Protocol for a Wnt reporter assay to measure its activity in human neural stem cells derived from induced pluripotent stem cells.

The canonical Wnt signaling is an essential pathway that regulates cellular proliferation, maturation, and differentiation during neurodevelopment and maintenance of adult tissue homeostasis. This pathway has been implicated with the pathophysiology of neuropsychiatric disorders and was associated with cognitive processes, such as learning and memory. However, the molecular investigation of the Wnt signaling in functional human neural cell lines might be challenging since brain biopsies are not possible and animal models may not represent the polygenic profile of some neurological and neurodevelopmental disorders. In this context, using induced pluripotent stem cells (iPSCs) has become a powerful tool to model disorders that affect the Central Nervous System (CNS) in vitro, by maintaining patients' genetic backgrounds. In this method paper, we report the development of a virus-free Wnt reporter assay in neural stem cells (NSCs) derived from human iPSCs from two healthy individuals, by using a vector containing a reporter gene (luc2P) under the control of a TCF/LEF (T-cell factor/lymphoid enhancer factor) responsive element. Dose-response curve analysis from this luciferase-based method might be useful when testing the activity of the Wnt signaling pathway after agonists (e.g. Wnt3a) or antagonists (e.g. DKK1) administration, comparing activity between cases and controls in distinct disorders. Using such a reporter assay method may help to elucidate whether neurological or neurodevelopmental mental disorders show alterations in this pathway, and testing whether targeted treatment may reverse these. Therefore, our established assay aims to help researchers on the functional and molecular investigation of the Wnt pathway in patient-specific cell types comprising several neuropsychiatric disorders.

Propranolol reduces viability and induces apoptosis in hemangioblastoma cells from von Hippel-Lindau patients.

BACKGROUND: Von Hippel-Lindau (VHL) disease is a rare oncological disease with an incidence of 1:36,000, and is characterized by the growth of different types of tumors: hemangioblastomas in the central nervous system (CNS) and retina, renal carcinoma, pheochromocytomas, pancreatic serous cystadenoma, and endolymphatic sac tumors. These tumors do not express VHL protein (pVHL). pVHL ubiquitinates hypoxia inducible factor (HIF) protein for degradation by the proteasome; in the absence of VHL, HIF translocates to the nucleus to activate the expression of its target genes. Targeting VHL-derived tumors with drugs that have reduced side effects is urgent to avoid repeat CNS surgeries. Recent reports have shown that propranolol, a ß-blocker used for the treatment of hypertension and other cardiac and neurological diseases, is the best option for infantile hemangioma (IH). Propranolol could be an efficient treatment to control hemangioblastoma growth in VHL disease because of its antiangiogenic effects demonstrated in IH and the hypothetical impact on HIF levels. METHODS: HeLa 9X (HRE) hypoxia responsive element cell line and primary hemangioblastoma-derived cells were subjected to propranolol treatment and cell viability and apoptosis were evaluated. HIF1-α and Hif-2α expression after propranolol treatment was analyzed by western blotting. Quantitative PCR was performed to study the mRNA expression of HIF target genes. Vascular endothelial growth factor (VEGF) was measured in culture supernatants by immunoassay. RESULTS: Propranolol downregulated HIF-dependent transcription in HeLa 9XHRE cells. Under hypoxic conditions, propranolol decreased the expression of HIF target genes in hemangioblastoma cells, which stopped proliferating and died following long-term treatment. These results suggests that propranolol treatment promoted reduced HIF protein expression and corresponding downregulation of HIF target genes, and inhibited cell proliferation in parallel with induction of cell death by apoptosis. CONCLUSIONS: Our results suggest that propranolol could reduce the growth of HIF-dependent tumors and may thus be a promising treatment to delay surgery in VHL patients.

Single-Cell Phenotypic Characterization of Human Pituitary GHomas and Non-Functioning Adenomas Based on Hormone Content and Calcium Responses to Hypothalamic Releasing Hormones.

Human pituitary tumors are generally benign adenomas causing considerable morbidity due to excess hormone secretion, hypopituitarism, and other tumor mass effects. Pituitary tumors are highly heterogeneous and difficult to type, often containing mixed cell phenotypes. We have used calcium imaging followed by multiple immunocytochemistry to type growth hormone secreting (GHomas) and non-functioning pituitary adenomas (NFPAs). Individual cells were typed for stored hormones and calcium responses to classic hypothalamic releasing hormones (HRHs). We found that GHomas contained growth hormone cells either lacking responses to HRHs or responding to all four HRHs. However, most GHoma cells were polyhormonal cells responsive to both thyrotropin-releasing hormone (TRH) and GH-releasing hormone. NFPAs were also highly heterogeneous. Some of them contained ACTH cells lacking responses to HRHs or polyhormonal gonadotropes responsive to LHRH and TRH. However, most NFPAs were made of cells storing no hormone and responded only to TRH. These results may provide new insights on the ontogeny of GHomas and NFPAs.

Coordinación de la asistencia entre un hospital comarcal y su centro de referencia: evaluación de dos modelos de atención neumológica / Coordination Between a District Hospital and a Reference Hospital: Evaluation of Chest Disease Care Models

No está bien definida el tipo de asistencia especializada que deben dar los hospitales comarcales. Paraevaluar diferentes opciones, se analizó retrospectivamente la atención neumológica en el año 2008 endos áreas comarcales de Galicia: Barbanza y Cee, con características poblacionales similares. El hospitalde Barbanza dispone de consulta de neumología atendida por especialistas del hospital de referencia 3días/semana, mientras que en Cee la atienden internistas del propio centro. En ambos casos, la hospitalizaciónestá a cargo del servicio de Medicina Interna. Los datos fueron proporcionados por los serviciosadministrativos de los hospitales y autonómicos.Las estancias medias para el agrupador CDM4 fueron similares en los dos comarcales, pero inferioresa las de los centros de referencia. No hubo diferencias en comorbilidad (Charlson) ni en reingresos almes de alta. Se realizaron más exploraciones funcionales en pacientes del Barbanza, tanto en el propiocentro (957 espirometrías vs 21; p < 0,0001) como en el hospital de referencia (214 determinaciones devolúmen/difusión vs 99; p < 0,001). La prevalencia de tratamientos con CPAP fue más elevada en el áreade Barbanza (3,9 vs 2/1.000 habitantes; p < 0,0001). No encontramos diferencias en la prevalencia deoxigenoterapia ni en ventilación mecánica domiciliaria. La mortalidad por patología respiratoria el año2007 fue similar en las dos áreas.Estos datos sugieren que en un modelo de hospital comarcal con neumólogos consultores, la consultaambulatoria facilita el acceso a una asistencia más especializada, más completa y, probablemente, de máscalidad, que los comarcales sin este tipo de consultoría(AU)

Specialised medical care at district hospitals has not been thoroughly defined. Respiratory care data from2008 in Barbanza and Cee hospitals (Galicia, Spain), were analysed to evaluate different approaches,as they are both similar. Barbanza hospital has a chest diseases clinic run by specialist doctors fromthe reference hospital three days per week, while Cee hospital is operated by the staff on site. In bothcases hospitalisation is the responsibility of the Internal Medicine department. Data was provided by theadministrative departments of each hospital and the regional government.Average CDM4 stays were similar for both district hospitals; however, they were lower than in thereference hospital. Charlson scores and re-admissions a month after discharge were similar in both.Barbanza’s hospital carried out more functional explorations, both at the centre (957 spirometries vs21; P<.0001) and at the reference hospital (214 volume/diffusion tests vs 99; P<.001). CPAP treatments were more prevalent in the Barbanza area (3.9 vs 2/1,000 habitants; P<.0001). No differences were foundin oxygen therapy and home mechanical ventilation. Mortality due to respiratory disease in 2007 wassimilar in both regions.Data suggests that in a district hospital scheme supported by chest disease consultants and outpatientclinics gives easier access to specialised, comprehensive and probably, higher quality care than districthospitals without them(AU)

Coordination between a district hospital and a reference hospital: evaluation of chest disease care models.

Specialised medical care at district hospitals has not been thoroughly defined. Respiratory care data from 2008 in Barbanza and Cee hospitals (Galicia, Spain), were analysed to evaluate different approaches, as they are both similar. Barbanza hospital has a chest diseases clinic run by specialist doctors from the reference hospital three days per week, while Cee hospital is operated by the staff on site. In both cases hospitalisation is the responsibility of the Internal Medicine department. Data was provided by the administrative departments of each hospital and the regional government. Average CDM4 stays were similar for both district hospitals; however, they were lower than in the reference hospital. Charlson scores and re-admissions a month after discharge were similar in both. Barbanza's hospital carried out more functional explorations, both at the centre (957 spirometries vs 21; P<.0001) and at the reference hospital (214 volume/diffusion tests vs 99; P<.001). CPAP treatments were more prevalent in the Barbanza area (3.9 vs 2/1,000 habitants; P<.0001). No differences were found in oxygen therapy and home mechanical ventilation. Mortality due to respiratory disease in 2007 was similar in both regions. Data suggests that in a district hospital scheme supported by chest disease consultants and outpatient clinics gives easier access to specialised, comprehensive and probably, higher quality care than district hospitals without them.

Dificultad en el diagnóstico y el tratamiento del carcinoma de paratiroides / Difficulty in the diagnosis and treatment of parathyroid carcinoma: a case report

El carcinoma de paratiroides es una causa poco frecuente de hiperparatiroidismo primario. El principal problema de esta afección es el diagnóstico diferencial inicial entre adenoma y carcinoma de paratiroides, dado que los criterios histológicos en estos casos es difícil que ofrezcan un diagnóstico definitivo, a diferencia de otras neoplasias. No obstante, se encuentran descritas series de signos clínicos y bioquímicos de alta sospecha inicial de malignidad. Asimismo, en los casos de hiperparatiroidismo primario más aparatosos estamos obligados a pensar en esta enfermedad, que se acaba confirmando en la mayor parte de las ocasiones, incluso años después por la aparición de metástasis a distancia. El objetivo de este artículo es la descripción de un caso de hiperparatiroidismo primario severo y persistente en un paciente varón de 40 años, que años después se confirmó como secundario a un carcinoma de paratiroides (AU)

Parathyroid carcinoma is an uncommon cause of primary hyperparathyroidism. Tha main difficulty is the initial differential diagnosis between adenoma and carcinoma of the parathyroid gland because, unlike in other neoplasms, histologic criteria do provide a definitive result. However, clinical and biochemical characteristics that are highly suspicious for malignancy have been described. Moreover, parathyroid carcinoma should be suspected when the primary hyperparathyroidism is severe. In some cases the diagnosis is made several years later with detection of distant metastases. We report the case of a 40-year-old man with severe and persistent primary hyperparathyroidism, which was confirmed years later as being secondary to parathyroid carcinoma (AU)

Cerebellar glioblastoma multiforme in an adult.

Cerebellar glioblastoma multiforme (GBM) is a rare tumor. This is the third case published in Brazilian literature and, the last one has been described more than 15 years ago. The aggressive behavior of GBM prompts for fast treatment, which can be hampered by the fact that the diagnosis of GBM requires a high degree of suspicion. We describe a case of GBM in a 46 years old man. In conjunction, we present a literature review including particular issues, clinical data, advances in imaging studies, pathological characteristics, treatment options and the behavior of such malignant tumor.

Cerebellar glioblastoma multiforme in an adult

Glioblastoma multiforme (GBM) cerebelar é um tumor raro. Apresentamos o terceiro caso revisado em literatura neurológica brasileira, sendo o último descrito há mais de 15 anos. Devido a sua agressividade, o tratamento deve ser instituído rapidamente, porém certa indefinição quanto à conduta pode ocorrer, pois o diagnóstico de GBM pode não ser lembrado. Apresentamos um caso de GBM cerebelar em um homem de 46 anos. Também fazemos uma revisão a respeito de seu comportamento, quadro clínico e avanços quanto à investigação por imagem, aspectos histopatológicos, formas de tratamento e suas características peculiares.