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1.
Hum Mol Genet ; 22(1): 61-73, 2013 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-23010472

RESUMEN

Niemann-Pick type C is a lysosomal storage disease associated with mutations in NPC1 or NPC2, resulting in an accumulation of cholesterol in the endosomal-lysosomal system. Niemann-Pick type C has a clinical spectrum that ranges from a neonatal rapidly fatal disorder to an adult-onset chronic neurodegenerative disease combined with remarkably, in some cases, hematological defects such as thrombocytopenia, anemia and petechial rash. A role of NPC1 in hematopoiesis was never shown. Here, we describe platelet function abnormalities in three unrelated patients with a proven genetic and biochemical NPC1 defect. Their platelets have reduced aggregations, P-selectin expression and ATP secretions that are compatible with the observed abnormal alpha and reduced dense granules as studied by electron microscopy and CD63 staining after platelet spreading. Their blood counts were normal. NPC1 expression was shown in platelets and megakaryocytes (MKs). In vitro differentiated MKs from NPC1 patients exhibit hyperproliferation of immature MKs with different CD63(+) granules and abnormal cellular accumulation of cholesterol as shown by filipin stainings. The role of NPC1 in megakaryopoiesis was further studied using zebrafish with GFP-labeled thrombocytes or DsRed-labeled erythrocytes. NPC1 depletion in zebrafish resulted in increased cell death in the brain and abnormal cellular accumulation of filipin. NPC1-depleted embryos presented with thrombocytopenia and mild anemia as studied by flow cytometry and real-time QPCR for specific blood cell markers. In conclusion, this is the first report, showing a role of NPC1 in platelet function and formation but further studies are needed to define how cholesterol storage interferes with these processes.


Asunto(s)
Plaquetas/fisiología , Proteínas Portadoras/fisiología , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/fisiología , Enfermedad de Niemann-Pick Tipo C/sangre , Proteínas de Pez Cebra/fisiología , Animales , Proteínas Portadoras/genética , Muerte Celular , Diferenciación Celular , Niño , Femenino , Humanos , Recién Nacido , Péptidos y Proteínas de Señalización Intracelular , Masculino , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/genética , Trombocitopenia/genética , Pez Cebra/embriología , Proteínas de Pez Cebra/genética
2.
Cancer Cell ; 11(2): 119-32, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17292824

RESUMEN

The IkappaB kinase (IKK) subunit NEMO/IKKgamma is essential for activation of the transcription factor NF-kappaB, which regulates cellular responses to inflammation. The function of NEMO in the adult liver remains elusive. Here we show that ablation of NEMO in liver parenchymal cells caused the spontaneous development of hepatocellular carcinoma in mice. Tumor development was preceded by chronic liver disease resembling human nonalcoholic steatohepatitis (NASH). Antioxidant treatment and genetic ablation of FADD demonstrated that death receptor-mediated and oxidative stress-dependent death of NEMO-deficient hepatocytes triggered disease pathogenesis in this model. These results reveal that NEMO-mediated NF-kappaB activation in hepatocytes has an essential physiological function to prevent the spontaneous development of steatohepatitis and hepatocellular carcinoma, identifying NEMO as a tumor suppressor in the liver.


Asunto(s)
Carcinoma Hepatocelular/etiología , Hígado Graso/etiología , Quinasa I-kappa B/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Neoplasias Hepáticas/etiología , Animales , Apoptosis , Bromodesoxiuridina , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/fisiología , Hígado Graso/metabolismo , Hígado Graso/patología , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica , Hepatocitos/metabolismo , Immunoblotting , Etiquetado Corte-Fin in Situ , Leucina Zippers , Hígado/lesiones , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , FN-kappa B/genética , FN-kappa B/metabolismo , Fosforilación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Ubiquitina/metabolismo
3.
J Pharmacol Exp Ther ; 346(3): 362-9, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23792410

RESUMEN

Anthracyclines are used to treat cancers during the second and third trimester of pregnancy. The chemotherapeutic effect of anthracyclines is associated with a dose- and time-dependent cardiotoxicity that is well described for infants and adults. However, data regarding fetal anthracycline-related cardiotoxicity after administration of chemotherapeutics during pregnancy are limited. In this study, we analyzed the acute effect of doxorubicin, an anthracycline derivative, on fetal and maternal rat myocardium. We injected 10 or 20 mg/kg i.v. doxorubicin to pregnant Wistar rats at day 18 of pregnancy; age-matched pregnant rats injected with physiologic saline served as controls. Maternal echocardiography and fetal Doppler scanning were performed before the injection and before sacrifice. Cesarean operation was performed at day 19 or 20, and maternal and fetal blood samples and heart biopsies were collected to measure apoptosis, the impact on cell proliferation, and structural cardiac damage. Acute maternal cardiotoxicity is associated with loss of body weight, moderately deteriorated left ventricular function, induction of apoptosis, and a decrease in cell turnover. Despite a 30% lower fetal body weight and elevated plasma B-type natriuretic peptide concentrations after doxorubicin administration, the fetal hearts had intact microstructure, an unaltered number of apoptotic cells, and preserved cell proliferation compared with controls. Our study suggests that acute treatment using anthracyclines during pregnancy impairs maternal cardiac function, whereas fetal hearts are protected.


Asunto(s)
Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/toxicidad , Doxorrubicina/farmacocinética , Doxorrubicina/toxicidad , Corazón Fetal/efectos de los fármacos , Cardiopatías/inducido químicamente , Animales , Apoptosis/efectos de los fármacos , Factor Natriurético Atrial/metabolismo , Peso Corporal/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Ecocardiografía , Femenino , Cardiopatías/diagnóstico por imagen , Etiquetado Corte-Fin in Situ , Inyecciones Intravenosas , Intercambio Materno-Fetal , Miocardio/metabolismo , Miosinas/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Embarazo , ARN/biosíntesis , ARN/genética , Ratas , Ratas Wistar , Transcripción Genética/efectos de los fármacos
4.
Genet Med ; 15(1): 55-63, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22899094

RESUMEN

PURPOSE: Aquaporin 7 (AQP7) belongs to the aquaglyceroporin family, which transports glycerol and water. AQP7-deficient mice develop obesity, insulin resistance, and hyperglyceroluria. However, AQP7's pathophysiologic role in humans is not yet known. METHODS: Three children with psychomotor retardation and hyperglyceroluria were screened for AQP7 mutations. The children were from unrelated families. Urine and plasma glycerol levels were measured using a three-step enzymatic approach. Platelet morphology and function were studied using electron microscopy, aggregations, and adenosine triphosphate (ATP) secretion tests. RESULTS: The index patients were homozygous for AQP7 G264V, which has previously been shown to inhibit transport of glycerol in Xenopus oocytes. We also detected a subclinical platelet secretion defect with reduced ATP secretion, and the absence of a secondary aggregation wave after epinephrine stimulation. Electron microscopy revealed round platelets with centrally located granules. Immunostaining showed AQP7 colocalization, with dense granules that seemed to be released after strong platelet activation. Healthy relatives of these patients, who were homozygous (not heterozygous) for G264V, also had hyperglyceroluria and platelet granule abnormalities. CONCLUSION: The discovery of an association between urine glycerol loss and a platelet secretion defect is a novel one, and our findings imply the involvement of AQPs in platelet secretion. Additional studies are needed to define whether AQP7 G264V is also a risk factor for mental disability.


Asunto(s)
Acuaporinas/genética , Trastornos de las Plaquetas Sanguíneas/genética , Homocigoto , Mutación , Adolescente , Adulto , Sustitución de Aminoácidos , Acuaporina 3/genética , Acuaporinas/metabolismo , Plaquetas/metabolismo , Plaquetas/ultraestructura , Niño , Preescolar , Codón , Femenino , Glicerol/sangre , Glicerol/orina , Humanos , Lactante , Masculino , Persona de Mediana Edad , Linaje , Transporte de Proteínas , Adulto Joven
5.
Hepatology ; 56(3): 1178-81, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22706971

RESUMEN

Heterozygous deletion or mutation in hepatocyte nuclear factor 1 homeobox B/transcription factor 2 (HNF1B/TCF2) causes renal cyst and diabetes syndrome (OMIM #137920). Mice with homozygous liver-specific deletion of Hnf1ß revealed that a complete lack of this factor leads to ductopenia and bile duct dysplasia, in addition to mild hepatocyte defects. However, little is known about the hepatic consequences of deficient HNF1B function in humans. Three patients with heterozygous HNF1B deficiency were found to have normal bile duct formation on radiology and routine liver pathology. Electron microscopy revealed a paucity or absence of normal primary cilia. Therefore, heterozygous HNF1B deficiency is associated with ciliary anomalies in cholangiocytes, and this may cause cholestasis.


Asunto(s)
Conductos Biliares/citología , Cilios , Células Epiteliales/patología , Factor Nuclear 1-beta del Hepatocito/deficiencia , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad
6.
J Cell Mol Med ; 16(9): 2085-93, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22151349

RESUMEN

The upper lamina propria (ULP) area of interstitial cells (IC) has been studied extensively in bladder, but is rather unexplored in the rest of the urinary tract. This cell layer is intriguing because of the localization directly underneath the urothelium, the intercellular contacts and the close relationship with nerve endings and capillaries. In this study, we examine the ULP layer of IC in human renal pelvis, ureter and urethra, and we make a comparison with ULP IC in bladder. Tissue was obtained from normal areas in nephrectomy, cystectomy and prostatectomy specimens, and processed for morphology, immunohistochemistry and electron microscopy. A morphological and immunohistochemical phenotype for the ULP IC was assessed and region-dependent differences were looked for. The ULP IC in renal pelvis, ureter and urethra had a similar ultrastructural phenotype, which differed somehow from that of bladder IC, that is, thinner and longer cytoplasmic processes, no peripheral actin filaments and presence of dense core granules and microtubules. Together with their immunohistochemical profile, these features are most compatible with the phenotype of telocytes, a recently discovered group of stromal cells. Based on their global ultrastructural and immunohistochemical phenotype, ULP IC in human bladder should also be classified as telocytes. The most striking immunohistochemical finding was the variable expression of oestrogen receptor (ER) and progesterone receptor (PR). The functional relevance of ULP telocytes in the urinary tract remains to be elucidated, and ER and PR might therefore be promising pharmacological research targets.


Asunto(s)
Membrana Mucosa/citología , Receptores de Estrógenos/análisis , Receptores de Progesterona/análisis , Uréter/citología , Uretra/citología , Adulto , Humanos , Inmunohistoquímica , Células Intersticiales de Cajal/citología , Pelvis Renal/citología , Pelvis Renal/ultraestructura , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Membrana Mucosa/ultraestructura , Fenotipo , Uréter/ultraestructura , Uretra/ultraestructura , Vejiga Urinaria/citología , Urotelio/citología , Urotelio/ultraestructura
7.
Hum Mol Genet ; 19(7): 1368-78, 2010 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-20071347

RESUMEN

Autism is a neurodevelopmental disorder characterized by impaired social reciprocity, impaired communication and stereotypical behaviors. Despite strong evidence for a genetic basis, few susceptibility genes have been identified. Here, we describe the positional cloning of SCAMP5, CLIC4 and PPCDC as candidate genes for autism, starting from a person with idiopathic, sporadic autism carrying a de novo chromosomal translocation. One of these genes, SCAMP5 is silenced on the derivative chromosome, and encodes a brain-enriched protein involved in membrane trafficking, similar to the previously identified candidate genes NBEA and AMISYN. Gene silencing of Nbea, Amisyn and Scamp5 in mouse beta-TC3 cells resulted in a 2-fold increase in stimulated secretion of large dense-core vesicles (LDCVs), while overexpression suppressed secretion. Moreover, ultrastructural analysis of blood platelets from the patients with haploinsufficieny of one of the three candidate genes, showed morphological abnormalities of dense-core granules, which closely resemble LDCVs. Taken together, this study shows that in three independent patients with autism three different negative regulators of LDCV secretion are affected, respectively, suggesting that in at least a subgroup of patients the regulation of neuronal vesicle trafficking may be involved in the pathogenesis of autism.


Asunto(s)
Trastorno Autístico/genética , Proteínas Portadoras/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Vesículas Secretoras/metabolismo , Adulto , Animales , Trastorno Autístico/sangre , Plaquetas/patología , Proteínas Portadoras/fisiología , Línea Celular , Cromosomas Humanos Par 15 , Silenciador del Gen , Humanos , Masculino , Proteínas de la Membrana/fisiología , Ratones , Translocación Genética
8.
Liver Transpl ; 18(12): 1495-507, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22987314

RESUMEN

The wider use of livers from expanded criteria donors and donation after circulatory death donors may help to improve access to liver transplantation. A prerequisite for safely using these higher risk livers is the development of objective criteria for assessing their condition before transplantation. Compared to simple cold storage, hypothermic machine perfusion (HMP) provides a unique window for evaluating liver grafts between procurement and transplantation. In this proof-of-concept study, we tested basic parameters during HMP that may reflect the condition of human liver grafts, and we assessed their morphology after prolonged HMP. Seventeen discarded human livers were machine-perfused. Eleven livers were nontransplantable (major absolute contraindications and severe macrovesicular steatosis in the majority of the cases). Six livers were found in retrospect to be transplantable but could not be allocated and served as controls. Metabolic parameters (pH, lactate, partial pressure of oxygen, and partial pressure of carbon dioxide), enzyme release in the perfusate [aspartate aminotransferase (AST) and lactate dehydrogenase (LDH)], and arterial/portal resistances were monitored during HMP. Nontransplantable livers released more AST and LDH than transplantable livers. In contrast, arterial/portal vascular resistances and metabolic profiles did not differ between the 2 groups. Morphologically, transplantable livers remained well preserved after 24 hours of HMP. In conclusion, HMP preserves the morphology of human livers for prolonged periods. A biochemical analysis of the perfusate provides information reflecting the extent of the injury endured.


Asunto(s)
Frío , Trasplante de Hígado/métodos , Hígado/efectos de los fármacos , Soluciones Preservantes de Órganos/farmacología , Preservación de Órganos/métodos , Perfusión , Donantes de Tejidos/provisión & distribución , Adenosina Trifosfato/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Alanina Transaminasa/metabolismo , Aspartato Aminotransferasas/metabolismo , Biomarcadores/metabolismo , Dióxido de Carbono/metabolismo , Diseño de Equipo , Femenino , Regulación de la Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , L-Lactato Deshidrogenasa/metabolismo , Ácido Láctico/metabolismo , Hígado/irrigación sanguínea , Hígado/metabolismo , Hígado/ultraestructura , Masculino , Persona de Mediana Edad , Preservación de Órganos/instrumentación , Oxígeno/metabolismo , Presión Parcial , Perfusión/instrumentación , ARN Mensajero/metabolismo , Factores de Tiempo , Resistencia Vascular , Adulto Joven
9.
J Cell Mol Med ; 15(12): 2586-93, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21251216

RESUMEN

The upper lamina propria (ULP) area of interstitial cells (IC) in bladder has been studied for more than a decade in several species including human beings. Nevertheless there is still lack of uniformity in terminology of this cell layer. The aim of the present study was to add new data to the morphological and immunohistochemical phenotype of these cells and to find out whether this phenotype is changed in bladders from patients with neurogenic detrusor overactivity (NDO) and bladder pain syndrome (BPS). Bladder tissue was obtained from a control group and from patients with NDO and BPS. Samples were processed for morphology, electron microscopy and immunohistochemistry. A morphological and immunohistochemical phenotype for the ULP IC was assessed and changes in this phenotype were looked for in samples from patients with NDO and BPS. The ULP IC were characterized ultrastructurally by the presence of actin filaments with densifications, many caveolae and abundant rough endoplasmic reticulum (RER); on immunohistochemistry ULP IC were immunoreactive for α-sma, vimentin, CD10 and podoplanin and categorized as interstitial Cajal-like cells (ICLC). In NDO and BPS bladders we found a phenotypical shift towards a fibroblastic phenotype which was even more pronounced in the NDO group. In both groups there was also an increased presence in ULP lymphocytes. The ULP area in the human bladder contains a population of ICLC with distinct ultrastructural morphology and immunohistochemical phenotype. Their unique α-sma(+) /desmin(-) /CD34(-) phenotype allows studying this population in various bladder disorders. In bladders form patients with BPS and NDO, we observed these ULP ICLC to shift towards a fibroblast phenotype.


Asunto(s)
Cistitis Intersticial/patología , Células Intersticiales de Cajal/patología , Membrana Mucosa/patología , Vejiga Urinaria Neurogénica/patología , Vejiga Urinaria Hiperactiva/patología , Vejiga Urinaria/citología , Adulto , Femenino , Humanos , Células Intersticiales de Cajal/ultraestructura , Masculino , Persona de Mediana Edad , Membrana Mucosa/ultraestructura , Fenotipo , Vejiga Urinaria/ultraestructura
10.
J Autoimmun ; 36(3-4): 239-52, 2011 May.
Artículo en Inglés | MEDLINE | ID: mdl-21376533

RESUMEN

Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a key enzyme involved in inflammatory, hematological, vascular and neoplastic diseases. In previous studies, we explored the intracellular substrate set or 'degradome' of MMP-9 and found many systemic autoantigens as novel intracellular gelatinase B substrates. Little is known, however, about the functional role of MMP-9 in the development of systemic autoimmunity in vivo. B6(lpr/lpr) mice with defective Fas-mediated apoptosis were used to investigate the functions of MMP-9 in lymphocyte proliferation and in the development of systemic autoimmunity. Combined Fas and gelatinase B deficiency resulted in extreme lymphoproliferative disease with enhanced lymphadenopathy and splenomegaly, and significantly reduced survival compared with single Fas deficiency. At the cellular level, this was corroborated by increased lymph node accumulation of 'double negative' T cells, B cells and myeloid cells. In addition, higher autoantibody titers and more pronounced autoimmune tissue injury were found in the absence of MMP-9, culminating in chronically enhanced systemic lupus erythematosus (SLE)-like autoimmunity. After cleavage by MMP-9 the SLE autoantigens U1snRNP A and ribosomal protein P0 were hardly recognized by plasma samples of both B6(lpr/lpr).MMP-9⁻/⁻ and B6(lpr/lpr).MMP-9+/+ mice, pointing to a destruction of B cell epitopes by MMP-9-mediated proteolysis. In addition, the same loss of immunodominant epitopes was observed with plasma samples from SLE patients, suggesting that MMP-9 suppresses systemic antibody-mediated autoimmunity by clearance of autoepitopes in immunogenic substrates. Thus, new protective functions for MMP-9 were revealed in the suppression of lymphoproliferation and dampening of systemic autoimmunity, cautioning against the long-term use of MMP inhibitors in autoimmune lymphoproliferative syndrome (ALPS) and SLE.


Asunto(s)
Enfermedades Autoinmunes/etiología , Lupus Eritematoso Sistémico/etiología , Trastornos Linfoproliferativos/etiología , Metaloproteinasa 9 de la Matriz/fisiología , Animales , Autoinmunidad , Epítopos de Linfocito B/inmunología , Femenino , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr
11.
Proc Natl Acad Sci U S A ; 105(28): 9733-8, 2008 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-18606991

RESUMEN

Inflammatory destruction of intrahepatic bile ducts is a common cause of vanishing bile duct syndrome and cholestasis, often progressing to biliary cirrhosis and liver failure. However, the molecular mechanisms underlying the pathogenesis of inflammatory biliary disease are poorly understood. Here, we show that the two IkappaB kinases, IKK1/IKKalpha and IKK2/IKKbeta, display distinct collaborative and specific functions that are essential to protect the liver from cytokine toxicity and bile duct disease. Combined conditional ablation of IKK1 and IKK2, but not of each kinase alone, sensitized the liver to in vivo LPS challenge, uncovering a redundant function of the two IkappaB kinases in mediating canonical NF-kappaB signaling in hepatocytes and protecting the liver from TNF-induced failure. Unexpectedly, mice with combined ablation of IKK1 and IKK2 or IKK1 and NEMO spontaneously developed severe jaundice and fatal cholangitis characterized by inflammatory destruction of small portal bile ducts. This bile duct disease was caused by the combined impairment of canonical NF-kappaB signaling together with inhibition of IKK1-specific functions affecting the bile-blood barrier. These results reveal a novel function of the two IkappaB kinases in cooperatively regulating liver immune homeostasis and bile duct integrity and suggest that IKK signaling may be implicated in human biliary diseases.


Asunto(s)
Enfermedades de los Conductos Biliares/etiología , Conductos Biliares/enzimología , Quinasa I-kappa B/fisiología , Hígado , Animales , Enfermedades de los Conductos Biliares/patología , Conductos Biliares/fisiología , Hepatocitos/metabolismo , Lipopolisacáridos/farmacología , Ratones , Ratones Noqueados , FN-kappa B/metabolismo
12.
Gut ; 59(2): 164-9, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19880965

RESUMEN

BACKGROUND: Oesophageal mucosa dilated intercellular spaces (DIS) may be important for symptom perception in non-erosive reflux disease (NERD). Patients with NERD might have DIS even in the proximal oesophagus. We aimed to assess the effect of oesophageal perfusions with acid and weakly acidic solutions on 'exposed' and 'non-exposed' oesophageal mucosa and its relationship to symptoms in healthy subjects. METHODS: 14 healthy volunteers underwent upper gastrointestinal endoscopy with biopsies at 3 and 13 cm proximal to the oesophagogastric junction (OGJ). In following sessions, subjects received 30 min perfusions with neutral, weakly acidic, acidic and acidic-bile acid solutions at 5 cm above the EGJ (separated 4 weeks). Biopsies were taken 20 min after perfusions. Electron microscopy was used to measure DIS. Subjects scored heartburn during perfusions using a visual analogue scale. RESULTS: (1) Oesophageal perfusion with acid solutions, with or without bile acids, provoked DIS in the 'exposed' oesophageal mucosa; (2) oesophageal perfusion with weakly acidic solutions provoked identical changes to those observed after perfusion with acid solutions; (3) distal oesophageal perfusions not only provoked changes in the 'exposed' but also in the more proximal 'non-exposed' mucosa; and (4) in spite of the presence of perfusion-induced DIS, most healthy subjects did not perceive heartburn during the experiments. CONCLUSIONS: The human oesophageal mucosa is very sensitive to continuous exposure with acidic and weakly acidic solutions. In spite of the presence of intraluminal acid and DIS, healthy subjects did not experience heartburn, suggesting that NERD patients should have other critical factors underlying their symptoms.


Asunto(s)
Ácidos/farmacología , Esófago/efectos de los fármacos , Adulto , Esófago/ultraestructura , Femenino , Pirosis/inducido químicamente , Pirosis/patología , Humanos , Concentración de Iones de Hidrógeno , Masculino , Microscopía Electrónica , Membrana Mucosa/efectos de los fármacos , Membrana Mucosa/ultraestructura , Dimensión del Dolor/métodos , Índice de Severidad de la Enfermedad , Adulto Joven
13.
Curr Urol ; 15(2): 126-128, 2021 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-34168533

RESUMEN

The male Wolffian tumor is an extremely rare case in male patients. Here, we report a patient with such malignancy and successful radical surgical treatment at 15-year follow-up. The clinicopathological, immunohistochemical, and ultrastructural features are described. The differential diagnosis of this tumor in a male patient is discussed.

14.
Int J Cancer ; 127(12): 2790-803, 2010 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-21351258

RESUMEN

Cutaneous squamous cell carcinoma (cSCC) is one of the most common cancers in the Caucasian population. Although early stages of skin cancer have a high curability and excellent prognosis, advanced cSCC shows resistance to chemotherapy, including cisplatin. The PI3-K/AKT pathway is known to have a role in both skin cancer development and resistance to therapeutic drugs. In this study, we used isogenic cell lines representing different stages of malignant transformation of the keratinocytes that were derived from dysplastic forehead skin (PM1), primary cutaneous SCC (MET1) and its lymph node metastasis (MET4) of an immunosuppressed patient. We show that skin tumor progression parallels enhanced AKT activation and increased resistance to cisplatin-induced apoptosis. Pharmacological AKT inhibition, or specific AKT1 knock down, sensitizes the apoptosis-resistant MET1 and, to a lesser extent, MET4 cells to cisplatin-mediated cell death. Concomitantly autophagy induction was observed in MET4, as demonstrated by accumulation of the autophagic protein marker LC3-II, by analysis of full autophagosome maturation process using tandem mRFP-GFP fluorescence microscopy and by electron microscopy. Counteracting the autophagic process by 3-methyladenine or specific ATG5 knock down enhanced cytotoxicity of cisplatin combined with AKT inhibitor, thus revealing a key role for autophagy in chemoresistance. Taken together, these results indicate that concomitant inhibition of autophagy is required to increase the therapeutic benefit of AKT inhibition for combination therapy with the standard chemotherapeutic agent cisplatin in advanced skin carcinoma.


Asunto(s)
Apoptosis , Autofagia , Carcinoma de Células Escamosas/secundario , Cisplatino/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias Cutáneas/patología , Adenina/análogos & derivados , Adenina/farmacología , Antineoplásicos/farmacología , Western Blotting , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Metástasis Linfática , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Polimorfismo de Nucleótido Simple/genética , Proteínas Proto-Oncogénicas c-akt/genética , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
15.
Hum Mol Genet ; 17(3): 357-66, 2008 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-17981813

RESUMEN

Controversy exists regarding the cause of the significantly increased blood loss during spinal surgery in Duchenne muscular dystrophy (DMD) patients compared with similar surgery in other patients. DMD is caused by a mutation in the cytoskeletal dystrophin, which binds to extracellular matrix laminin and which has been described as a G-protein-coupled receptor. We hypothesized that disturbed cytoskeleton organization in DMD patients would alter Gs protein and collagen signalling in platelets, leading to dysfunctional platelets and a haemorrhagic tendency during surgery. In the present study, we found that platelets and skin fibroblasts, respectively, express the Dp71 and Dp116 dystrophin isoforms. Absent or decreased expression of these isoforms in DMD patients correlates with significant Gs alpha upregulation. Moreover, dysfunctional dystrophin in these cells is accompanied with increased Gs signalling and higher cAMP levels after Gs stimulation. Functional analysis showed that DMD platelets responded slower to collagen with an extensive shape change in the aggregometer and with a significantly reduced platelet adhesion to coated collagen under flow. The decreased collagen activation was shown to result from both Gs activation and cytoskeletal disruption and not from decreased expression of platelet membrane receptors or impaired von Willebrand factor (vWF) activity. In conclusion, DMD platelets have a disorganized cytoskeleton and manifest Gs hyperactivity and reduced platelet collagen reactivity. Their increased bleeding during surgery will, at least partly, result from the increased platelet Gs activity after the release of natural Gs agonists as prostacyclin during surgery and an ineffective reactivity to collagen.


Asunto(s)
Plaquetas/metabolismo , Distrofina/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/sangre , Distrofia Muscular de Duchenne/sangre , Distrofia Muscular de Duchenne/genética , Fusión Vertebral/efectos adversos , Pérdida de Sangre Quirúrgica/fisiopatología , Plaquetas/efectos de los fármacos , Plaquetas/ultraestructura , Estudios de Casos y Controles , Niño , Colágeno/farmacología , Citoesqueleto/metabolismo , Humanos , Técnicas In Vitro , Masculino , Mutación , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/genética , Transducción de Señal
16.
Pediatr Res ; 67(3): 314-9, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19924028

RESUMEN

We describe a patient, who developed during the first week of life petechiae and hematomas caused by severe thrombocytopenia and gastrointestinal bleeding due to multiple small gastric hemangiomata. Bone marrow examination showed hypermegakaryocytosis and dysmegakaryopoiesis. Alloimmune thrombocytopenia was excluded. Only 3 y later, platelet counts normalized and bleedings disappeared but small skin hemangiomata remained. Electron microscopy showed enlarged round platelets with a paucity of alpha granules similar as in GATA1-deficient patients but no GATA1 mutation was found. Immunoblot analysis showed a strong interaction between patient Igs and recombinant GATA1, GATA2, and the N finger (Nf) of GATA1. The lymphocyte transformation test with recombinant GATA1Nf was positive. In vitro culturing of normal CD34 cells with purified patient Igs showed a decreased number of megakaryocyte colonies but an increased overall size of the colonies compared with control Igs. Mice injected with patient Igs showed a reduced platelet count compared with mice injected with control Igs. Thrombopoiesis was also reduced after injection of patient Igs in transgenic zebrafish compared with control Igs. In conclusion, this study is the first report of an anti-GATA1 autoantibody leading to severe thrombocytopenia and gastrointestinal bleeding from multiple pinpoint hemangiomata.


Asunto(s)
Autoanticuerpos/sangre , Autoinmunidad , Factor de Transcripción GATA1/inmunología , Hemorragia Gastrointestinal/inmunología , Neoplasias Gastrointestinales/inmunología , Hemangioma/inmunología , Trombocitopenia/inmunología , Animales , Animales Modificados Genéticamente , Células Cultivadas , Preescolar , Femenino , Factor de Transcripción GATA1/genética , Hemorragia Gastrointestinal/sangre , Hemorragia Gastrointestinal/terapia , Neoplasias Gastrointestinales/sangre , Neoplasias Gastrointestinales/terapia , Hemangioma/sangre , Humanos , Recién Nacido , Linfocitos/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Recuento de Plaquetas , Índice de Severidad de la Enfermedad , Trombocitopenia/sangre , Trombocitopenia/terapia , Trombopoyesis , Transfección , Pez Cebra/genética
17.
Cancer Genomics Proteomics ; 17(6): 669-685, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33099469

RESUMEN

BACKGROUND: Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. Mice lacking the tumor-suppressive protein phosphatase 2A subunit B56δ (Ppp2r5d) spontaneously develop HCC, correlating with increased c-MYC oncogenicity. MATERIALS AND METHODS: We used two-dimensional difference gel electrophoresis-coupled matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to identify differential proteomes of livers from wild-type, non-cancerous and HCC-affected B56δ knockout mice. RESULTS: A total of 23 proteins were differentially expressed/regulated in liver between wild-type and non-cancerous knockout mice, and 119 between non-cancerous and HCC knockout mice ('cancer proteins'). Overlap with our reported differential transcriptome data was poor. Overall, 56% of cancer proteins were reported before in HCC proteomics studies; 44% were novel. Gene Ontology analysis revealed cancer proteins mainly associated with liver metabolism (18%) and mitochondria (15%). Ingenuity Pathway Analysis identified 'cancer' and 'gastrointestinal disease' as top hits. CONCLUSION: We identified several proteins for further exploration as novel potential HCC biomarkers, and independently underscored the relevance of Ppp2r5d knockout mice as a valuable hepatocarcinogenesis model.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteína Fosfatasa 2/fisiología , Proteoma/análisis , Proteoma/metabolismo , Animales , Apoptosis , Carcinoma Hepatocelular/patología , Proliferación Celular , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Tumorales Cultivadas
18.
Am J Physiol Lung Cell Mol Physiol ; 297(5): L903-11, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19734320

RESUMEN

In vivo, translocation of inhaled nanoparticles to the circulation has been demonstrated. However, the interaction of nanoparticles with the lung epithelium is not understood. In this study, we investigated, in vitro, the translocation of nano-sized quantum dots (QDs; 25 pmol/ml) through a tight monolayer of primary isolated rat alveolar epithelial cells. The influence of surface charge on translocation was examined using nonfunctionalized QDs, amine-QDs, and carboxyl-QDs. The interaction between nanoparticles and the lung epithelium was monitored by repeatedly measuring the transepithelial electrical resistance (TEER) and by examining the cell layer with confocal microscopy. The effect of oxidative stress was tested by incubating the cells with tert-butyl hydroperoxide (t-BOOH; 75 microM or 1 or 10 mM); the antioxidant N-acetyl-L-cysteine was also used to assess the role of particle-mediated oxidative stress. No translocation through a tight monolayer of primary rat alveolar epithelial cells was observed for any of the different types of QDs. In general, an increase in TEER was found after incubation with QDs. A condition of low oxidative stress did not enhance translocation. In contrast, conditions of high stress (1 or 10 mM t-BOOH or due to QDs toxicity) with disruption of the cell layer, as shown in a decreased TEER, resulted in substantial translocation. In conclusion, no translocation of QDs was found through a tight monolayer of primary rat alveolar epithelial cells, regardless of the QDs surface charge. QDs did not impair the barrier function of the epithelial cells. In conditions with disruption of the cell-cell barrier, translocation was demonstrated.


Asunto(s)
Estrés Oxidativo , Puntos Cuánticos , Animales , Transporte Biológico/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Células Cultivadas , Impedancia Eléctrica , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Masculino , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Tamaño de la Partícula , Alveolos Pulmonares/citología , Ratas , Ratas Wistar , terc-Butilhidroperóxido/farmacología
19.
Gastroenterology ; 134(3): 781-92, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18325391

RESUMEN

BACKGROUND & AIMS: The Rho small guanosine triphosphatase Cdc42 is critical for diverse cellular functions, including regulation of actin organization, cell polarity, intracellular membrane trafficking, transcription, cell-cycle progression, and cell transformation. This implies that Cdc42 might be required for liver function. METHODS: Mice in which Cdc42 was ablated in hepatocytes and bile duct cells were generated by Cre-loxP technology. Livers were examined by histologic, immunohistochemical, ultrastructural, and serum analysis to define the effect of loss of Cdc42 on liver structure. RESULTS: Mice lacking Cdc42 in their hepatocytes were born at Mendelian ratios. They did not show increased mortality but showed chronic jaundice. They developed hepatomegaly soon after birth, and signs of liver transformation, such as formation of nodules and tumors, became visible macroscopically at age 6 months. Hepatocellular carcinoma was observed 8 months after birth. Tumors grew slowly and lacked expression of nuclear beta-catenin. Lung metastases were observed at the late stage of carcinogenesis. Immunofluorescent examination and electron microscopy revealed severe defects in the liver. At the age of 2 months, the canaliculi between hepatocytes were greatly enlarged, although the tight junctions flanking the canaliculi appeared normal. Regular liver plates were absent. E-cadherin expression pattern and gap junction localization were distorted. Analysis of serum samples indicated cholestasis. CONCLUSIONS: We describe a mouse model in which chronic liver disease leads to hepatocarcinogenesis.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Transformación Celular Neoplásica/metabolismo , Ictericia Obstructiva/complicaciones , Neoplasias Hepáticas/metabolismo , Hígado/metabolismo , Neoplasias Pulmonares/metabolismo , Lesiones Precancerosas/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Conductos Biliares/metabolismo , Conductos Biliares/ultraestructura , Cadherinas/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Moléculas de Adhesión Celular/metabolismo , Proteínas de Ciclo Celular , Polaridad Celular , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Enfermedad Crónica , Progresión de la Enfermedad , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , Hepatomegalia , Uniones Intercelulares/metabolismo , Uniones Intercelulares/ultraestructura , Ictericia Obstructiva/genética , Ictericia Obstructiva/metabolismo , Ictericia Obstructiva/patología , Hígado/enzimología , Hígado/ultraestructura , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tamaño de los Órganos , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Proteína Quinasa C/metabolismo , Factores de Tiempo , Proteína de Unión al GTP cdc42/deficiencia , Proteína de Unión al GTP cdc42/genética
20.
Crit Care Med ; 37(4): 1355-64, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19242345

RESUMEN

OBJECTIVE: In critically ill patients, preventing hyperglycemia (HG) with insulin therapy partially prevented organ dysfunction and protected mitochondria. A study in a rabbit model of critical illness indicated that lower blood glucose level, rather than higher insulinemia, is a key factor in such organ protection. In this model, we now investigated the impact of blood glucose lowering vs. hyperinsulinemia (HI) on mitochondria in relation to organ damage. We assessed whether such effects on mitochondria are mediated indirectly via organ perfusion or directly via reducing cellular glucose toxicity. DESIGN: Prospective, randomized laboratory investigation. SETTING: University laboratory. SUBJECTS: Three-month-old male rabbits. INTERVENTIONS: After induction of critical illness by burn injury, followed by fluid-resuscitation and parenteral nutrition, rabbits were allocated to four groups, each a combination of normal or elevated blood glucose levels with normal or elevated insulin levels. This required alloxan administration, immediately followed by intravenous insulin and glucose infusions titrated to the respective targets. MEASUREMENTS AND MAIN RESULTS: In liver, the reduced damage by glucose lowering was not explained by better perfusion/oxygen delivery. Abnormal mitochondrial ultrastructure and function was present in the two hyperglycemic groups, most pronounced with concomitant HI. Affected mitochondrial respiratory chain enzyme activities were reduced to 25% to 62% of values in healthy rabbits, in the presence of up to five-fold increased tissue levels of glucose. This was accompanied by elevated levels of dicarbonyls, which may mediate direct toxicity of cellular glucose overload and accelerated glycolysis. The abnormalities were also present in myocardium, although to a lesser extent, and absent in skeletal muscle. CONCLUSIONS: In a rabbit model of critical illness, HG evokes cellular glucose overload in liver and myocardium inducing mitochondrial dysfunction, which explained the HG-induced organ damage. Maintenance of normoglycemia, but not HI, protects against such mitochondrial and organ damage.


Asunto(s)
Glucemia , Quemaduras/metabolismo , Mitocondrias , Animales , Glucemia/análisis , Quemaduras/sangre , Quemaduras/complicaciones , Enfermedad Crítica , Modelos Animales de Enfermedad , Glucosa/toxicidad , Hiperglucemia/etiología , Hiperinsulinismo/etiología , Masculino , Conejos
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