[Study of the expression of neural stem cell markers in neuroblastoma tumor samples and correlation with prognostic factors]. / Estudio de expresión de marcadores de células madre neurales en neuroblastoma y correlación con factores pronóstico.

INTRODUCTION: The existence of cancer stem cells (CSC) in neuroblastoma (NB) has been associated with the development of metastasis, resistance to chemotherapy and recurrence. Our objective is to analyze the expression of proliferation and differentiation markers of neural progenitor cells in NB samples, and to correlate this expression with clinical variables such as histology, genetics and response to conventional therapy. MATERIAL AND METHODS: We performed a retrospective-experimental study with neuroblastoma samples obtained from biopsies or tumor resections between 2010-2012 in our Hospital. Fluorescence immunohistochemistry was used to analyze the expression of the different markers: CD44, CD74, CD133, tyrosine hydroxylase, endothelin receptors type A (ETA) and B (ETB), p75, nestina y and Phox2b, all of them related to neural stem cell biology. The level of expression of the markers was then correlated with clinical variables. RESULTS: Nestin expression was positive in 72.2% of samples and ETA in 66.7%. PHOX2B and CD74 expression were lower, being positive in less than 30%. The markers CD44, ETB and PHOX2B were expressed in more aggressive tumors. ETA expression correlated significantly with unfavorable histology tumors (p= 0.01), N-myc amplification (p= 0.05) and recurrence/progression (p= 0.05). CONCLUSION: The expression of CD44, ETB and ETA was associated with more aggressive tumors and poor prognostic factors. These markers are in the membrane of neural stem cells and may be useful to identify and isolate by flow cytometry CSCs of NB for the study of new therapeutic targets.

The carotid body, a neurogenic niche in the adult peripheral nervous system.

We have described a new population of adult neural stem cells residing in the carotid body, a chemoreceptor organ in the peripheral nervous system. These progenitor cells support neurogenesis in vivo in response to physiological stimuli like hypoxemia, and give rise to multipotent neurospheres in culture. Studying the biology of CB stem cells helps to understand the physiological adaptations of the organ, and might shed light on the pathogenesis of CB tumors. Understanding proliferation and differentiation of these cells will enable their use for cell therapy against neurodegenerative diseases.

Carotid body oxygen sensing.

The carotid body (CB) is a neural crest-derived organ whose major function is to sense changes in arterial oxygen tension to elicit hyperventilation in hypoxia. The CB is composed of clusters of neuron-like glomus, or type-I, cells enveloped by glia-like sustentacular, or type-II, cells. Responsiveness of CB to acute hypoxia relies on the inhibition of O(2)-sensitive K(+) channels in glomus cells, which leads to cell depolarisation, Ca(2+) entry and release of transmitters that activate afferent nerve fibres. Although this model of O(2) sensing is generally accepted, the molecular mechanisms underlying K(+) channel modulation by O(2) tension are unknown. Among the putative hypoxia-sensing mechanisms there are: the production of oxygen radicals, either in mitochondria or reduced nicotinamide adenine dinucleotide phosphate oxidases; metabolic mitochondrial inhibition and decrease of intracellular ATP; disruption of the prolylhydroxylase/hypoxia inducible factor pathway; or decrease of carbon monoxide production by haemoxygenase-2. In chronic hypoxia, the CB grows with increasing glomus cell number. The current authors have identified, in the CB, neural stem cells, which can differentiate into glomus cells. Cell fate experiments suggest that the CB progenitors are the glia-like sustentacular cells. The CB appears to be involved in the pathophysiology of several prevalent human diseases.

Collapse of conductance is prevented by a glutamate residue conserved in voltage-dependent K(+) channels.

Voltage-dependent K(+) channel gating is influenced by the permeating ions. Extracellular K(+) determines the occupation of sites in the channels where the cation interferes with the motion of the gates. When external [K(+)] decreases, some K(+) channels open too briefly to allow the conduction of measurable current. Given that extracellular K(+) is normally low, we have studied if negatively charged amino acids in the extracellular loops of Shaker K(+) channels contribute to increase the local [K(+)]. Surprisingly, neutralization of the charge of most acidic residues has minor effects on gating. However, a glutamate residue (E418) located at the external end of the membrane spanning segment S5 is absolutely required for keeping channels active at the normal external [K(+)]. E418 is conserved in all families of voltage-dependent K(+) channels. Although the channel mutant E418Q has kinetic properties resembling those produced by removal of K(+) from the pore, it seems that E418 is not simply concentrating cations near the channel mouth, but has a direct and critical role in gating. Our data suggest that E418 contributes to stabilize the S4 voltage sensor in the depolarized position, thus permitting maintenance of the channel open conformation.

Stem cell self-renewal and cancer cell proliferation are regulated by common networks that balance the activation of proto-oncogenes and tumor suppressors.

Networks of proto-oncogenes and tumor suppressors that control cancer cell proliferation also regulate stem cell self-renewal and possibly stem cell aging. Proto-oncogenes promote regenerative capacity by promoting stem cell function but must be balanced with tumor suppressor activity to avoid neoplastic proliferation. Conversely, tumor suppressors inhibit regenerative capacity by promoting cell death or senescence in stem cells. For example, the polycomb family proto-oncogene, Bmi-1, is consistently required for the self-renewal of diverse adult stem cells, as well as for the proliferation of cancer cells in the same tissues. Bmi-1 promotes stem cell self-renewal partly by repressing the expression of Ink4a and Arf, tumor suppressor genes that are commonly deleted in cancer. Despite ongoing Bmi-1 expression, Ink4a expression increases with age, potentially reducing stem cell frequency and function. Increased tumor suppressor activity during aging therefore may partly account for age-related declines in stem cell function. Thus, networks of proto-oncogenes and tumor suppressors have evolved to coordinately regulate stem cell function throughout life. Imbalances within such networks cause cancer or premature declines in stem cell activity that resemble accelerated aging.

[Pathogeny of paranoid delirium]. / Contribución a la patogenia del delirio paranoide.

Revisiting the old discussion on the intellectual, or emotional genesis of non-hallucinatory delusions, the author collects the opinion of classical authors on this essential subject of Psychiatry. Based on Kraepelin's description of the constitutive elements of paranoiac delusion, and relating such descriptions to the current advances in Neurobiology--especially Goldar's, on the participation of the limbic brain to the dynamics of personality--the author discusses his position regarding delirious verbal performances, and intends a subdivision of such disorders according to their own pathogenesis.

Cellular mechanism of oxygen sensing.

O2 sensing is a fundamental biological process necessary for adaptation of living organisms to variable habitats and physiological situations. Cellular responses to hypoxia can be acute or chronic. Acute responses rely mainly on O2-regulated ion channels, which mediate adaptive changes in cell excitability, contractility, and secretory activity. Chronic responses depend on the modulation of hypoxia-inducible transcription factors, which determine the expression of numerous genes encoding enzymes, transporters and growth factors. O2-regulated ion channels and transcription factors are part of a widely operating signaling system that helps provide sufficient O2 to the tissues and protect the cells against damage due to O2 deficiency. Despite recent advances in the molecular characterization of O2-regulated ion channels and hypoxia-inducible factors, several unanswered questions remain regarding the nature of the O2 sensor molecules and the mechanisms of interaction between the sensors and the effectors. Current models of O2 sensing are based on either a heme protein capable of reversibly binding O2 or the production of oxygen reactive species by NAD(P)H oxidases and mitochondria. Complete molecular characterization of the hypoxia signaling pathways will help elucidate the differential sensitivity to hypoxia of the various cell types and the gradation of the cellular responses to variable levels of PO2. A deeper understanding of the cellular mechanisms of O2 sensing will facilitate the development of new pharmacological tools effective in the treatment of diseases such as stroke or myocardial ischemia caused by localized deficits of O2.

Secretory responses of intact glomus cells in thin slices of rat carotid body to hypoxia and tetraethylammonium.

We have developed a thin-slice preparation of whole rat carotid body that allows us to perform patch-clamp recording of membrane ionic currents and to monitor catecholamine secretion by amperometry in single glomus cells under direct visual control. In normoxic conditions (P(O(2)) approximately 140 mmHg; 1 mmHg = 133 Pa), most glomus cells did not have measurable secretory activity, but exposure to hypoxia (P(O(2)) approximately 20 mmHg) elicited the appearance of a large number of spike-like exocytotic events. This neurosecretory response to hypoxia was fully reversible and required extracellular Ca(2+) influx. The average charge of single quantal events was 46 +/- 25 fC (n = 218), which yields an estimate of approximately 140,000 catecholamine molecules per vesicle. Addition of tetraethylammonium (TEA; 2-5 mM) to the extracellular solution induced in most (>95%) cells tested (n = 32) a secretory response similar to that elicited by low P(O(2)). Cells nonresponsive to hypoxia but activated by exposure to high external K(+) were also stimulated by TEA. A secretory response similar to the responses to hypoxia and TEA was also observed after treatment of the cells with iberiotoxin to block selectively Ca(2+)- and voltage-activated maxi-K(+) channels. Our data further show that membrane ion channels are critically involved in sensory transduction in the carotid body. We also show that in intact glomus cells inhibition of voltage-dependent K(+) channels can contribute to initiation of the secretory response to low P(O(2)).

K+ and Ca2+ channel activity and cytosolic [Ca2+] in oxygen-sensing tissues.

Ion channels are known to participate in the secretory or mechanical responses of chemoreceptor cells to changes in oxygen tension (P(O2)). We review here the modifications of K+ and Ca2+ channel activity and the resulting changes in cytosolic [Ca2+] induced by low P(O2) in glomus cells and arterial smooth muscle which are well known examples of O2-sensitive cells. Glomus cells of the carotid body behave as presynaptic-like elements where hypoxia produces a reduction of K+ conductance leading to enhanced membrane excitability, Ca2+ entry and release of dopamine and other neurotransmitters. In arterial myocytes, hypoxia can inhibit or potentiate Ca2+ channel activity, thus regulating cytosolic [Ca2+] and contraction. Ca2+ channel inhibition is observed in systemic myocytes and most conduit pulmonary myocytes, whereas potentiation is seen in a population of resistance pulmonary myocytes. The mechanism whereby O2 modulates ion channel activity could depend on either the direct allosteric modulation by O2-sensing molecules or redox modification by reactive chemical species.

Estudio de expresión de marcadores de células madre neurales en neuroblastoma y correlación con factores pronóstico / Study of the expression of neural Stem Cell markers in neuroblastoma tumor samples and correlation with prognostic factors

INTRODUCCIÓN: En el neuroblastoma (NB), la existencia de células madre cancerosas (CMC) se ha relacionado con la presencia de metástasis, resistencia al tratamiento quimioterápico y recidiva. Nuestro objetivo es analizar la expresión de marcadores relacionados con proliferación y diferenciación de células progenitoras neurales en muestras de NB, y correlacionarlo con parámetros clínicos, histología, genética y respuesta al tratamiento. MATERIAL Y MÉTODOS: Realizamos un estudio experimental retrospectivo con muestras de neuroblastoma obtenidas mediante biopsia o exéresis tumoral entre 2010 y 2012 en nuestro hospital. Mediante inmunohistoquímica de fluorescencia analizamos la expresión de los marcadores: CD44, CD74, CD133, tirosina hidroxilasa, receptor de endotelina A (REA) y endotelina B (REB), p75, nestina y Phox2b, todos relacionados con la biología de células madre neurales. Posteriormente, relacionamos los niveles de expresión con variables clínicas. RESULTADOS: La expresión de nestina fue positiva en el 72,2% de las muestras y el REA en el 66,7%. Phox2b y CD74 fueron de menor expresión, siendo positiva en menos del 30%. Los marcadores CD44, REB y Phox2b se expresaban en tumores más agresivos. La expresión de REA se correlacionó de forma significativa con tumores de histología desfavorable (p= 0,01), amplificación del N-myc (p= 0,05) y recidiva/ progresión (p= 0,05). [Conclusión] La expresión de CD44, REB y REA se asoció con tumores más agresivos y factores de mal pronóstico. Estos marcadores están presentes en la membrana de células madre neurales, pudiendo ser útiles para identificar y aislar por citometría de flujo las CMCs del NB y para el estudio de nuevas dianas terapéuticas

INTRODUCTION: The existence of cancer stem cells (CSC) in neuroblastoma (NB) has been associated with the development of metastasis, resistance to chemotherapy and recurrence. Our objective is to analyze the expression of proliferation and differentiation markers of neural progenitor cells in NB samples, and to correlate this expression with clinical variables such as histology, genetics and response to conventional therapy. MATERIAL AND METHODS: We performed a retrospective-experimental study with neuroblastoma samples obtained from biopsies or tumor resections between 2010-2012 in our Hospital. Fluorescence immunohistochemistry was used to analyze the expression of the different markers: CD44, CD74, CD133, tyrosine hydroxylase, endothelin receptors type A (ETA) and B (ETB), p75, nestina y and Phox2b, all of them related to neural stem cell biology. The level of expression of the markers was then correlated with clinical variables. [Results] Nestin expression was positive in 72.2% of samples and ETA in 66.7%. PHOX2B and CD74 expression were lower, being positive in less than 30%. The markers CD44, ETB and PHOX2B were expressed in more aggressive tumors. ETA expression correlated significantly with unfavorable histology tumors (p= 0.01), N-myc amplification (p= 0.05) and recurrence/progression (p= 0.05). [Conclusion] The expression of CD44, ETB and ETA was associated with more aggressive tumors and poor prognostic factors. These markers are in the membrane of neural stem cells and may be useful to identify and isolate by flow cytometry CSCs of NB for the study of new therapeutic targets