosíntesis , Lipoproteínas/farmacología , Lipoproteínas LDL/metabolismo , Ácido Mevalónico/farmacología , Microscopía Electrónica , Regiones Promotoras Genéticas , ARN Mensajero/efectos de los fármacos , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
The objective of this work was to determine markers of endothelial cell activation in valves from patients with antiphospholipid syndrome (APS) and heart valve involvement, in order to establish a role for endothelial cells in the pathogenesis of the valvular disease. Sixteen valves from ten patients with APS, obtained from autopsies or removed during valve replacement, were studied. Two groups of valves were used as controls. One group included seven normal valves from patients who died from non-cardiac diseases. The other group of valves were obtained from patients with bacterial endocarditis during autopsies or valve replacement operations. Immunoperoxidase and immunofluorescence stainings with antibodies to human immunoglobulins, endothelial cells, alpha3beta1 integrin, collagen IV, laminin and fibronectin were employed. Three histopathological patterns were apparent: normal valves, valves with verrucous endocarditis and valves with fibrocalcific changes. In all the valves with verrucous endocarditis the following findings were observed: (1) increased expression of the alpha3beta1 integrin on the endothelial cells, (2) increased amount of collagen IV, laminin and fibronectin, (3) proliferation of blood vessels and (4) linear subendothelial deposition of immunoglobulins and complement. The valves with fibrocalcific changes were deformed and showed a thick layer of collagen IV, laminin and fibronectin, yet in two valves the indothelial cells showed an expression of the alpha3beta1 integrin. The control valves did not express the integrin and had only a thin subendothelial band of collagen IV. In valves from patients with APS, 1 markers of endothelial cell activation are upregulated while the inflammatory exudate is scant. There is also a prominent deposition of immunoglobulins in the valves from patients with APS, suggesting a possible association between the deposition of the antibodies and the activation of the endothelial cells in APS.
Asunto(s)
Síndrome Antifosfolípido/metabolismo , Endocarditis Bacteriana/metabolismo , Enfermedades de las Válvulas Cardíacas/metabolismo , Integrinas/biosíntesis , Actinas/análisis , Actinas/biosíntesis , Adulto , Anciano , Anticuerpos Anticardiolipina/inmunología , Antígenos CD/análisis , Antígenos CD/biosíntesis , Antígenos de Diferenciación Mielomonocítica/análisis , Antígenos de Diferenciación Mielomonocítica/biosíntesis , Síndrome Antifosfolípido/inmunología , Membrana Basal/inmunología , Membrana Basal/metabolismo , Colágeno/análisis , Colágeno/biosíntesis , Endocarditis Bacteriana/inmunología , Endotelio/química , Endotelio/inmunología , Endotelio/metabolismo , Exudados y Transudados , Femenino , Fibronectinas/análisis , Fibronectinas/biosíntesis , Enfermedades de las Válvulas Cardíacas/inmunología , Humanos , Inmunoglobulina A/análisis , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Integrina alfa3beta1 , Integrinas/análisis , Laminina/análisis , Laminina/biosíntesis , Masculino , Persona de Mediana Edad , Receptores de Laminina/análisis , Receptores de Laminina/biosíntesisRESUMEN
Calcified concretions are a normal and constant finding in the anterior pituitary gland of fetuses and newborns. Their light and electron microscopic characteristics have been recently reported by the authors. In this study, undecalcified and decalcified sections from 20 neonatal and 60 fetal anterior pituitary glands were studied by histochemical and immunohistochemical methods to further clarify their nature and mechanism of formation. All the glands revealed homogeneous and/or laminar calcifications located either within the interstitium or follicular structures. They were composed of a diastase-resistant periodic acid-Schiff-positive carbohydrate-rich matrix. The Feulgen method for DNA was negative. Their core frequently reacted to Alcian blue and epithelial membrane antigen (EMA). EMA also stained the apical membranes of adjacent epithelial cells. Other immunostains (vimentin, keratin, and pituitary hormones) were negative. The positive staining for Alcian blue and EMA and the negative staining with the Feulgen method for DNA suggest that the core of the calcifications consists of acidic mucosubstances and EMA-positive proteinaceous material previously secreted by viable pituitary cells. The EMA-negative periphery of the concretions probably develops from further extracellular peripheral mineralization that leads to larger, sometimes laminated psammoma bodies. The occurrence of pituitary calcifications in states of adult physiological and pathological hyperprolactinemia suggests that the marked proliferation of lactotrophs occurring during the fetal life play an important role in the pathogenesis of the fetal and neonatal concretions.
Asunto(s)
Calcinosis , Feto/patología , Enfermedades de la Hipófisis/patología , Biomarcadores/análisis , Histocitoquímica , Humanos , Inmunohistoquímica , Recién Nacido , Adenohipófisis/química , Adenohipófisis/patologíaRESUMEN
Apoptosis in cells of different lineages is restrained by survival signals which depend upon cell-to-cell communication. The aim of this study was to determine whether colonic cells deprived of crypt ambient are doomed to die prior to their normal chronological demise. Apoptosis was studied in rat whole colonic tissue, in isolated intact crypts, and in colonic cell populations collected from the crypt axis at different stages of proliferation and differentiation. In a number of experiments, cell harvest was performed in the presence of either a tetrapeptide (YVAD-CMK) inhibitor of interleukin-1beta-converting enzyme (ICE), or tyrphostin A25, a protein tyrosine kinase inhibitor, or sodium-orthovanadate, a phosphatase inhibitor. DNA fragmentation was assessed by electrophoretic and nonisotopic-labeling procedures. The ultrastructure of colonic tissue specimens and isolated cells was examined by transmission electron microscopy. Apoptosis in whole colonic tissue and in isolated crypts was confined predominantly to cells resident in the upper crypt regions. In contrast, extensive apoptotic death was observed in isolated colonic cells, irrespective of their developmental stage and positional hierarchy within the crypt continuum at harvest time. An apoptotic gradient, however, was evident. Exposure to YVAD-CMK resulted in a marked decrease in the number of apoptotic cells. Treatment with tyrphostin A25 caused a sharp rise in the apoptotic index; conversely, vanadate significantly impeded apoptosis. Cumulatively, these results indicate that disordered intercellular communication provokes unscheduled ICE-mediated apoptosis of colonocytes, and that local signals along the crypt continuum control both the reprieve from death and the timely demise of distinct colonic cell populations. Attenuation of tyrosine phosphorylation may be a contributory event in the acquisition of the apoptotic phenotype.
Asunto(s)
Apoptosis/fisiología , Colon/fisiología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Caspasa 1/efectos de los fármacos , Caspasa 1/metabolismo , Separación Celular , Colon/citología , Masculino , Fosforilación , Ratas , Tirosina/metabolismoRESUMEN
Transforming growth factor beta1 (TGF-beta1) is a cytokine known to play a key role in the control of cell growth. TGF-beta1 potently inhibits the proliferation of human and rodent-derived epithelial cells. Colonic precancerous and moderately differentiated cancer cells are responsive to TGF-beta1, whereas malignant colon cancer cells are resistant to the inhibitory action of the cytokine. These observations have been derived exclusively from in vitro studies. Therefore, the main aim of our study was to determine whether TGF-beta1 exerts a growth-restraining action on colon carcinogenesis in vivo. TGF-beta1 was sequestered into ethylene acetate copolymer matrices and "loaded" preparations were implanted intraperitoneally (i.p.) in rats. One week later, the animals were treated with dimethylhydrazine (DMH), a colon procarcinogen. Empty matrices devoid of TGF-beta1 but containing bovine serum albumin (BSA) carrier served as the appropriate control preparations. The number of aberrant crypt foci (ACF), considered to be preneoplastic lesions of the colon, was scored. Tumor formation and size were assessed at the appropriate times. TGF-beta1 released in a sustained manner from copolymer matrices: (i) markedly inhibited colonic ACF formation and the number of aberrant crypts and (ii) significantly reduced colonic tumor formation and size.