Influence of bradykinin on diacylglycerol and phosphatidic acid accumulation in cultured bovine adrenal chromaffin cells.

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Owen, P J; Boarder, M R

1991-09-01

Earlier studies have shown that bradykinin stimulated release of catecholamines from chromaffin cells by an influx of calcium through dihydropyridine-insensitive channels, and also that bradykinin stimulated (poly)phosphoinositide hydrolysis. To investigate membrane-bound second messengers in chromaffin cells, and to elucidate any role these may play in stimulus-secretion coupling, we have studied the influence of bradykinin on diacylglycerol and phosphatidic acid (PA). Using equilibrium labelling of primary cultures of chromaffin cells with [3H]arachidonic acid or [3H]glycerol, we found no influence of bradykinin (10 nM) on labelled diacylglycerol formation, either in the presence or absence of inhibitors of diacylglycerol lipase or kinase. However, when we used cells prelabelled with 32Pi for 2.5 h, we found that bradykinin produced a substantial stimulation of label found in PA, with an EC50 value of about 1 nM. This bradykinin stimulation of [32P]PA formation was only partially dependent on extracellular calcium, in contrast to the smaller response to nicotine, which was completely dependent on extracellular calcium. Short (10 min) pretreatment with tetradecanoylphorbol acetate (TPA) almost completely eliminated the bradykinin-stimulated formation of inositol phosphates, but failed to affect bradykinin stimulation of label in PA, suggesting that PA production in response to bradykinin is not downstream of phospholipase C activation. TPA alone failed to stimulate [32P]PA substantially, whereas long-term (24 or 48 h) treatment with TPA failed to attenuate the response to bradykinin. Diacylglycerol kinase inhibitors were also without effect on the bradykinin stimulation of [32P]PA. These results suggest that bradykinin stimulates PA production by a mechanism independent of the activation of protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)

Surface-modified CMOS IC electrochemical sensor array targeting single chromaffin cells for highly parallel amperometry measurements.

Science.gov (United States)

Huang, Meng; Delacruz, Joannalyn B; Ruelas, John C; Rathore, Shailendra S; Lindau, Manfred

2018-01-01

Amperometry is a powerful method to record quantal release events from chromaffin cells and is widely used to assess how specific drugs modify quantal size, kinetics of release, and early fusion pore properties. Surface-modified CMOS-based electrochemical sensor arrays allow simultaneous recordings from multiple cells. A reliable, low-cost technique is presented here for efficient targeting of single cells specifically to the electrode sites. An SU-8 microwell structure is patterned on the chip surface to provide insulation for the circuitry as well as cell trapping at the electrode sites. A shifted electrode design is also incorporated to increase the flexibility of the dimension and shape of the microwells. The sensitivity of the electrodes is validated by a dopamine injection experiment. Microwells with dimensions slightly larger than the cells to be trapped ensure excellent single-cell targeting efficiency, increasing the reliability and efficiency for on-chip single-cell amperometry measurements. The surface-modified device was validated with parallel recordings of live chromaffin cells trapped in the microwells. Rapid amperometric spikes with no diffusional broadening were observed, indicating that the trapped and recorded cells were in very close contact with the electrodes. The live cell recording confirms in a single experiment that spike parameters vary significantly from cell to cell but the large number of cells recorded simultaneously provides the statistical significance.

Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements.

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Daungruthai Jarukanont

Full Text Available Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles' arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We

Vesicle Motion during Sustained Exocytosis in Chromaffin Cells: Numerical Model Based on Amperometric Measurements.

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Jarukanont, Daungruthai; Bonifas Arredondo, Imelda; Femat, Ricardo; Garcia, Martin E

2015-01-01

Chromaffin cells release catecholamines by exocytosis, a process that includes vesicle docking, priming and fusion. Although all these steps have been intensively studied, some aspects of their mechanisms, particularly those regarding vesicle transport to the active sites situated at the membrane, are still unclear. In this work, we show that it is possible to extract information on vesicle motion in Chromaffin cells from the combination of Langevin simulations and amperometric measurements. We developed a numerical model based on Langevin simulations of vesicle motion towards the cell membrane and on the statistical analysis of vesicle arrival times. We also performed amperometric experiments in bovine-adrenal Chromaffin cells under Ba2+ stimulation to capture neurotransmitter releases during sustained exocytosis. In the sustained phase, each amperometric peak can be related to a single release from a new vesicle arriving at the active site. The amperometric signal can then be mapped into a spike-series of release events. We normalized the spike-series resulting from the current peaks using a time-rescaling transformation, thus making signals coming from different cells comparable. We discuss why the obtained spike-series may contain information about the motion of all vesicles leading to release of catecholamines. We show that the release statistics in our experiments considerably deviate from Poisson processes. Moreover, the interspike-time probability is reasonably well described by two-parameter gamma distributions. In order to interpret this result we computed the vesicles' arrival statistics from our Langevin simulations. As expected, assuming purely diffusive vesicle motion we obtain Poisson statistics. However, if we assume that all vesicles are guided toward the membrane by an attractive harmonic potential, simulations also lead to gamma distributions of the interspike-time probability, in remarkably good agreement with experiment. We also show that

The hemodynamically-regulated vascular microenvironment promotes migration of the steroidogenic tissue during its interaction with chromaffin cells in the zebrafish embryo.

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Chih-Wei Chou

Full Text Available BACKGROUND: While the endothelium-organ interaction is critical for regulating cellular behaviors during development and disease, the role of blood flow in these processes is only partially understood. The dorsal aorta performs paracrine functions for the timely migration and differentiation of the sympatho-adrenal system. However, it is unclear how the adrenal cortex and medulla achieve and maintain specific integration and whether hemodynamic forces play a role. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, the possible modulation of steroidogenic and chromaffin cell integration by blood flow was investigated in the teleostean counterpart of the adrenal gland, the interrenal gland, in the zebrafish (Danio rerio. Steroidogenic tissue migration and angiogenesis were suppressed by genetic or pharmacologic inhibition of blood flow, and enhanced by acceleration of blood flow upon norepinephrine treatment. Repressed steroidogenic tissue migration and angiogenesis due to flow deficiency were recoverable following restoration of flow. The regulation of interrenal morphogenesis by blood flow was found to be mediated through the vascular microenvironment and the Fibronectin-phosphorylated Focal Adhesion Kinase (Fn-pFak signaling. Moreover, the knockdown of krüppel-like factor 2a (klf2a or matrix metalloproteinase 2 (mmp2, two genes regulated by the hemodynamic force, phenocopied the defects in migration, angiogenesis, the vascular microenvironment, and pFak signaling of the steroidogenic tissue observed in flow-deficient embryos, indicating a direct requirement of mechanotransduction in these processes. Interestingly, epithelial-type steroidogenic cells assumed a mesenchymal-like character and downregulated β-Catenin at cell-cell junctions during interaction with chromaffin cells, which was reversed by inhibiting blood flow or Fn-pFak signaling. Blood flow obstruction also affected the migration of chromaffin cells, but not through

Mobilisation of store Ca2+ activates tyrosine hydroxylase in bovine adrenal chromaffin cells

International Nuclear Information System (INIS)

McKenzie, S.; Marley, P.D.

2001-01-01

Full text: Many receptor agonists are able to activate tyrosine hydroxylase (TOH) in bovine adrenal chromaffin cells. The majority of these are dependent on extracellular Ca 2+ for this action. Entry of extracellular Ca 2+ through voltage-operated Ca 2+ channels is very effective at activating TOH. The contribution of the intracellular Ca 2+ stores to TOH activation however is not known. Previous studies have shown that mobilisation of intracellular Ca 2+ stores is effective at increasing phosphorylation of TOH, but its effect on TOH activity has not been studied. Therefore, in the present study, the effect of mobilisation of store Ca 2+ on TOH activity was investigated using primary cultures of bovine adrenal chromaffin cells. Cells were prepared from abattoir tissue and cultured for 3-6 days. TOH activity was determined over 10 minutes, measuring the 14 CO 2 produced following the hydroxylation and rapid decarboxylation of 14 C-tyrosine offered to intact cells. Caffeine increased TOH activity in a concentration-dependent manner with a maximum response of 100% increase at 20mM. This effect was not due to osmolarity since 20mM sucrose had no effect.Nor was it due to inhibition of phosphodiesterases, since the effect of caffeine was still seen in the presence of 1mM IBMX. However,caffeine-induced TOH activation was substantially reduced in the absence of extracellular Ca 2+ . The results suggest that TOH activity can be increased by mobilising intracellular Ca 2+ stores, but that this effect involves extracellular Ca 2+ influx, possibly through store-operated channels. Copyright (2001) Australian Neuroscience Society

Protein thiophosphorylation associated with secretory inhibition in permeabilized chromaffin cells

International Nuclear Information System (INIS)

Brooks, J.C.; Brooks, M.

1985-01-01

Permeabilized cells treated with the adenosine triphosphate analog, ( 35 S)adenosine-5'-0-3(3-thiotriphosphate) ((γ- 35 S)ATP), showed thiophosphorylation of a small number of cellular proteins. A 54 kilodalton (kDa) protein was heavily thiophosphorylated in unstimulated control cells and a 43 kilodalton protein was more heavily thiophosphorylated in calcium stimulated cells. Intact cells incorporated 35 S into a series of higher molecular weight proteins. Stimulation of prelabelled, permeabilized cells resulted in a loss of 35 S from the cells over a 20 min period. Treatment of permeabilized cells with ATPγS inhibited secretion and 35 S incorporation into the cells. Pretreatment with ATPγS resulted in subsequent inhibition of both secretion and the ability of the cells to incorporate 35 S from (γ- 35 S)ATP. These results indicate that the sites normally available for phosphorylation were inactivated by thiophosphorylation and were unavailable to participate in the secretory process. The inhibition of secretion associated with thiophosphorylation of these proteins suggests that they may play a role in the control of secretion by chromaffin cells. 15 references, 1 figure, 3 tables

Adrenal Chromaffin Cells Exposed to 5-ns Pulses Require Higher Electric Fields to Porate Intracellular Membranes than the Plasma Membrane: An Experimental and Modeling Study.

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Zaklit, Josette; Craviso, Gale L; Leblanc, Normand; Yang, Lisha; Vernier, P Thomas; Chatterjee, Indira

2017-10-01

Nanosecond-duration electric pulses (NEPs) can permeabilize the endoplasmic reticulum (ER), causing release of Ca 2+ into the cytoplasm. This study used experimentation coupled with numerical modeling to understand the lack of Ca 2+ mobilization from Ca 2+ -storing organelles in catecholamine-secreting adrenal chromaffin cells exposed to 5-ns pulses. Fluorescence imaging determined a threshold electric (E) field of 8 MV/m for mobilizing intracellular Ca 2+ whereas whole-cell recordings of membrane conductance determined a threshold E-field of 3 MV/m for causing plasma membrane permeabilization. In contrast, a 2D numerical model of a chromaffin cell, which was constructed with internal structures representing a nucleus, mitochondrion, ER, and secretory granule, predicted that exposing the cell to the same 5-ns pulse electroporated the plasma and ER membranes at the same E-field amplitude, 3-4 MV/m. Agreement of the numerical simulations with the experimental results was obtained only when the ER interior conductivity was 30-fold lower than that of the cytoplasm and the ER membrane permittivity was twice that of the plasma membrane. A more realistic intracellular geometry for chromaffin cells in which structures representing multiple secretory granules and an ER showed slight differences in the thresholds necessary to porate the membranes of the secretory granules. We conclude that more sophisticated cell models together with knowledge of accurate dielectric properties are needed to understand the effects of NEPs on intracellular membranes in chromaffin cells, information that will be important for elucidating how NEPs porate organelle membranes in other cell types having a similarly complex cytoplasmic ultrastructure.

Permissive effect of dexamethasone on the increase of proenkephalin mRNA induced by depolarization of chromaffin cells

International Nuclear Information System (INIS)

Naranjo, J.R.; Mocchetti, I.; Schwartz, J.P.; Costa, E.

1986-01-01

In cultured bovine chromaffin cells, changes in the dynamic state of enkephalin stores elicited experimentally were studied by measuring cellular proenkephalin mRNA, as well as enkephalin precursors and authentic enkephalin content of cells and culture media. In parallel, tyrosine hydroxylase mRNA and catecholamine cell content were also determined. Low concentrations (0.5-100 pM) of dexamethasone increased the cell contents of proenkephalin mRNA and enkephalin-containing peptides. High concentrations of the hormone(1 μM) were required to increase the cell contents of tyrosine hydroxylase mRNA and catecholamines. Depolarization of the cells with 10 μM veratridine resulted in a depletion of enkephalin and catecholamine stores after 24 hr. The enkephalin, but not the catecholamine, content was restored by 48 hr. An increase in proenkephalin mRNA content might account for the recovery; this increase was curtailed by tetrodotoxin and enhanced by 10 pM dexamethasone. Tyrosine hydroxylase mRNA content was not significantly modified by depolarization, even in the presence of 1 μM dexamethasone. Aldosterone, progesterone, testosterone, or estradiol (1 μM) failed to change proenkephalin mRNA. Hence, dexamethasone appears to exert a specific permissive action on the stimulation of the proenkephalin gene elicited by depolarization. Though the catecholamines and enkephalins are localized in the same chromaffin granules and are coreleased by depolarization, the genes coding for the processes that are rate limiting in the production of these neuromodulators can be differentially regulated

Evidence of chromaffin oxygen sensing in neuroblastoma.

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Hedborg, F; Franklin, G; Norrman, J; Grimelius, L; Wassberg, E; Hero, B; Schilling, F; Berthold, F; Harms, D; Sandstedt, B

2001-01-01

With the aid of IGF2 and VEGF in situ hybridization; tyrosine hydroxylase, chromogranin A, and Ki67 immunohistochemistry; and TUNEL staining applied to a large series of clinical neuroblastomas and to an animal model, we show here that stroma-poor neuroblastomas show evidence of chromaffin differentiation similar to that of type 1 small intensely fluorescent (SIF) cells and that this occurs in a vascular-dependent fashion, indicating a role for local tumor hypoxia in the differentiation process.

The BAR Domain Protein PICK1 Controls Vesicle Number and Size in Adrenal Chromaffin Cells

DEFF Research Database (Denmark)

da Silva Pinheiro, Paulo César; Jansen, Anna M; de Wit, Heidi

2014-01-01

, a marker for immature granules. In chromaffin cells isolated from a PICK1 knockout (KO) mouse the amount of exocytosis was reduced, while release kinetics and Ca(2+) sensitivity were unaffected. Vesicle-fusion events had a reduced frequency and released lower amounts of transmitter per vesicle (i...... in vesicle number and size, whereas the fusion competence of generated vesicles was unaffected by the absence of PICK1. Viral rescue experiments demonstrated that long-term re-expression of PICK1 is necessary to restore normal vesicular content and secretion, while short-term overexpression is ineffective...

Nanosecond electric pulses differentially affect inward and outward currents in patch clamped adrenal chromaffin cells.

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Lisha Yang

Full Text Available This study examined the effect of 5 ns electric pulses on macroscopic ionic currents in whole-cell voltage-clamped adrenal chromaffin cells. Current-voltage (I-V relationships first established that the early peak inward current was primarily composed of a fast voltage-dependent Na+ current (INa, whereas the late outward current was composed of at least three ionic currents: a voltage-gated Ca2+ current (ICa, a Ca2+-activated K+ current (IK(Ca, and a sustained voltage-dependent delayed rectifier K+ current (IKV. A constant-voltage step protocol was next used to monitor peak inward and late outward currents before and after cell exposure to a 5 ns pulse. A single pulse applied at an electric (E-field amplitude of 5 MV/m resulted in an instantaneous decrease of ~4% in peak INa that then declined exponentially to a level that was ~85% of the initial level after 10 min. Increasing the E-field amplitude to 8 or 10 MV/m caused a twofold greater inhibitory effect on peak INa. The decrease in INa was not due to a change in either the steady-state inactivation or activation of the Na+ channel but instead was associated with a decrease in maximal Na+ conductance. Late outward current was not affected by a pulse applied at 5 MV/m. However, for a pulse applied at the higher E-field amplitudes of 8 and 10 MV/m, late outward current in some cells underwent a progressive ~22% decline over the course of the first 20 s following pulse exposure, with no further decline. The effect was most likely concentrated on ICa and IK(Ca as IKV was not affected. The results of this study indicate that in whole-cell patch clamped adrenal chromaffin cells, a 5 ns pulse differentially inhibits specific voltage-gated ionic currents in a manner that can be manipulated by tuning E-field amplitude.

Mapping organelle motion reveals a vesicular conveyor belt spatially replenishing secretory vesicles in stimulated chromaffin cells.

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Maucort, Guillaume; Kasula, Ravikiran; Papadopulos, Andreas; Nieminen, Timo A; Rubinsztein-Dunlop, Halina; Meunier, Frederic A

2014-01-01

How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this "conveyor belt" towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation.

Chromaffin paraganglioma

International Nuclear Information System (INIS)

Ocampo, Jose; Trejos, Javier; Morales, Mariano; Encalada, Jose; Canizares, Manuela

2006-01-01

We present the case of a 37 year old male patient with a left sub maxillary region tumoral mass, submitted to exploratory cervicotomy; the pathologic diagnosis, was Limpho-hemangioma and lymphatic node with reactive follicular hyperplasia. The patient had a tumoral recidive, with a big ganglial tumor that occupied the left side of triangle of the neck, anterior and posterior, with larynx and trachea displacement; after tracheostomy and surgical biopsy, the pathologic diagnosis was chromaffin paraganglioma (carotid glomus quemodectoma). Paragangliomas are uncommon tumors, these are located at abdomen, chest, head and neck; computer tomography and nuclear magnetic resonance determine their tumoral extension. Surgery and radiotherapy are specific treatment. (The author)

Augmentation of catecholamine release elicited by an Eugenia punicifolia extract in chromaffin cells

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Ricardo de Pascual

2011-10-01

Full Text Available Plant extracts of Eugenia punicifolia (Kunth DC., Myrtaceae, are used in Amazon region of Brazil to treat diarrhea and stomach disturbances, and as hypoglycemic medicine. We have recently shown that an aqueous extract of E. punicifolia augmented cholinergic neurotransmission in a rat phrenic nerve-diaphragm preparation. In this study, we investigated the effects of an E. punicifolia dichloromethane extract (EPEX in a neuronal model of cholinergic neurotransmission, the bovine adrenal chromaffin cell. EPEX augmented the release of catecholamine triggered by acetylcholine (ACh pulses but did not enhance ACh-evoked inward currents, which were inhibited by 30%. Since EPEX did not cause a blockade of acetylcholinesterase or butyrylcholinesterase, it seems that EPEX is not directly activating the cholinergic system. EPEX also augmented K+-elicited secretion without enhancing the whole-cell inward calcium current. This novel and potent effect of EPEX in enhancing exocytosis might help to identify the active component responsible for augmenting exocytosis. When elucidated, the molecular structure of this active principle could serve as a template to synthesise novel compounds to regulate the exocytotic release of neurotransmitters.

Direct visualization of secretion from single bovine adrenal chromaffin cells by laser-induced native fluorescence imaging microscopy

Energy Technology Data Exchange (ETDEWEB)

Tong, W.; Yeung, E.S. [Ames Laboratory---USDOE and Department of Chemistry, Iowa State University, Ames, Iowa 50011 (United States)

1998-03-01

Direct visualization of the secretion process of individual bovine adrenal chromaffin cells was achieved with laser-induced native fluorescence imaging microscopy. By monitoring the native fluorescence of catecholamines excited by the 275 nm laser line with an intensified charge-coupled-device (CCD) camera, we obtained good temporal and spatial resolution simultaneously without using additional fluorescent probes. Large variations were found among individual cells in terms of the amounts of catecholamines secreted and the rates of secretion. Different regions of a cell also behave differently during the secretion process. However, the degree of this local heterogeneity is smaller than in neurons and neuralgia. The influence of deep-ultraviolet (UV) laser excitation on cells is also discussed. This quantitative imaging technique provides a useful noninvasive approach for the study of dynamic cellular changes and the understanding of the molecular mechanisms of secretory processes. {copyright} {ital 1998} {ital Society for Applied Spectroscopy}

Ciguatoxin-induced catecholamine secretion in bovine chromaffin cells: mechanism of action and reversible inhibition by brevenal.

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Nguyen-Huu, Truong D; Mattei, César; Wen, Peter J; Bourdelais, Andrea J; Lewis, Richard J; Benoit, Evelyne; Baden, Daniel G; Molgó, Jordi; Meunier, Frédéric A

2010-10-01

Ciguatoxin (P-CTX-1B) from the dinoflagellate Gambierdiscus toxicus, belongs to the family of polyether neurotoxins responsible for the neurological poisoning disorder ciguatera. Although it is the most widespread marine-borne disease affecting humans, there is no current FDA-approved treatment available except for symptomatic therapies. In this paper, we report that P-CTX-1B promotes catecholamine secretion from bovine chromaffin cells, an effect that is insensitive to concomitant activation of capacitative Ca(2+) entry. Moreover, we confirm that brevenal, a polyether from the dinoflagellate Karenia brevis, blocks P-CTX-1B-induced catecholamine secretion. This effect is partially reversible. Our results therefore raise the prospect of finding functional antagonists for P-CTX-1B that could be useful for the treatment of ciguatera. Copyright 2009 Elsevier Ltd. All rights reserved.

The affection of APA microcapsulation on catecholamine and leucine-enkephalin secretion from the bovine adrenal medullary chromaffin cells

International Nuclear Information System (INIS)

Guo Shuilong; Cui Xin; Luo Yun; Xue Yilong

2002-01-01

The affection of alginate-polylysine-alginate (APA) microcapsulation on catecholamine (CA) and leucine-enkephalin (L-EK) secretion from bovine adrenal medullary chromaffin cells was analysed. Encapsulating BCCs with the APA microcapsulation, the secretion of CA and L-EK in encapsulated BCCs was detected by high-performance liquid chromatography-electrochemical assay and radioimmunoassay, respectively. There is little difference between the encapsulated BCCs and the non-encapsulated BCCs in the secretion of epinephrine (E) and noradrenaline (NE) but the secretion of dopamine (DA) and L-EK in several points decline. The studies indicated that APA microcapsulation of BCCs didn't affect the secretion of E and NE, but did the secretion DA and L-EK in forepart

Regulation of Ca2+ influx by a protein kinase C activator in chromaffin cells: differential role of P/Q- and L-type Ca2+ channels.

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Sena, C M; Santos, R M; Boarder, M R; Rosário, L M

1999-02-05

Phorbol esters reduce depolarization-evoked Ca2+ influx in adrenal chromaffin cells, suggesting that voltage-sensitive Ca2+ channels (VSCCs) are inhibited by protein kinase C-mediated phosphorylation. We now address the possibility that L- and P/Q-type Ca2+ channel subtypes might be differentially involved in phorbol ester action. In bovine chromaffin cells, short-term (10 min) incubations with phorbol 12-myristate 13-acetate (PMA) inhibited early high K+-evoked rises in cytosolic free Ca2+ concentration ([Ca2+]i) and the early component of the depolarization-evoked Mn2+ quenching of fura-2 fluorescence in a dose-dependent manner (IC50: 18 and 7 nM; maximal inhibitions: 45 and 48%, respectively). The protein kinase C inhibitor staurosporine (100 nM) reverted the inhibitory action of PMA. PMA (0.1-1 microM) inhibited the early and late phases of the ionomycin (2 microM)-evoked [Ca2+]i transients by 14-23%. Omega-agatoxin IVA, a blocker of P/Q-type Ca2+ channels, inhibited high K+-evoked [Ca2+]i rises in a dose-dependent fashion (IC50 = 50 nM). In contrast, 0.1 microM omega-conotoxin GVIA, a blocker of N-type channels, was without effect. A sizeable (< 45%) component of early Ca2+ influx persisted in the combined presence of omega-agatoxin IVA (100 nM) and nitrendipine (1 microM). Simultaneous exposure to omega-agatoxin IVA and PMA inhibited both the early [Ca2+]i transients and Mn2+ quenching to a much greater extent than each drug separately. Inhibition of the [Ca2+]i transients by nitrendipine and PMA did not significantly exceed that produced by PMA alone. It is concluded that phorbol ester-mediated activation of protein kinase C inhibits preferentially L-type VSCCs over P/Q type channels in adrenal chromaffin cells. However, the possibility cannot be ruled out that dihydropyridine-resistant, non-P/Q type channels might also be negatively regulated by protein kinase C. This may represent an important pathway for the specific control of VSCCs by protein kinase C

Biological amine transport in chromaffin ghosts. Coupling to the transmembrane proton and potential gradients.

Science.gov (United States)

Johnson, R G; Pfister, D; Carty, S E; Scarpa, A

1979-11-10

The effect of the transmembrane proton gradient (delta pH) and potential gradient (delta psi) upon the rate and extent of amine accumulation was investigated in chromaffin ghosts. The chromaffin ghosts were formed by hypo-osmotic lysis of isolated bovine chromaffin granules and extensive dialysis in order to remove intragranular binding components and dissipate the endogenous electrochemical gradients. Upon ATP addition to suspensions of chromaffin ghosts, a transmembrane proton gradient alone, a transmembrane gradient alone, or both, could be established, depending upon the compositions of the media in which the ghosts were formed and resuspended. When chloride was present in the medium, addition of ATP resulted in the generation of a transmembrane proton gradient, acidic inside of 1 pH unit (measured by [14C]methylamine distribution), and no transmembrane potential (measured by [14C]-thiocyanate distribution). When ATP was added to chromaffin ghosts suspended in a medium in which chloride was substituted by isethionate, a transmembrane potential, inside positive, of 45 mV and no transmembrane proton gradient, was measured. In each medium, the addition of agents known to affect proton or potential gradients, respectively, exerted a predictable mechanism of action. Accumulation of [14C]epinephrine or [14C]5-hydroxytryptamine was over 1 order of magnitude greater in the presence of the transmembrane proton gradient or the transmembrane potential than in the absence of any gradient and, moreover, was related to the magnitude of the proton or potential gradient in a dose-dependent manner. When ghosts were added to a medium containing chloride and isethionate, both a delta pH and delta psi could be generated upon addition of ATP. In this preparation, the maximal rate of amine accumulation was observed. The results indicate that amine accumulation into chromaffin ghosts can occur in the presence of either a transmembrane proton gradient, or a transmembrane potential

Brevenal inhibits pacific ciguatoxin-1B-induced neurosecretion from bovine chromaffin cells.

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César Mattei

Full Text Available Ciguatoxins and brevetoxins are neurotoxic cyclic polyether compounds produced by dinoflagellates, which are responsible for ciguatera and neurotoxic shellfish poisoning (NSP respectively. Recently, brevenal, a natural compound was found to specifically inhibit brevetoxin action and to have a beneficial effect in NSP. Considering that brevetoxin and ciguatoxin specifically activate voltage-sensitive Na+ channels through the same binding site, brevenal has therefore a good potential for the treatment of ciguatera. Pacific ciguatoxin-1B (P-CTX-1B activates voltage-sensitive Na+ channels and promotes an increase in neurotransmitter release believed to underpin the symptoms associated with ciguatera. However, the mechanism through which slow Na+ influx promotes neurosecretion is not fully understood. In the present study, we used chromaffin cells as a model to reconstitute the sequence of events culminating in ciguatoxin-evoked neurosecretion. We show that P-CTX-1B induces a tetrodotoxin-sensitive rise in intracellular Na+, closely followed by an increase in cytosolic Ca2+ responsible for promoting SNARE-dependent catecholamine secretion. Our results reveal that brevenal and beta-naphtoyl-brevetoxin prevent P-CTX-1B secretagogue activity without affecting nicotine or barium-induced catecholamine secretion. Brevenal is therefore a potent inhibitor of ciguatoxin-induced neurotoxic effect and a potential treatment for ciguatera.

Brevenal inhibits pacific ciguatoxin-1B-induced neurosecretion from bovine chromaffin cells.

Science.gov (United States)

Mattei, César; Wen, Peter J; Nguyen-Huu, Truong D; Alvarez, Martha; Benoit, Evelyne; Bourdelais, Andrea J; Lewis, Richard J; Baden, Daniel G; Molgó, Jordi; Meunier, Frédéric A

2008-01-01

Ciguatoxins and brevetoxins are neurotoxic cyclic polyether compounds produced by dinoflagellates, which are responsible for ciguatera and neurotoxic shellfish poisoning (NSP) respectively. Recently, brevenal, a natural compound was found to specifically inhibit brevetoxin action and to have a beneficial effect in NSP. Considering that brevetoxin and ciguatoxin specifically activate voltage-sensitive Na+ channels through the same binding site, brevenal has therefore a good potential for the treatment of ciguatera. Pacific ciguatoxin-1B (P-CTX-1B) activates voltage-sensitive Na+ channels and promotes an increase in neurotransmitter release believed to underpin the symptoms associated with ciguatera. However, the mechanism through which slow Na+ influx promotes neurosecretion is not fully understood. In the present study, we used chromaffin cells as a model to reconstitute the sequence of events culminating in ciguatoxin-evoked neurosecretion. We show that P-CTX-1B induces a tetrodotoxin-sensitive rise in intracellular Na+, closely followed by an increase in cytosolic Ca2+ responsible for promoting SNARE-dependent catecholamine secretion. Our results reveal that brevenal and beta-naphtoyl-brevetoxin prevent P-CTX-1B secretagogue activity without affecting nicotine or barium-induced catecholamine secretion. Brevenal is therefore a potent inhibitor of ciguatoxin-induced neurotoxic effect and a potential treatment for ciguatera.

Phosphatidic acid accumulation and catecholamine release in adrenal chromaffin cells: stimulation by high potassium and by nicotine, and effect of a diacylglycerol kinase inhibitor R 59 022.

Science.gov (United States)

Owen, P J; Jones, J A; Boarder, M R

1991-09-01

Using primary cultures of bovine adrenal chromaffin cells labelled with 32Pi, we show that stimulation with bradykinin, nicotine, or a depolarising concentration of potassium stimulates the accumulation of [32P]phosphatidic acid. The effects of nicotine and potassium are smaller than the effect of bradykinin, and are dependent entirely on extracellular calcium. The diacylglycerol kinase inhibitor R 59 022 attenuates the formation of phosphatidic acid by nicotine and depolarising concentrations of potassium. This inhibitor also blocks the nicotine and potassium stimulation of noradrenaline release from chromaffin cells. Using 45Ca2+ influx studies, we show that the nicotine-evoked calcium influx is also attenuated by R 59 022. These observations contrast with those in another report in which we showed that bradykinin stimulation of either [32P]phosphatidic acid accumulation or noradrenaline release is not affected by R 59 022. It is likely that the calcium influx produced by nicotine and depolarising potassium is blocked by R 59 022 by a mechanism that is independent of its ability to block diacylglycerol kinase. The nicotine- and potassium-stimulated [32P]phosphatidic acid accumulation is a consequence of this calcium influx and presumably reflects calcium activation of either phospholipase C or phospholipase D.

Differential regulation of histamine- and bradykinin-stimulated phospholipase C in adrenal chromaffin cells: evidence for involvement of different protein kinase C isoforms.

Science.gov (United States)

Sena, C M; Rosário, L M; Parker, P J; Patel, V; Boarder, M R

1996-03-01

In this report we investigate the isoforms of protein kinase C (PKC) present in cultured adrenal chromaffin cells with respect to their modulation by treatment with phorbol ester and their possible differential involvement in the regulation of responses to histamine and bradykinin. The presence of individual isoforms of PKC was investigated by using eight isoform specific antisera, as a result of which PKC-alpha, epsilon, and zeta were identified. To characterize down-regulation of these enzymes, cells were incubated for 6-48 h with 1 microM phorbol myristate acetate (PMA). PKC-epsilon down-regulated more rapidly than PKC-alpha. At 12 h, PMA pretreatment, for example, PKC-epsilon was maximally down-regulated (23 +/- 4% of controls), whereas PKC-alpha was unchanged. PKC-alpha showed partial down-regulation by 24 h of PMA pretreatment. PKC-zeta did not down-regulate at any of the times tested. Translocation from cytosol to membrane in response to PMA was also more rapid for PKC-epsilon than for PKC-alpha. The accumulation of total 3H-inositol (poly) phosphates in response to bradykinin or histamine was essentially abolished by prior treatment with 10-min PMA treatment (1 microM). However, with 12-h exposure to PMA, the bradykinin response was restored to the level seen with no prior PMA exposure. The histamine response showed no recovery by 12 h of PMA, but showed partial recovery by 24 h of PMA pretreatment. These observations showed that the restoration of the response to bradykinin corresponds to the loss of PKC-epsilon, whereas the restoration of the histamine response corresponds to the loss of PKC-alpha. This picture was confirmed with further studies on cytosolic Ca2+. The results show that chromaffin cells exhibit an unusual pattern of down-regulation of PKC isoforms on prolonged exposure to PMA, and that there is a differential effect of exposure to PMA on the histamine and bradykinin responses, suggesting that different PLC-linked receptors in chromafin

Asthma pregnancy alters postnatal development of chromaffin cells in the rat adrenal medulla.

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Xiu-Ming Wu

Full Text Available Adrenal neuroendocrine plays an important role in asthma. The activity of the sympathoadrenal system could be altered by early life events. The effects of maternal asthma during pregnancy on the adrenal medulla of offspring remain unknown.This study aims to explore the influence of maternal asthma during pregnancy on the development and function of adrenal medulla in offspring from postnatal day 3 (P3 to postnatal day 60 (P60. Asthmatic pregnant rats (AP, nerve growth factor (NGF-treated pregnant rats (NP and NGF antibody-treated pregnant rats (ANP were sensitized and challenged with ovalbumin (OVA; NP and ANP were treated with NGF and NGF antibody respectively. Offspring rats from the maternal group were divided into four groups: offspring from control pregnant rats (OCP, offspring from AP (OAP, offspring from NP (ONP, and offspring from ANP (OANP. The expressions of phenylethanolamine N-methyltransferase (PNMT protein in adrenal medulla were analyzed. The concentrations of epinephrine (EPI, corticosterone and NGF in serum were measured. Adrenal medulla chromaffin cells (AMCC were prone to differentiate into sympathetic nerve cells in OAP and ONP. Both EPI and PNMT were decreased in OAP from P3 to P14, and then reached normal level gradually from P30 to P60, which were lower from birth to adulthood in ONP. Corticosterone concentration increased significantly in OAP and ONP.Asthma pregnancy may promote AMCC to differentiate into sympathetic neurons in offspring rats and inhibit the synthesis of EPI, resulting in dysfunction of bronchial relaxation.

Src kinases regulate de novo actin polymerization during exocytosis in neuroendocrine chromaffin cells.

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María José Olivares

Full Text Available The cortical actin network is dynamically rearranged during secretory processes. Nevertheless, it is unclear how de novo actin polymerization and the disruption of the preexisting actin network control transmitter release. Here we show that in bovine adrenal chromaffin cells, both formation of new actin filaments and disruption of the preexisting cortical actin network are induced by Ca2+ concentrations that trigger exocytosis. These two processes appear to regulate different stages of exocytosis; whereas the inhibition of actin polymerization with the N-WASP inhibitor wiskostatin restricts fusion pore expansion, thus limiting the release of transmitters, the disruption of the cortical actin network with cytochalasin D increases the amount of transmitter released per event. Further, the Src kinase inhibitor PP2, and cSrc SH2 and SH3 domains also suppress Ca2+-dependent actin polymerization, and slow down fusion pore expansion without disturbing the cortical F-actin organization. Finally, the isolated SH3 domain of c-Src prevents both the disruption of the actin network and the increase in the quantal release induced by cytochalasin D. These findings support a model where a rise in the cytosolic Ca2+ triggers actin polymerization through a mechanism that involves Src kinases. The newly formed actin filaments would speed up the expansion of the initial fusion pore, whereas the preexisting actin network might control a different step of the exocytosis process.

Catecholamine secretion by chemical hypoxia in guinea-pig, but not rat, adrenal medullary cells: differences in mitochondria.

Science.gov (United States)

Harada, K; Endo, Y; Warashina, A; Inoue, M

2015-08-20

The effects of mitochondrial inhibitors (CN(-), a complex IV inhibitor and CCCP, protonophore) on catecholamine (CA) secretion and mitochondrial function were explored functionally and biochemically in rat and guinea-pig adrenal chromaffin cells. Guinea-pig chromaffin cells conspicuously secreted CA in response to CN(-) or CCCP, but rat cells showed a little, if any, secretory response to either of them. The resting metabolic rates in rat adrenal medullae did not differ from those in guinea-pig adrenal medullae. On the other hand, the time course of depolarization of the mitochondrial membrane potential (ΔΨm) in guinea-pig chromaffin cells in response to CN(-) was slower than that in rat chromaffin cells, and this difference was abolished by oligomycin, an F1F0-ATPase inhibitor. The extent of CCCP-induced decrease in cellular ATP in guinea-pig chromaffin cells, which was indirectly measured using a Mg(2+) indicator, was smaller than that in rat chromaffin cells. Relative expression levels of F1F0-ATPase inhibitor factor in guinea-pig adrenal medullae were smaller than in rat adrenal medullae, and the opposite was true for F1F0-ATPase α subunit. The present results indicate that guinea-pig chromaffin cells secrete more CA in response to a mitochondrial inhibitor than rat chromaffin cells and this higher susceptibility in the former is accounted for by a larger extent of reversed operation of F1F0-ATPase with the consequent decrease in ATP under conditions where ΔΨm is depolarized. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Evidence for a dihydropyridine-sensitive and conotoxin-insensitive release of noradrenaline and uptake of calcium in adrenal chromaffin cells.

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Owen, P. J.; Marriott, D. B.; Boarder, M. R.

1989-01-01

1. It has been suggested that neuronal voltage-sensitive calcium channels (VSCC) may be divided into dihydropyridine (DHP)-sensitive (L) and DHP-insensitive (N and T), and that both the L and the N type channels are attenuated by the peptide blocker omega-conotoxin. Here the effects of omega-conotoxin on release of noradrenaline and uptake of calcium in bovine adrenal chromaffin cells were investigated. 2. Release of noradrenaline in response to 25 mM K+, 65 mM K+, 10 nM bradykinin or 10 microM prostaglandin E1 was not affected by omega-conotoxin in the range 10 nM-1 microM. 3. 45Ca2+ uptake stimulated by high K+ and prostaglandin was attenuated by 1 microM nitrendipine and enhanced by 1 microM Bay K 8644; these calcium fluxes were not modified by 20 nM omega-conotoxin. 4. With superfused rat brain striatal slices in the same medium as the above cell studies, release of dopamine in response to 25 mM K+ was attenuated by 20 nM omega-conotoxin. 5. These results show that in these neurone-like cells, release may be effected by calcium influx through DHP-sensitive but omega-conotoxin-insensitive VSCC, a result inconsistent with the suggestion that omega-conotoxin blocks both L-type and N-type neuronal calcium channels. PMID:2470457

Transforming growth factor-beta, but not ciliary neurotrophic factor, inhibits DNA synthesis of adrenal medullary cells in vitro

DEFF Research Database (Denmark)

Wolf, N; Krohn, K; Bieger, S

1999-01-01

by the neuroendocrine chromaffin cells, which also express the transforming growth factor-beta receptor type II. In contrast to the developmentally related sympathetic neurons, chromaffin cells continue to proliferate throughout postnatal life. Using 5-bromo-2'-deoxyuridine pulse labeling and tyrosine hydroxylase...... immunocytochemistry as a marker for young postnatal rat chromaffin cells, we show that treatment with fibroblast growth factor-2 (1 nM) and insulin-like growth factor-II (10 nM) increased the fraction of 5-bromo-2'-deoxyuridine-labeled nuclei from 1% to about 40% of the cells in the absence of serum. In the presence...... of fibroblast growth factor-2 and insulin-like growth factor-II, transforming growth factor-beta1 (0.08 nM) reduced 5-bromo-2'-deoxyuridine labeling by about 50%, without interfering with chromaffin cell survival or death. Doses lower and higher than 0.08 nM were less effective. Similar effects were seen...

Transbilayer transport of a propyltrimethylammonium derivative of diphenylhexatriene (TMAP-DPH) in bovine blood platelets and adrenal chromaffin cells.

Science.gov (United States)

Kitagawa, Shuji; Tachikawa, Eiichi; Kashimoto, Takashi

2002-12-01

The membrane fluorescent probe N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl)trimethylammonium (TMAP-DPH) has an additional three-carbon spacer between the fluorophore and the trimethylammonium substituent of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). As a basic study to clarify the transport mechanism of amphiphilic quaternary ammoniums, we observed the characteristics of the transbilayer transport of TMAP-DPH in bovine blood platelets and bovine adrenal chromaffin cells using the albumin extraction method. We compared these inward transport rates with those of TMA-DPH. TMAP-DPH crossed into the cytoplasmic layers of the membranes more slowly than TMA-DPH after rapid binding to the outer halves of the plasma membranes. The transport rate markedly depended on temperature. Time to reach the half-maximal incorporated amount of TMAP-DPH increased threefold accompanied by an increase in the concentration from 0.2 to 1.5 microM. The transport was stimulated significantly by various types of membrane perturbations such as modification of sulfhydryl-groups by N-ethylmaleimide and benzyl alcohol-induced increase in the fluidity of the lipid bilayer. The saturation phenomenon suggested the presence of the regulatory process in the transbilayer transport of TMAP-DPH.

Effect of forskolin and prostaglandin E1 on stimulus secretion coupling in cultured bovine adrenal chromaffin cells.

Science.gov (United States)

Marriott, D; Adams, M; Boarder, M R

1988-02-01

Treatment of adrenal chromaffin cells with forskolin (0.1-10 microM) stimulated cyclic AMP levels, reduced the maximal stimulation of release of noradrenaline by nicotine, and increased release in response to elevated external potassium and the calcium ionophore A23187. The presence of the phosphodiesterase inhibitor Ro 20-17-24 with forskolin potentiated both the stimulation of cyclic AMP and the inhibition of nicotine-induced noradrenaline release. Dibutyryl cyclic AMP, and the elevation of cyclic AMP with prostaglandin E1, also attenuated nicotine-stimulated release. However, when the stimulation of intracellular cyclic AMP production by prostaglandin E1 was potentiated by low levels of forskolin, there was not a concomitant potentiation of effect on noradrenaline release. Dideoxyforskolin, an analogue of forskolin which does not stimulate adenylate cyclase, inhibited both potassium- and nicotine-stimulated release, probably by a mechanism unrelated to the action of forskolin in these experiments. Using Fura-2 to estimate free intracellular calcium levels, both forskolin and dideoxyforskolin (at 10 microM) reduced the calcium transient in response to nicotine. These results support a model in which elevation of cyclic AMP inhibits the activation of nicotinic receptors, but augments stimulus secretion coupling downstream of calcium entry. The data, however, do not indicate a simple relationship between total intracellular cyclic AMP levels and the attenuation of nicotinic stimulation of release.

Early fetal acquisition of the chromaffin and neuronal immunophenotype by human adrenal medullary cells. An immunohistological study using monoclonal antibodies to chromogranin A, synaptophysin, tyrosine hydroxylase, and neuronal cytoskeletal proteins.

NARCIS (Netherlands)

Molenaar, W M; Lee, V M; Trojanowski, J Q

1990-01-01

The development of chromaffin and neuronal features in the adrenal medulla was studied in normal human fetuses with gestational ages (GAs) of 6-34 weeks. Monoclonal antibodies specific for chromogranin A, synaptophysin, and tyrosine hydroxylase; for different subunits and phosphoisoforms of

Imidacloprid, a neonicotinoid insecticide, facilitates tyrosine hydroxylase transcription and phenylethanolamine N-methyltransferase mRNA expression to enhance catecholamine synthesis and its nicotine-evoked elevation in PC12D cells.

Science.gov (United States)

Kawahata, Ichiro; Yamakuni, Tohru

2018-02-01

Imidacloprid is a neonicotinoid insecticide acting as an agonist of nicotinic acetylcholine receptors (nAChRs) in the target insects. However, questions about the safety to mammals, including human have emerged. Overactivation of mammalian peripheral catecholaminergic systems leads to onset of tachycardia, hypertension, vomiting, etc., which have been observed in acutely imidacloprid-poisoned patients as well. Physiological activation of the nAChRs is known to drive catecholamine biosynthesis and secretion in mammalian adrenal chromaffin cells. Yet, the impacts of imidacloprid on the catecholaminergic function of the chromaffin cells remain to be evaluated. In this study using PC12D cells, a catecholaminergic cell line derived from the medulla chromaffin-cell tumors of rat adrenal gland, we examined whether imidacloprid itself could impact the catecholamine-synthesizing ability. Imidacloprid alone did facilitate tyrosine hydroxylase (TH) transcription via activation of α3β4 nAChR and the α7 subunit-comprising receptor. The insecticide showed the TH transcription-facilitating ability at the concentrations of 3 and 30 μM, at which acetylcholine is known to produce physiological responses, including catecholamine secretion through the nAChRs in adrenal chromaffin cells. The insecticide-facilitated TH transcription was also dependent on PKA- and RhoA-mediated signaling pathways. The insecticide coincidentally raised levels of TH and phenylethanolamine N-methyltransferase (PNMT) mRNA, and as a consequence, increased catecholamine production, although the efficacy of the neonicotinoid was lesser than that of nicotine, indicating its partial agonist-like action. Intriguingly, in cultured rat adrenal chromaffin cells, imidacloprid did increase levels of TH and PNMT protein. When the chromaffin cells were treated with nicotine in the presence of the insecticide, nicotine-elevated adrenaline production was enhanced due to facilitation of nicotine-increased TH and PNMT

Rapid relief of block by mecamylamine of neuronal nicotinic acetylcholine receptors of rat chromaffin cells in vitro: an electrophysiological and modeling study.

Science.gov (United States)

Giniatullin, R A; Sokolova, E M; Di Angelantonio, S; Skorinkin, A; Talantova, M V; Nistri, A

2000-10-01

The mechanism responsible for the blocking action of mecamylamine on neuronal nicotinic acetylcholine receptors (nAChRs) was studied on rat isolated chromaffin cells recorded under whole-cell patch clamp. Mecamylamine strongly depressed (IC(50) = 0.34 microM) inward currents elicited by short pulses of nicotine, an effect slowly reversible on wash. The mecamylamine block was voltage-dependent and promptly relieved by a protocol combining membrane depolarization with a nicotine pulse. Either depolarization or nicotine pulses were insufficient per se to elicit block relief. Block relief was transient; response depression returned in a use-dependent manner. Exposure to mecamylamine failed to block nAChRs if they were not activated by nicotine or if they were activated at positive membrane potentials. These data suggest that mecamylamine could not interact with receptors either at rest or at depolarized level. Other nicotinic antagonists like dihydro-beta-erythroidine or tubocurarine did not share this action of mecamylamine although proadifen partly mimicked it. Mecamylamine is suggested to penetrate and block open nAChRs that would subsequently close and trap this antagonist. Computer modeling indicated that the mechanism of mecamylamine blocking action could be described by assuming that 1) mecamylamine-blocked receptors possessed a much slower, voltage-dependent isomerization rate, 2) the rate constant for mecamylamine unbinding was large and poorly voltage dependent. Hence, channel reopening plus depolarization allowed mecamylamine escape and block relief. In the presence of mecamylamine, therefore, nAChRs acquire the new property of operating as coincidence detectors for concomitant changes in membrane potential and receptor occupancy.

Secretion of [Met]enkephalyl-Arg6-Phe7-related peptides and catecholamines from bovine adrenal chromaffin cells: modification by changes in cyclic AMP and by treatment with reserpine.

Science.gov (United States)

Adams, M; Boarder, M R

1987-07-01

Investigations into the effects of culturing bovine adrenal chromaffin cells in the presence (72 h) of dibutyryl cyclic AMP, forskolin, and reserpine on the level and release of [Met]enkephalyl-Arg6-Phe7 immunoreactivity, noradrenaline, and adrenaline are reported. The assay for [Met]enkephalyl-Arg6-Phe7 immunoreactivity recognises both peptide B, the 31-amino acid carboxy-terminal segment of proenkephalin, and its heptapeptide fragment, [Met]enkephalyl-Arg6-Phe7. Treatments that elevate cyclic AMP increase the amount of peptide immunoreactivity in these cells; this is predominantly peptide B-like immunoreactivity in both control cells and cyclic AMP-elevated cells. Treatment with reserpine gives no change in total immunoreactivity levels, but does not result in increased accumulation of the heptapeptide [Met]enkephalyl-Arg6-Phe7 at the expense of immunoreactivity that elutes with its immediate precursor, peptide B. Cyclic AMP treatment causes either no change or a decrease in levels of accumulated noradrenaline and adrenaline. However, the release of [Met]enkephalin-Arg6-Phe7 immunoreactivity, noradrenaline, and adrenaline is increased by 72-h pretreatment with forskolin or dibutyryl cyclic AMP, whether release is stimulated by nicotine or elevated potassium. In each case the molecular form of [Met]enkephalyl-Arg6-Phe7 immunoreactivity that is released approximately reflects the cell content. Pretreatment with reserpine has no effect on the total [Met]enkephalyl-Arg6-Phe7 immunoreactivity released, but does result in an increased release of the heptapeptide and a decrease in release of peptide B-like immunoreactivity. The studies suggest that the levels of [Met]enkephalyl-Arg6-Phe7 and peptide B available for release are controlled both at the level of proenkephalin synthesis and at the level of double-basic residue proteolysis.

Incorporation of radioactive sulfate (Na235SO4) by mouse adrenal medullary cells as shown by radioautography

International Nuclear Information System (INIS)

Munhoz, C.O.G.; Merzel, J.

1977-01-01

Preliminary radioautographic results, observed in the adreno-medullary cells of mice injected with radiosulfate, suggested that the cells might synthetize sulfur-containing compounds. Only further studies could make-clear if sulfate groups are linked to carbohydrate molecules and/or chromaffin granules [pt

Transplantation of Reprogrammed Autologous Stem Cells for Chronic Pain and Drug Abuse

Science.gov (United States)

2016-07-01

were removed for more detailed examination. No enlarged lymph node was detected. Subsequent immunohistochemical examination was conducted for...100 UI/ml peni - cillin, 100 mg/ml streptomycin, and 0.125 ml/ml fungi- zone (PSF; Sigma-Aldrich) for transport. Preparation of chromaffin cells was

Identifying Genes Involved in Paraganglioma Genesis

NARCIS (Netherlands)

J. Gaal (José)

2010-01-01

textabstractThe paraganglion system is composed of a collection of chromaffin cells that is distributed throughout the body. Embryonically, chromaffin cells arise from the neuroectodermal tissue of the neural crest and are thought to migrate along the innervating nerves or vasculature towards

Numerical study of induced current perturbations in the vicinity of excitable cells exposed to extremely low frequency magnetic fields

International Nuclear Information System (INIS)

Hassan, Noha; Chatterjee, Indira; Publicover, Nelson G; Craviso, Gale L

2003-01-01

Realistic three-dimensional cell morphologies were modelled to determine the current density induced in excitable cell culture preparations exposed to 60 Hz magnetic fields and to identify important factors that can influence the responses of cells to these fields. Cell morphologies representing single spherical adrenal chromaffin cells, single elongated smooth muscle cells and chromaffin cell aggregates in a Petri dish containing culture medium were modelled using the finite element method. The computations for a spherical cell revealed alterations in the magnitude and spatial distribution of the induced current density in the immediate vicinity of the cell. Maxima occurred at the equatorial sides and minima at the poles. Proximity of cells to each other as well as cell aggregate shape, size and orientation with respect to the induced current influenced the magnitude and spatial distribution of the induced current density. For an elongated cell, effects on the induced current density were highly dependent on cell orientation with respect to the direction of the induced current. These results provide novel insights into the perturbations in induced current that occur in excitable cell culture preparations and lay a foundation for understanding the mechanisms of interaction with extremely low frequency magnetic fields at the tissue level

Adrenocortical and adrenomedullary homologs in eight species of adult and developing teleosts: morphology, histology, and immunohistochemistry.

Science.gov (United States)

Grassi Milano, E; Basari, F; Chimenti, C

1997-12-01

Morphology, histology, and immunohistochemistry of the adrenocortical and adrenomedullary homologs (adrenal glands) of the following developing and adult teleosts were examined: Salmoniformes-Oncorhynchus mykiss (rainbow trout), Salmo trutta fario (brown trout), Coregonus lavaretus (white fish); Cyprinodontiformes-Gambusia affinis (mosquito fish). Perciformes-Dicentrarchus labrax (sea bass), Sparus aurata (sea bream), Diplodus sargus (white bream), Oblada melanura (saddled bream). The anatomical relationships of the gland with the renal system and venous vessels were also noted. In adults of all species steroidogenic and catecholaminergic chromaffin cells were found in the head kidney, which is pronephric in origin and subsequently transformed into a hematopoietic lymphatic organ. In Perciformes, chromaffin cells are distributed around the anterior and posterior cardinal veins and ducts of Cuvier; in Salmoniformes, around the posterior cardinal veins and in the hematopoietic tissue; and in G. affinis, around the ducts of Cuvier and posterior cardinal veins, while a few are visible also around the sinus venosus. In Perciformes and Salmoniformes, numerous chromaffin cells are also present in the posterior kidney, derived from the opisthonephros, in contact with the caudal vein. Steroidogenic cells are always confined to the head kidney. During development chromaffin and steroidogenic cells appear early after hatching in the pronephric kidney, at the level of the ducts of Cuvier and of the cephalic part of the posterior cardinal veins. Later, chromaffin cells in Perciformes reach the anterior cardinal veins, and subsequently, in both Perciformes and Salmoniformes, they reach the developing posterior kidney. Their localization along the posterior kidney is still in progress about 4 months after hatching and is completed about a year after hatching. These findings support the concept that the structure of the adrenal gland in teleosts is intermediate between that of the

The purified ATPase from chromaffin granule membranes is an anion-dependent proton pump.

Science.gov (United States)

Moriyama, Y; Nelson, N

1987-07-05

The proton-ATPase of chromaffin granules was purified so as to maintain its proton-pumping activity when reconstituted into phospholipid vesicles. The purification procedure involved solubilization with polyoxyethylene 9 lauryl ether, hydroxylapatite column, precipitation by ammonium sulfate, and glycerol gradient centrifugation. The protease inhibitor mixture used in previous studies inhibited the proton-pumping activity of the enzyme; therefore, the protein was stabilized by pepstatin A and leupeptin. The enzyme was purified at least 50-fold with respect to both ATPase and proton-pumping activity. The ATP-dependent proton uptake activity of the reconstituted enzyme was absolutely dependent on the presence of Cl- or Br- outside the vesicles, whereas sulfate, acetate, formate, nitrate, and thiocyanate were inhibitory. Sulfate inhibition seems to be due to competition with Cl- on the anion-binding site outside the vesicles, whereas nitrate and thiocyanate inhibited only from the internal side. As with the inhibition by N-ethylmaleimide, the proton-pumping activity was much more sensitive to nitrate than the ATPase activity. About 20 mM nitrate were sufficient for 90% inhibition of the proton-pumping activity while 100 mM inhibited only 50% of the ATPase activity both in situ and in the reconstituted enzyme. The possible regulatory effect of anions on the ATP-dependent proton uptake in secretory granules is discussed.

Cell-to-cell communication in bilateral macronodular adrenal hyperplasia causing hypercortisolism

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Herve eLefebvre

2015-04-01

Full Text Available It has been well established that, in the human adrenal gland, cortisol secretion is not only controlled by circulating corticotropin but is also influenced by a wide variety of bioactive signals, including conventional neurotransmitters and neuropeptides, released within the cortex by various cell types such as chromaffin cells, neurons, cells of the immune system, adipocytes and endothelial cells. These different types of cells are present in bilateral macronodular adrenal hyperplasia, a rare etiology of primary adrenal Cushing’s syndrome, where they appear intermingled with adrenocortical cells in the hyperplastic cortex. In addition, the genetic events which cause the disease favor abnormal adrenal differenciation that results in illicit expression of paracrine regulatory factors and their receptors in adrenocortical cells. All these defects constitute the molecular basis for aberrant autocrine/paracrine regulatory mechanisms which are likely to play a role in the pathophysiology of bilateral macronodular adrenal hyperplasia-associated hypercortisolism. The present review summarizes the current knowledge on this topic as well as the therapeutic perspectives offered by this new pathophysiological concept.

Cytochemical identification of endocrine thymus of chicken in relation to aging

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Uma Kanta Mishra

2013-09-01

Full Text Available Age related cytochemical changes of thymic endocrine cells were studied in 78 day old chicks at five day interval to age of day 60 employing a panel of cytochemical stains. Methenamine silver revealed cell morphology including cell processes distinctly while diamine silver revealed a stronger argentaffinity in these cells. The cells had greater affinity for diamine silver compared to methenamine silver (Argentaffin, followed by formaldehyde induced auto- fluorescence, argyrophilia, lead hematoxylin and HCl-toluidine. The chromaffin reaction was the weakest. Cytochemically, three different endocrine cell populations i.e. argentaffin cells, argyrophilic cells and amine precursor uptake and decarboxylation series (APUD/chromaffin cells, formed the resident population of thymic endocrine cells. Occurrence of numerous serotonin storing cells, moderately frequent APUD cells, and fewer chromaffin as well as mast cells suggests for a conspicuous reservoir of amine storing cells in thymus. Morphologically argentaffin cells were of four types i.e. the peripherally granulated spherical cells (Type-I, densely granulated oval cells (Type-II, pyramidal argentaffin cells (Type-III and diffusely granulated elongated cells (Type-IV. The type-II argentaffin cells were most frequent in the medulla followed by the type-I cells and the type-III cells. The type-IV cells were least in frequency. The age related changes in frequency of these cells are also discussed.

HIF2A and IGF2 Expression Correlates in Human Neuroblastoma Cells and Normal Immature Sympathetic Neuroblasts

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Sofie Mohlin

2013-03-01

Full Text Available During normal sympathetic nervous system (SNS development, cells of the ganglionic lineage can malignantly transform and develop into the childhood tumor neuroblastoma. Hypoxia-inducible transcription factors (HIFs mediate cellular responses during normal development and are central in the adaptation to oxygen shortage. HIFs are also implicated in the progression of several cancer forms, and high HIF-2α expression correlates with disseminated disease and poor outcome in neuroblastoma. During normal SNS development, HIF2A is transiently expressed in neuroblasts and chromaffin cells. SNS cells can, during development, be distinguished by distinct gene expression patterns, and insulin-like growth factor 2 (IGF2 is a marker of sympathetic chromaffin cells, whereas sympathetic neuroblasts lack IGF2 expression. Despite the neuronal derivation of neuroblastomas, we show that neuroblastoma cell lines and specimens express IGF2 and that expression of HIF2A and IGF2 correlates, with the strongest correlation in high-stage tumors. In neuroblastoma, both IGF2 and HIF2A are hypoxia-driven and knocking down IGF2 at hypoxia resulted in downregulated HIF2A levels. HIF-2α and IGF2 were strongly expressed in subsets of immature neuroblastoma cells, suggesting that these two genes could be co-expressed also at early stages of SNS development. We show that IGF2 is indeed expressed in sympathetic chain ganglia at embryonic week 6.5, a developmental stage when HIF-2α is present. These findings provide a rationale for the unexpected IGF2 expression in neuroblastomas and might suggest that IGF2 and HIF2A positive neuroblastoma cells are arrested at an embryonic differentiation stage corresponding to the stage when sympathetic chain ganglia begins to coalesce.

Internal Ca2+ mobilization and secretion in bovine adrenal chromaffin cells

DEFF Research Database (Denmark)

Cheek, T R; Thastrup, Ole

1989-01-01

and the sesquiterpene lactone thapsigargin (TG), which releases internal Ca2+ without concomitant breakdown of inositol lipids or protein kinase C activation, to examine the events which follow depletion of the releasable Ca2+ store in these cells. Monitoring [Ca2+]i using Fura-2 demonstrated that TG released Ca2+ from......-state that was dependent on the continual entry of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)...

Expression and Role of the BDNF Receptor-TrkB in Rat Adrenal Gland under Acute Immobilization Stress

International Nuclear Information System (INIS)

Kondo, Yusuke; Saruta, Juri; To, Masahiro; Shiiki, Naoto; Sato, Chikatoshi; Tsukinoki, Keiichi

2010-01-01

We reported that plasma brain-derived neurotrophic factor (BDNF) was maximally elevated following a 60-min period of acute immobilization stress and that salivary glands were the main source of plasma BDNF under this stress condition. However, the expression pattern of the BDNF receptor, Tyrosine receptor kinase B (TrkB), under this condition has yet to be determined. We therefore investigated the effect of this stress on the expression level of TrkB in various rat organs using real-time PCR. No significant differences were found between controls and 60 min-stressed rats with respect to TrkB level in various organs. Only adrenal glands showed significantly increased TrkB mRNA levels after 60 min of stress. TrkB mRNA and protein were observed to localize in chromaffin cells. In addition, we investigated whether BDNF-TrkB interaction influences the release of stress hormones from PC12 cells, derived from chromaffin cells. Truncated receptor, TrkB-T1, was identified in PC12 cells using RT-PCR. Exposure of PC12 cells to BDNF induced the release of catecholamine. This BDNF-evoked release was totally blocked by administration of the K252a in which an inhibitor of Trk receptors. Thus, BDNF-TrkB interactions may modulate catecholamine release from adrenal chromaffin cells under acute stress conditions

The adrenal gland of the African buffalo, Syncerus caffer : A light and ...

African Journals Online (AJOL)

The medulla is characterized by two distinct regions: an outer region of cells with granules of varying electron density which do not stain specifically with the Masson-Fontana technique and an inner zone of cells containing intensely electron dense granules which are chromaffin positive. These cells are adrenaline- and ...

The Gata3 transcription factor is required for the survival of embryonic and adult sympathetic neurons

NARCIS (Netherlands)

K. Tsarovina (Konstantina); T. Reiff (Tobias); J. Stubbusch (Jutta); D. Kurek (Dorota); F.G. Grosveld (Frank); R. Parlato (Rosanna); G. Schütz (Günther); H. Rohrer (Hermann)

2010-01-01

textabstractThe transcription factor Gata3 is essential for the development of sympathetic neurons and adrenal chromaffin cells. As Gata3 expression is maintained up to the adult stage, we addressed its function in differentiated sympathoadrenal cells at embryonic and adult stages by conditional

Review: the role of neural crest cells in the endocrine system.

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Adams, Meghan Sara; Bronner-Fraser, Marianne

2009-01-01

The neural crest is a pluripotent population of cells that arises at the junction of the neural tube and the dorsal ectoderm. These highly migratory cells form diverse derivatives including neurons and glia of the sensory, sympathetic, and enteric nervous systems, melanocytes, and the bones, cartilage, and connective tissues of the face. The neural crest has long been associated with the endocrine system, although not always correctly. According to current understanding, neural crest cells give rise to the chromaffin cells of the adrenal medulla, chief cells of the extra-adrenal paraganglia, and thyroid C cells. The endocrine tumors that correspond to these cell types are pheochromocytomas, extra-adrenal paragangliomas, and medullary thyroid carcinomas. Although controversies concerning embryological origin appear to have mostly been resolved, questions persist concerning the pathobiology of each tumor type and its basis in neural crest embryology. Here we present a brief history of the work on neural crest development, both in general and in application to the endocrine system. In particular, we present findings related to the plasticity and pluripotency of neural crest cells as well as a discussion of several different neural crest tumors in the endocrine system.

Somatic RET mutation in a patient with pigmented adrenal pheochromocytoma

NARCIS (Netherlands)

Maison, Nicole; Korpershoek, Esther; Eisenhofer, Graeme; Robledo, Mercedes; de Krijger, Ronald; Beuschlein, Felix

UNLABELLED: Pheochromocytomas (PCC) and paraganglioma (PGL) are rare neuroendocrine tumors arising from chromaffin cells of the neural crest. Mutations in the RET-proto-oncogene are associated with sporadic pheochromocytoma, familial or sporadic medullary thyroid carcinoma (MTC) and multiple

Nature of rate-limiting steps in a compartmentalized enzyme system. Quantitation of dopamine transport and hydroxylation rates in resealed chromaffin granule ghosts

International Nuclear Information System (INIS)

Ahn, N.G.; Klinman, J.P.

1989-01-01

Using isolated chromaffin granule ghosts from bovine adrenal medullae, we have studied the kinetics of dopamine beta-monooxygenase (D beta M) activity as it is linked to dopamine transport. Measurements of the initial rates of transport and of transport-linked norepinephrine formation suggested that enzyme activity may be partially rate-limiting in the coupled carrier/enzyme system. This was confirmed by (i) measurements of initial rates of norepinephrine formation using deuterated substrate, which gave isotope effects greater than 2.0, and (ii) kinetic measurements using ghosts pulsed with varying concentrations of labeled dopamine, which indicated substantial substrate accumulation in the vesicle interior as a function of time. Initial rates of product formation, when combined with approximations of internal substrate concentrations, allowed estimates of Kcat and Km for intravesicular D beta M. Activation by external reductant was apparent in both initial rate parameters and the measurements of transients. Under conditions of optimal D beta M activity, the enzyme rate parameters (kcat = 0.31 nmol/s.mg and Km = 2 mM) indicated partial rate limitation compared to dopamine transport (kcat = 0.38 nmol/s.mg and Km = 32 microM). Compartmental analysis of the time curves, performed using numerical nonlinear least squares methods, gave least squares estimates of rate constants for a simple carrier mechanism and kcat values for D beta M which were consistent with estimates from initial rates

Serotonin and Serotonin Transporters in the Adrenal Medulla: A Potential Hub for Modulation of the Sympathetic Stress Response.

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Brindley, Rebecca L; Bauer, Mary Beth; Blakely, Randy D; Currie, Kevin P M

2017-05-17

Serotonin (5-HT) is an important neurotransmitter in the central nervous system where it modulates circuits involved in mood, cognition, movement, arousal, and autonomic function. The 5-HT transporter (SERT; SLC6A4) is a key regulator of 5-HT signaling, and genetic variations in SERT are associated with various disorders including depression, anxiety, and autism. This review focuses on the role of SERT in the sympathetic nervous system. Autonomic/sympathetic dysfunction is evident in patients with depression, anxiety, and other diseases linked to serotonergic signaling. Experimentally, loss of SERT function (SERT knockout mice or chronic pharmacological block) has been reported to augment the sympathetic stress response. Alterations to serotonergic signaling in the CNS and thus central drive to the peripheral sympathetic nervous system are presumed to underlie this augmentation. Although less widely recognized, SERT is robustly expressed in chromaffin cells of the adrenal medulla, the neuroendocrine arm of the sympathetic nervous system. Adrenal chromaffin cells do not synthesize 5-HT but accumulate small amounts by SERT-mediated uptake. Recent evidence demonstrated that 5-HT 1A receptors inhibit catecholamine secretion from adrenal chromaffin cells via an atypical mechanism that does not involve modulation of cellular excitability or voltage-gated Ca 2+ channels. This raises the possibility that the adrenal medulla is a previously unrecognized peripheral hub for serotonergic control of the sympathetic stress response. As a framework for future investigation, a model is proposed in which stress-evoked adrenal catecholamine secretion is fine-tuned by SERT-modulated autocrine 5-HT signaling.

Metabologenomics of Phaeochromocytoma and Paraganglioma: An Integrated Approach for Personalised Biochemical and Genetic Testing.

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Eisenhofer, Graeme; Klink, Barbara; Richter, Susan; Lenders, Jacques Wm; Robledo, Mercedes

2017-04-01

The tremendous advances over the past two decades in both clinical genetics and biochemical testing of chromaffin cell tumours have led to new considerations about how these aspects of laboratory medicine can be integrated to improve diagnosis and management of affected patients. With germline mutations in 15 genes now identified to be responsible for over a third of all cases of phaeochromocytomas and paragangliomas, these tumours are recognised to have one of the richest hereditary backgrounds among all neoplasms. Depending on the mutation, tumours show distinct differences in metabolic pathways that relate to or even directly impact clinical presentation. At the same time, there has been improved understanding about how catecholamines are synthesised, stored, secreted and metabolised by chromaffin cell tumours. Although the tumours may not always secrete catecholamines it has become clear that almost all continuously produce and metabolise catecholamines. This has not only fuelled changes in laboratory medicine, but has also assisted in recognition of genotype-biochemical phenotype relationships important for diagnostics and clinical care. In particular, differences in catecholamine and energy pathway metabolomes can guide genetic testing, assist with test interpretation and provide predictions about the nature, behaviour and imaging characteristics of the tumours. Conversely, results of genetic testing are important for guiding how routine biochemical testing should be employed and interpreted in surveillance programmes for at-risk patients. In these ways there are emerging needs for modern laboratory medicine to seamlessly integrate biochemical and genetic testing into the diagnosis and management of patients with chromaffin cell tumours.

Intrastriatal Grafting of Chromospheres: Survival and Functional Effects in the 6-OHDA Rat Model of Parkinson's Disease.

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Alejandra Boronat-García

Full Text Available Cell replacement therapy in Parkinson's disease (PD aims at re-establishing dopamine neurotransmission in the striatum by grafting dopamine-releasing cells. Chromaffin cell (CC grafts produce some transitory improvements of functional motor deficits in PD animal models, and have the advantage of allowing autologous transplantation. However, CC grafts have exhibited low survival, poor functional effects and dopamine release compared to other cell types. Recently, chromaffin progenitor-like cells were isolated from bovine and human adult adrenal medulla. Under low-attachment conditions, these cells aggregate and grow as spheres, named chromospheres. Here, we found that bovine-derived chromosphere-cell cultures exhibit a greater fraction of cells with a dopaminergic phenotype and higher dopamine release than CC. Chromospheres grafted in a rat model of PD survived in 57% of the total grafted animals. Behavioral tests showed that surviving chromosphere cells induce a reduction in motor alterations for at least 3 months after grafting. Finally, we found that compared with CC, chromosphere grafts survive more and produce more robust and consistent motor improvements. However, further experiments would be necessary to determine whether the functional benefits induced by chromosphere grafts can be improved, and also to elucidate the mechanisms underlying the functional effects of the grafts.

Met-enkephalyl-Arg6-Phe7 immunoreactivity in a human neuroblastoma cell line: effect of dibutyryl 3':5'-cyclic AMP and reserpine.

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Boarder, M R; Marriott, D; Adams, M

1986-12-30

The carboxy terminal part of the proenkephalin A sequence is the 31 amino acid peptide B, which has as its final seven amino acids the sequence of the opioid peptide Met-enkephalyl-Arg6-Phe7. Using a radioimmunoassay which recognises both these peptides we have investigated the relative amounts of peptide B and Met-enkephalyl-Arg6-Phe7 in a human neuroblastoma cell line. We show that these cells contain peptide B-like immunoreactivity but not its heptapeptide fragment. This may be due to lack of proteolytic activity cleaving Met-enkephalyl-Arg6-Phe7 from its precursor, peptide B. On treatment with dibutyryl cyclic AMP the level of immunoreactivity approximately doubles, due to increased amounts of peptide B-like immunoreactivity. Treatment with reserpine, which increases conversion of peptide B to the heptapeptide in bovine chromaffin cells in culture does not stimulate the accumulation of Met-enkephalyl-Arg6-Phe7 in the human neuroblastoma cells. The results are discussed with respect to peptide processing.

Bases fisiológicas para una interacción entre las células cromafines y las endoteliales de la glándula adrenal Physiological bases for an interaction between chromaffin and endothelial cells from the adrenal gland

Directory of Open Access Journals (Sweden)

MARIO LUXORO

2001-03-01

Full Text Available En este trabajo tratamos de investigar las posibles interacciones entre las células endoteliales de la glándula adrenal y aquellas sustancias relacionadas con la secreción de las células cromafines. Para lo anterior, estudiamos el efecto de acetilcolina (ACh, o de catecolaminas (CA tanto en el nivel de Ca2+ citoplasmático ([Ca2+]i, como en el potencial de membrana de las células endoteliales. Nuestros resultados muestran que tanto la ACh como la nicotina, pero no la muscarina, son capaces de inducir un aumento del [Ca2+]i y una despolarización de la membrana plasmática de las células endoteliales. El antagonista nicotínico, hexametonium, bloquea tanto el efecto de la ACh como de la nicotina lo que sugiere la presencia de receptores nicotínicos. Por otra parte, las CA (tanto adrenalina como noradrenalina o agonistas a1-adrenérgicos también producen un aumento del [Ca2+]i en las células endoteliales aunque no despolarización evidente. En este caso, el aumento es bifásico siendo la primera fase de un pico rápido e independiente del Ca2+ extracelular en tanto que la segunda se presenta con oscilaciones y depende tanto de que los canales de Ca2+ no estén bloqueados como de la presencia de ese ión en el medio externo. Dado que se ha demostrado que el aumento del [Ca2+]i en las células endoteliales desencadena la secreción de sustancias vasodilatadoras (prostaciclina y óxido nítrico, proponemos que éste sería un mecanismo compensatorio del sistema para contrarestar el enorme efecto vasoconstrictor de las CA secretadas por las células cromafinesIn this work we investigated the possible interactions between the endothelial cells from the adrenal gland and the substances related with the secretion from the chromaffin cells. In order to do so, we studied the effect of acetyl-choline (ACh or of catecholamines (CA on the level of the membrane potential and the cytoplasmic concentration of free calcium ([Ca2+]i in the endothelial

Review of Pediatric Pheochromocytoma and Paraganglioma

Directory of Open Access Journals (Sweden)

Reshma Bholah

2017-07-01

Full Text Available Pheochromocytoma (PCC and paraganglioma (PGL are rare chromaffin cell tumors which secrete catecholamines and form part of the family of neuroendocrine tumors. Although a rare cause of secondary hypertension in pediatrics, the presentation of hypertension in these patients is characteristic, and treatment is definitive. The gold standard for diagnosis is via measurement of plasma free metanephrines, with imaging studies performed for localization, identification of metastatic lesions and for surgical resection. Preoperative therapy with alpha-blocking agents, beta blockers, and potentially tyrosine hydroxylase inhibitors aid in a safe pre-, intra- and postoperative course. PCC and PGL are inherited in as much as 80% of pediatric cases, and all patients with mutations should be followed closely given the risk of recurrence and malignancy. While the presentation of chromaffin cell tumors has been well described with multiple endocrine neoplasia, NF1, and Von Hippel–Lindau syndromes, the identification of new gene mutations leading to chromaffin cell tumors at a young age is changing the landscape of how clinicians approach such cases. The paraganglioma–pheochromocytoma syndromes (SDHx comprise familial gene mutations, of which the SDHB gene mutation carries a high rate of malignancy. Since the inheritance rate of such tumors is higher than previously described, genetic screening is recommended in all patients, and lifelong follow-up for recurrent tumors is a must. A multidisciplinary team approach allows for optimal health-care delivery in such children. This review serves to provide an overview of pediatric PCC and PGL, including updates on the preferred methods of imaging, guidelines on gene testing as well as management of hypertension in such patients.

Functional modifications of the enteric nervous system following radiation exposure: short and long term effects; Modifications du fonctionnement du systeme nerveux enterique suite a une exposition aux rayonnements ionisants: effets precoces et a long terme

Energy Technology Data Exchange (ETDEWEB)

Ropenga, A

2003-09-15

Exposure of the gastrointestinal tract to ionising radiation induces at short or at long term, digestive dysfunctions, including nausea, diarrhoea, constipation and eventually abdominal pain. The mechanisms implicated remain incompletely understood, but may involve at long term functional modifications of the enteric nervous system (ENS). The mediator 5-Hydroxytryptamine (5-HT, serotonin) is present in entero-chromaffin cells and the ENS and plays an important role in digestive functions. The aim of this work was to follow between 3 days and 3 months after an hemi-body irradiation (10 Gy, X rays) radiation-induced modifications of 5-HT content, 5-HT receptor expression and effects on electrolyte movement in rat distal colon. At 3 days following irradiation, a reduction of total epithelial cells was observed along with a diminution of 5-HT transporter expression. Receptors 5-HT{sub 1A} and 5-HT{sub 2A} expression was diminished concomitant with a reduced response to 5-HT or neural stimulation and an increased importance of the receptor 5-HT{sub 3}. At 7 days crypt total cell number was increased and the importance of receptors 5-HT{sub 2A} and 5-HT{sub 3} in the secretory response was also increased. At later times, between 28 and 43 days, irradiation increased mucosal 5-HT content. This increase can be related to an increase of the number of entero-chromaffin cells at 28 days and is concomitant with the diminution of the importance of the receptor 5-HT{sub 2A} in the secretory response. In conclusion, this project has established for the first time differential expression of 5-HT receptors in the mucosal and muscle layers in the distal colon. Moreover, irradiation induces modifications in 5-HT receptor expression and importance in secretory epithelial responses. Irradiation also disturbs the equilibrium of different cell types by the epithelium in increasing the number of entero-chromaffin cells containing 5-HT. (author)

Secretagogue stimulation of neurosecretory cells elicits filopodial extensions uncovering new functional release sites.

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Papadopulos, Andreas; Martin, Sally; Tomatis, Vanesa M; Gormal, Rachel S; Meunier, Frederic A

2013-12-04

Regulated exocytosis in neurosecretory cells relies on the timely fusion of secretory granules (SGs) with the plasma membrane. Secretagogue stimulation leads to an enlargement of the cell footprint (surface area in contact with the coverslip), an effect previously attributed to exocytic fusion of SGs with the plasma membrane. Using total internal reflection fluorescence microscopy, we reveal the formation of filopodia-like structures in bovine chromaffin and PC12 cells driving the footprint expansion, suggesting the involvement of cortical actin network remodeling in this process. Using exocytosis-incompetent PC12 cells, we demonstrate that footprint enlargement is largely independent of SG fusion, suggesting that vesicular exocytic fusion plays a relatively minor role in filopodial expansion. The footprint periphery, including filopodia, undergoes extensive F-actin remodeling, an effect abolished by the actomyosin inhibitors cytochalasin D and blebbistatin. Imaging of both Lifeact-GFP and the SG marker protein neuropeptide Y-mCherry reveals that SGs actively translocate along newly forming actin tracks before undergoing fusion. Together, these data demonstrate that neurosecretory cells regulate the number of SGs undergoing exocytosis during sustained stimulation by controlling vesicular mobilization and translocation to the plasma membrane through actin remodeling. Such remodeling facilitates the de novo formation of fusion sites.

Assessment of functional changes in nanoparticle-exposed neuroendocrine cells with amperometry: exploring the generalizability of nanoparticle-vesicle matrix interactions.

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Love, Sara A; Haynes, Christy L

2010-09-01

Using two of the most commonly synthesized noble metal nanoparticle preparations, citrate-reduced Au and Ag, the impacts of short-term accidental nanoparticle exposure are examined in primary culture murine adrenal medullary chromaffin cells. Transmission electron microscopy (TEM), inductively coupled plasma atomic emission spectroscopy (ICP-AES) and Alamar Blue viability studies revealed that nanoparticles are taken up by cells but do not decrease cell viability within 48 hours of exposure. Carbon-fiber microelectrode amperometry (CFMA) examination of exocytosis in nanoparticle-exposed cells revealed that nanoparticle exposure does lead to decreased secretion of chemical messenger molecules, of up to 32.5% at 48 hours of Au exposure. The kinetics of intravesicular species liberation also slows after nanoparticle exposure, between 30 and 50% for Au and Ag, respectively. Repeated stimulation of exocytosis demonstrated that these effects persisted during subsequent stimulations, meaning that nanoparticles do not interfere directly with the vesicle recycling machinery but also that cellular function is unable to recover following vesicle content expulsion. By comparing these trends with parallel studies done using mast cells, it is clear that similar exocytosis perturbations occur across cell types following noble metal nanoparticle exposure, supporting a generalizable effect of nanoparticle-vesicle interactions.

Refractive Index Imaging of Cells with Variable-Angle Near-Total Internal Reflection (TIR) Microscopy.

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Bohannon, Kevin P; Holz, Ronald W; Axelrod, Daniel

2017-10-01

The refractive index in the interior of single cells affects the evanescent field depth in quantitative studies using total internal reflection (TIR) fluorescence, but often that index is not well known. We here present method to measure and spatially map the absolute index of refraction in a microscopic sample, by imaging a collimated light beam reflected from the substrate/buffer/cell interference at variable angles of incidence. Above the TIR critical angle (which is a strong function of refractive index), the reflection is 100%, but in the immediate sub-critical angle zone, the reflection intensity is a very strong ascending function of incidence angle. By analyzing the angular position of that edge at each location in the field of view, the local refractive index can be estimated. In addition, by analyzing the steepness of the edge, the distance-to-substrate can be determined. We apply the technique to liquid calibration samples, silica beads, cultured Chinese hamster ovary cells, and primary culture chromaffin cells. The optical technique suffers from decremented lateral resolution, scattering, and interference artifacts. However, it still provides reasonable results for both refractive index (~1.38) and for distance-to-substrate (~150 nm) for the cells, as well as a lateral resolution to about 1 µm.

Hydralazine administration activates sympathetic preganglionic neurons whose activity mobilizes glucose and increases cardiovascular function.

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Parker, Lindsay M; Damanhuri, Hanafi A; Fletcher, Sophie P S; Goodchild, Ann K

2015-04-16

Hypotensive drugs have been used to identify central neurons that mediate compensatory baroreceptor reflex responses. Such drugs also increase blood glucose. Our aim was to identify the neurochemical phenotypes of sympathetic preganglionic neurons (SPN) and adrenal chromaffin cells activated following hydralazine (HDZ; 10mg/kg) administration in rats, and utilize this and SPN target organ destination to ascribe their function as cardiovascular or glucose regulating. Blood glucose was measured and adrenal chromaffin cell activation was assessed using c-Fos immunoreactivity (-ir) and phosphorylation of tyrosine hydroxylase, respectively. The activation and neurochemical phenotype of SPN innervating the adrenal glands and celiac ganglia were determined using the retrograde tracer cholera toxin B subunit, in combination with in situ hybridization and immunohistochemistry. Blood glucose was elevated at multiple time points following HDZ administration but little evidence of chromaffin cell activation was seen suggesting non-adrenal mechanisms contribute to the sustained hyperglycemia. 16±0.1% of T4-T11 SPN contained c-Fos and of these: 24.3±1.4% projected to adrenal glands and 29±5.5% projected to celiac ganglia with the rest innervating other targets. 62.8±1.4% of SPN innervating adrenal glands were activated and 29.9±3.3% expressed PPE mRNA whereas 53.2±8.6% of SPN innervating celiac ganglia were activated and 31.2±8.8% expressed PPE mRNA. CART-ir SPN innervating each target were also activated and did not co-express PPE mRNA. Neurochemical coding reveals that HDZ administration activates both PPE+SPN, whose activity increase glucose mobilization causing hyperglycemia, as well as CART+SPN whose activity drive vasomotor responses mediated by baroreceptor unloading to raise vascular tone and heart rate. Copyright © 2015 Elsevier B.V. All rights reserved.

Measurement of the internal pH of mast cell granules using microvolumetric fluorescence and isotopic techniques

International Nuclear Information System (INIS)

De Young, M.B.; Nemeth, E.F.; Scarpa, A.

1987-01-01

The intragranular pH of isolated mast cell granules was measured. Because of the minute amounts of isolated granules available, two techniques were developed by modifying aminoacridine fluorescence and [ 14 C]methylamine accumulation techniques to permit measurements with microliter sample volumes. Granule purity was demonstrated by electron microscopy, ruthenium red exclusion, and biochemical (histamine, mast cell granule protease) analysis. The internal pH was determined to be 5.55 +/- 0.06, indicating that the pH environment within mast cell granules is not significantly different from that of previously studied granule types (i.e., chromaffin, platelet, pancreatic islet, and pituitary granules). Collapse of the pH gradient by NH+4 was demonstrated with both techniques. No evidence of Cl-/OH- or specific cation/H+ transport was found, and major chloride permeability could not be unequivocably demonstrated. Ca 2+ and Cl- at concentrations normally present extracellularly destabilized granules in the presence of NH+4, but this phenomenon does not necessarily indicate a role for these ions in the exocytotic release of granule contents from intact cells. The pH measurement techniques developed for investigating the properties of granules in mast cells may be useful for studying other granules that can be obtained only in limited quantities

Imaging findings of a primary paraganglioma of the liver: A case report

Energy Technology Data Exchange (ETDEWEB)

Ji, Seung Woo; Kang, Ung Rae; Park, Jae Bok [Daegu Catholic University Medical Center, Catholic University of Daegu College of Medicine, Daegu (Korea, Republic of)

2016-11-15

Primary hepatic paraganglioma is an extremely rare type of tumor originating from extra-adrenal chromaffin cells. We report a case of primary intrahepatic paraganglioma in a 52-year-old man, with pathologic confirmation through right hepatectomy. An imaging study indicated a predominately hemorrhagic septated cystic mass and peripheral marked enhancement of the solid portions, which showed persistent enhancement.

Hemi-fused structure mediates and controls fusion and fission in live cells.

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Zhao, Wei-Dong; Hamid, Edaeni; Shin, Wonchul; Wen, Peter J; Krystofiak, Evan S; Villarreal, Seth A; Chiang, Hsueh-Cheng; Kachar, Bechara; Wu, Ling-Gang

2016-06-23

Membrane fusion and fission are vital for eukaryotic life. For three decades, it has been proposed that fusion is mediated by fusion between the proximal leaflets of two bilayers (hemi-fusion) to produce a hemi-fused structure, followed by fusion between the distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion or hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed. Here we report the observation of a hemi-fused Ω-shaped structure in live neuroendocrine chromaffin cells and pancreatic β-cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, the transition to full fusion or fission is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key missing evidence in support of the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion and fission, as fusion and fission mechanisms compete to determine the transition to fusion or fission.

[Adrenal tumours in childhood].

Science.gov (United States)

Martos-Moreno, G A; Pozo-Román, J; Argente, J

2013-09-01

This special article aims to summarise the current knowledge regarding the two groups of tumours with their origin in the adrenal gland: 1) adrenocortical tumours, derived from the cortex of the adrenal gland and 2) phaeochromocytomas and paragangliomas, neuroendocrine tumours derived from nodes of neural crest derived cells symmetrically distributed at both sides of the entire spine (paragangliomas [PG]). These PGs can be functioning tumors that secrete catecholamines, which confers their typical dark colour after staining with chromium salts (chromaffin tumors). Among these, the term phaeochromocytoma (PC) is restricted to those PGs derived from the chromaffin cells in the adrenal medulla (intra-adrenal PGs), whereas the term PG is used for those sympathetic or parasympathetic ones in an extra-adrenal location. We analyse the state of the art of their pathogenic and genetic bases, as well as their clinical signs and symptoms, the tests currently available for performing their diagnosis (biochemical, hormonal, imaging and molecular studies) and management (surgery, pre- and post-surgical medical treatment), considering the current and developing strategies in chemo- and radiotherapy. Copyright © 2013 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.

Examining changes in cellular communication in neuroendocrine cells after noble metal nanoparticle exposure.

Science.gov (United States)

Love, Sara A; Liu, Zhen; Haynes, Christy L

2012-07-07

As nanoparticles enjoy increasingly widespread use in commercial applications, the potential for unintentional exposure has become much more likely during any given day. Researchers in the field of nanotoxicity are working to determine the physicochemical nanoparticle properties that lead to toxicity in an effort to establish safe design rules. This work explores the effects of noble metal nanoparticle exposure in murine chromaffin cells, focusing on examining the effects of size and surface functionality (coating) in silver and gold, respectively. Carbon-fibre microelectrode amperometry was utilized to examine the effect of exposure on exocytosis function, at the single cell level, and provided new insights into the compromised functions of cells. Silver nanoparticles of varied size, between 15 and 60 nm diameter, were exposed to cells and found to alter the release kinetics of exocytosis for those cells exposed to the smallest examined size. Effects of gold were examined after modification with two commonly used 'bio-friendly' polymers, either heparin or poly (ethylene glycol), and gold nanoparticles were found to induce altered cellular adhesion or the number of chemical messenger molecules released, respectively. These results support the body of work suggesting that noble metal nanoparticles perturb exocytosis, typically altering the number of molecules and kinetics of release, and supports a direct disruption of the vesicle matrix by the nanoparticle. Overall, it is clear that various nanoparticle physicochemical properties, including size and surface coating, do modulate changes in cellular communication via exocytosis.

[Effect of drinking boron on microtructure of adrenal gland in rats].

Science.gov (United States)

Li, Shenghe; Wang, Jue; Zhou, Jinxing; Jin, Guangming; Gu, Youfang; Xu, Wanxiang

2012-09-01

The effects of drinking boron exposure on the mass, organ indexes and structure of adrenal gland were studied in the paper. Methods 192 Sprague-Dawley rats (28 +/- 2 days) with no bacteria infecting were divided into six groups (n = 32, male = female) randomly. Treated rats drunk the distilled water which supplemented with boron of 0, 40, 80, 160, 320 and 640 mg/L, respectively, for 60 days. At the 30th and the 60th day of experiment, 16 rats (n = 8, male = female) of each group were selected and made into narcosis with 10% Chloral Hydrate. The adrenal glands were obtained, weighted and fixed after dissection, then the samples were made into paraffin sections, stained with HE stain and chromaffin, observed and photographed by Olympus CH-30 microphotograph system. Compared with control group, the average mass of adrenal gland of male rats in each experiment group decreased significantly or most significantly at the 30th day of experiment (P boron at 60th day of experiment increased significantly (P boron were better obviously than control group, and the numbers of chromaffin granules in chromaffin cell increased obviously. The histopathological changes of different degree could be observed in the group of 80 to 640 mg/L boron, and they became remarkable with the boron supplementation. By comparative observation, the damage of cells in adrenal medulla appeared ahead of them in adrenal cortex, and the pathological change of adrenal gland in male rats were obvious than female rats. Drinking supplemented with 40 mg/L boron could prompt the structure of adrenal gland in rats, but could cause different degree damage, or even obvious toxic effect when the concentration of boron supplementation in drinking from 80 to 640 mg/L.

Beta-amyloid peptides undergo regulated co-secretion with neuropeptide and catecholamine neurotransmitters.

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Toneff, Thomas; Funkelstein, Lydiane; Mosier, Charles; Abagyan, Armen; Ziegler, Michael; Hook, Vivian

2013-08-01

Beta-amyloid (Aβ) peptides are secreted from neurons, resulting in extracellular accumulation of Aβ and neurodegeneration of Alzheimer's disease. Because neuronal secretion is fundamental for the release of neurotransmitters, this study assessed the hypothesis that Aβ undergoes co-release with neurotransmitters. Model neuronal-like chromaffin cells were investigated, and results illustrate regulated, co-secretion of Aβ(1-40) and Aβ(1-42) with peptide neurotransmitters (galanin, enkephalin, and NPY) and catecholamine neurotransmitters (dopamine, norepinephrine, and epinephrine). Regulated secretion from chromaffin cells was stimulated by KCl depolarization and nicotine. Forskolin, stimulating cAMP, also induced co-secretion of Aβ peptides with peptide and catecholamine neurotransmitters. These data suggested the co-localization of Aβ with neurotransmitters in dense core secretory vesicles (DCSV) that store and secrete such chemical messengers. Indeed, Aβ was demonstrated to be present in DCSV with neuropeptide and catecholamine transmitters. Furthermore, the DCSV organelle contains APP and its processing proteases, β- and γ-secretases, that are necessary for production of Aβ. Thus, Aβ can be generated in neurotransmitter-containing DCSV. Human IMR32 neuroblastoma cells also displayed regulated secretion of Aβ(1-40) and Aβ(1-42) with the galanin neurotransmitter. These findings illustrate that Aβ peptides are present in neurotransmitter-containing DCSV, and undergo co-secretion with neuropeptide and catecholamine neurotransmitters that regulate brain functions. Copyright © 2013 Elsevier Inc. All rights reserved.

The F-Actin Binding Protein Cortactin Regulates the Dynamics of the Exocytotic Fusion Pore through its SH3 Domain

Science.gov (United States)

González-Jamett, Arlek M.; Guerra, María J.; Olivares, María J.; Haro-Acuña, Valentina; Baéz-Matus, Ximena; Vásquez-Navarrete, Jacqueline; Momboisse, Fanny; Martinez-Quiles, Narcisa; Cárdenas, Ana M.

2017-01-01

Upon cell stimulation, the network of cortical actin filaments is rearranged to facilitate the neurosecretory process. This actin rearrangement includes both disruption of the preexisting actin network and de novo actin polymerization. However, the mechanism by which a Ca2+ signal elicits the formation of new actin filaments remains uncertain. Cortactin, an actin-binding protein that promotes actin polymerization in synergy with the nucleation promoting factor N-WASP, could play a key role in this mechanism. We addressed this hypothesis by analyzing de novo actin polymerization and exocytosis in bovine adrenal chromaffin cells expressing different cortactin or N-WASP domains, or cortactin mutants that fail to interact with proline-rich domain (PRD)-containing proteins, including N-WASP, or to be phosphorylated by Ca2+-dependent kinases, such as ERK1/2 and Src. Our results show that the activation of nicotinic receptors in chromaffin cells promotes cortactin translocation to the cell cortex, where it colocalizes with actin filaments. We further found that, in association with PRD-containing proteins, cortactin contributes to the Ca2+-dependent formation of F-actin, and regulates fusion pore dynamics and the number of exocytotic events induced by activation of nicotinic receptors. However, whereas the actions of cortactin on the fusion pore dynamics seems to depend on the availability of monomeric actin and its phosphorylation by ERK1/2 and Src kinases, cortactin regulates the extent of exocytosis by a mechanism independent of actin polymerization. Together our findings point out a role for cortactin as a critical modulator of actin filament formation and exocytosis in neuroendocrine cells. PMID:28522963

Regulation of alternative splicing of Slo K+ channels in adrenal and pituitary during the stress-hyporesponsive period of rat development.

Science.gov (United States)

Lai, Guey-Jen; McCobb, David P

2006-08-01

Stress triggers release of ACTH from the pituitary, glucocorticoids from the adrenal cortex, and epinephrine from the adrenal medulla. Although functions differ, these hormone systems interact in many ways. Previous evidence indicates that pituitary and steroid hormones regulate alternative splicing of the Slo gene at the stress axis-regulated exon (STREX), with functional implications for the calcium-activated K+ channels prominent in adrenal medullary and pituitary cells. Here we examine the role of corticosterone in Slo splicing regulation in pituitary and adrenal tissues during the stress-hyporesponsive period of early rat postnatal life. The sharp drop in plasma corticosterone (CORT) that defines this period offers a unique opportunity to test CORT's role in Slo splicing. We report that in both adrenal and pituitary tissues, the percentage of Slo transcripts having STREX declines and recovers in parallel with CORT. Moreover, addition of 500 nm CORT to cultures of anterior pituitary cells from 13-, 21-, and 30-d postnatal animals increased the percentage of Slo transcripts with STREX, whereas 20 microm CORT reduced STREX representation. Applied to adrenal chromaffin cells, 20 microm CORT decreased STREX inclusion, whereas neither 500 nm nor 2 microm had any effect. The mineralocorticoid receptor antagonist RU28318 abolished the effect of 500 nm CORT on splicing in pituitary cells, whereas the glucocorticoid receptor antagonist RU38486 blocked the effect of 20 microm CORT on adrenal chromaffin cells. These results support the hypothesis that the abrupt, transient drop in CORT during the stress-hyporesponsive period drives the transient decline in STREX splice variant representation in pituitary, but not adrenal.

Developmental programming of O2 sensing by neonatal intermittent hypoxia via epigenetic mechanisms

OpenAIRE

Nanduri, Jayasri; Prabhakar, Nanduri R.

2012-01-01

Recurrent apnea with intermittent hypoxia (IH) is a major clinical problem in infants born preterm. Carotid body chemo-reflex and catecholamine secretion from adrenal medullary chromaffin cells (AMC) are important for maintenance of cardio-respiratory homeostasis during hypoxia. This article highlights studies on the effects of IH on O2 sensing by the carotid body and AMC in neonatal rodents. Neonatal IH augments hypoxia-evoked carotid body sensory excitation and catecholamine secretion from ...

Responses of proenkephalin Peptide F to aerobic exercise stress in the plasma and white blood cell biocompartments.

Science.gov (United States)

Kraemer, William J; Fragala, Maren S; van Henegouwen, Wendy R H Beijersbergen; Gordon, Scott E; Bush, Jill A; Volek, Jeff S; Triplett, N Travis; Dunn-Lewis, Courtenay; Comstock, Brett A; Szivak, Tunde K; Flanagan, Shawn D; Hooper, David R; Luk, Hui-Ying; Mastro, Andrea M

2013-04-01

Proenkephalin Peptide F [107-140] is an enkephalin-containing peptide found predominantly within the adrenal medulla, co-packaged with epinephrine within the chromaffin granules. In vivo studies indicate that Peptide F has classic opioid analgesia effects; in vitro studies suggest potential immune cell interactions. In this investigation we examined patterns of Peptide F concentrations in different bio-compartments of the blood at rest and following sub-maximal cycle exercise to determine if Peptide F interacts with the white blood cell (WBC) bio-compartment during aerobic exercise. Eight physically active men (n=8) performed sub-maximal (80-85% V˙O2peak) cycle ergometer exercise for 30 min. Plasma Peptide F and WBC Peptide F immunoreactivity were examined pre-exercise, mid-exercise and immediately post-, 5-min post-, 15-min post-, 30-min post- and 60-min post-exercise and at similar time-points during a control condition (30 min rest). Peptide F concentrations significantly (pexercise, compared to pre-exercise concentrations. No significant increases in Peptide F concentrations in the WBC fraction were observed during or after exercise. However, a significant decrease was observed at 30 min post-exercise. An ultradian pattern of Peptide F distribution was apparent during rest. Furthermore, concentrations of T cells, B cells, NK cells, and total WBCs demonstrated significant changes in response to aerobic exercise. Data indicated that Peptide F was bound in significant molar concentrations in the WBC fraction and that this biocompartment may be one of the tissue targets for binding interactions. These data indicate that Peptide F is involved with immune cell modulation in the white blood circulatory biocompartment of blood. Copyright © 2013. Published by Elsevier Inc.

Receptor stimulated formation of inositol phosphates in cultures of bovine adrenal medullary cells: the effects of bradykinin, bombesin and neurotensin.

Science.gov (United States)

Bunn, S J; Marley, P D; Livett, B G

1990-04-01

The ability of a number of drugs and neuropeptides to stimulate phosphoinositide metabolism in cultured bovine adrenal medullary cells has been assessed. Low concentrations (10 nM) of angiotensin II, bradykinin, histamine, arginine-vasopressin, and bombesin, and high (10 microM) concentrations of oxytocin, prostaglandins E1, and E2, beta-endorphin, and neurotensin stimulated significant accumulation of [3H]inositol phosphates in adrenal medullary cells preloaded with [3H)]inositol. Bradykinin stimulated a significant response at concentration as low as 10pM, with an EC50 of approximately 0.5 nM. The response was markedly inhibited by the bradykinin B2 antagonist [Thi5,8,D-Phe7] bradykinin but not the B1 antagonist [Des-Arg9,Leu8] bradykinin. Higher concentrations of bombesin and neurotensin were required to elicit a response (10 nM and 10 microM respectively). The bombesin response was sensitive to inhibition by the bombesin antagonist [D-Arg1,D-Pro2,D-Trp7,9Leu11]-substance P. In contrast, the neurotensin response was not reduced by the NT1 antagonist [D-Trp11]-neurotensin. These results indicate there are a number of agents that can stimulate phosphatidylinositide hydrolysis in the adrenal medullary cells by acting on different classes of receptors. Such a range of diverse agonists that stimulate inositol phosphate formation will facilitate further analysis of the phosphatidylinositide breakdown in chromaffin cell function.

All-carbon multi-electrode array for real-time in vitro measurements of oxidizable neurotransmitters

Science.gov (United States)

Picollo, Federico; Battiato, Alfio; Bernardi, Ettore; Plaitano, Marilena; Franchino, Claudio; Gosso, Sara; Pasquarelli, Alberto; Carbone, Emilio; Olivero, Paolo; Carabelli, Valentina

2016-02-01

We report on the ion beam fabrication of all-carbon multi electrode arrays (MEAs) based on 16 graphitic micro-channels embedded in single-crystal diamond (SCD) substrates. The fabricated SCD-MEAs are systematically employed for the in vitro simultaneous amperometric detection of the secretory activity from populations of chromaffin cells, demonstrating a new sensing approach with respect to standard techniques. The biochemical stability and biocompatibility of the SCD-based device combined with the parallel recording of multi-electrodes array allow: i) a significant time saving in data collection during drug screening and/or pharmacological tests over a large number of cells, ii) the possibility of comparing altered cell functionality among cell populations, and iii) the repeatition of acquisition runs over many cycles with a fully non-toxic and chemically robust bio-sensitive substrate.

Effect of α-bungarotoxin and etorphine on acetylcholine-evoked release of endogenous and radiolabeled catecholamines from primary culture of adrenal chromaffin cells

International Nuclear Information System (INIS)

Kageyama, H.; Guidotti, A.

1984-01-01

Cell cultures of adrenal medulla have become an important research tool to study basic processes that regulate catecholamine storage, release and synthesis. Release has been studied either by labeling with [ 3 H]norepinephrine and measuring release of radioactivity or by measuring the endogenous catecholamines released with HPLC. Acetylcholine (5X10 -6 -10 -4 M) appears to release preferentially norepinephrine, although the cells store more epinephrine than norepinephrine. Etorphine and α-bungarotoxin antagonize the release of catecholamines elicited by acetylcholine. This inhibitory action appears to be greater when the measurement of endogenous catecholamines rather than radioactive norepinephrine is used to monitor the action of acetylcholine. The data suggest that the measurement of endogenous catecholamines by HPLC is preferable to the [ 3 H]NE loading and release technique, especially when analyzing the effects of low concentrations of drugs that are thought to affect nicotinic receptor function. (Auth.)

Characterization of endothelin receptors on a human neuroblastoma cell line: evidence for the ETA subtype.

Science.gov (United States)

Wilkes, L C; Boarder, M R

1991-11-01

1. Specific binding sites for synthetic endothelin (ET) isoforms were studied on intact cells of the SK-N-MC cell line, derived from a human neuroblastoma. 2. [125I]-ET-1 (2.5 x 10(-11) M) specifically bound to a single class of binding sites on these cells (Hill coefficient of 1.06 +/- 0.04, n = 3) with an apparent Kd of 1.4 +/- 0.3 x 10(-9) M and a Bmax of 3.1 +/- 1.0 pmol mg-1 protein. [125I]-ET-3 (2.5 x 10(-11) M), did not specifically bind to SK-N-MC cells. 3. The binding of [125I]-ET-1 was competitively inhibited by other ET isoforms, the order of potency being ET-1 greater than sarafotoxin S6b greater than ET-3. 4. Association of 1 nM [125I]-ET-1 at 37 degrees C reached apparent equilibrium at 60-80 min, with half-maximal binding being achieved at 12 min. 5. Dissociation was measured after both 10 min and 60 min of association with 64% and 30% respectively of specifically bound [125I]-ET-1 dissociating. The actual amounts of [125I]-ET-1 dissociated were similar in both cases. 6. Incubation of [125I]-ET-3 with SK-N-MC cells at 37 degrees C for 60 min did not result in significant degradation of this peptide. However, [125I]-ET-1 was broken down by incubation with SK-N-MC cells, the pattern of degradation of dissociable [125I]-ET-1 (and that found in the supernatant) being different from that of non-dissociable [125I]-ET-1. 7. ET-1 concentration-dependently induced an increase in total inositol phosphate accumulation in subconfluent (but not in confluent) cultures of SK-N-MC cells (EC50 = 6.43 +/- 1.9 x 1010M). ET-3 was without effect. 8. These results show that ET-1 specifically binds to SK-N-MC cells with the characteristics of an ETA receptor. Our earlier finding that adrenal chromaffin cells express an ETB receptor indicates the existence of multiple ET receptor types on neuronal cells.

Adrenal medullary hyperplasia. Hyperplasia-pheochromocytoma sequence.

Science.gov (United States)

Kurihara, K; Mizuseki, K; Kondo, T; Ohoka, H; Mannami, M; Kawai, K

1990-09-01

We present a case of unilateral adrenal medullary hyperplasia in a 63-year-old woman with clinical signs and symptoms of pheochromocytoma unassociated with multiple endocrine neoplasia. The surgically removed adrenal gland revealed diffuse medullary hyperplasia with multiple micronodules measuring up to 2 mm. The micronodules were composed of enlarged chromaffin cells with atypia, histologically similar to those of pheochromocytoma, forming small solid alveolar patterns separated by a fibrovascular stroma. Removal of the hyperplastic adrenal gland resulted in disappearance of paroxysmal nocturnal hypertension and palpitation. These results suggest that diffuse and nodular medullary hyperplasia is the precursor of pheochromocytoma.

Synaptotagmin interaction with SNAP-25 governs vesicle docking, priming, and fusion triggering

DEFF Research Database (Denmark)

Mohrmann, Ralf; de Wit, Heidi; Connell, Emma

2013-01-01

ramifications of proposed SNAP-25 × synaptotagmin-1 interaction in mouse chromaffin cells. We demonstrate that the postulated central binding domain surrounding layer zero covers both SNARE motifs of SNAP-25 and is essential for vesicle docking, priming, and fast fusion-triggering. Mutation of this site caused...... but is required for the full size of the readily releasable pool. Intriguingly, we also found that mutation of synaptotagmin-1 interaction sites led to more pronounced phenotypes in the context of the adult neuronal isoform SNAP-25B than in the embryonic isoform SNAP-25A. Further experiments demonstrated...

Autoimmunity and Gastric Cancer

Science.gov (United States)

Bizzaro, Nicola; Antico, Antonio; Villalta, Danilo

2018-01-01

Alterations in the immune response of patients with autoimmune diseases may predispose to malignancies, and a link between chronic autoimmune gastritis and gastric cancer has been reported in many studies. Intestinal metaplasia with dysplasia of the gastric corpus-fundus mucosa and hyperplasia of chromaffin cells, which are typical features of late-stage autoimmune gastritis, are considered precursor lesions. Autoimmune gastritis has been associated with the development of two types of gastric neoplasms: intestinal type and type I gastric carcinoid. Here, we review the association of autoimmune gastritis with gastric cancer and other autoimmune features present in gastric neoplasms. PMID:29373557

The kunitz protease inhibitor form of the amyloid precursor protein (KPI/APP) inhibits the proneuropeptide processing enzyme prohormone thiol protease (PTP). Colocalization of KPI/APP and PTP in secretory vesicles.

Science.gov (United States)

Hook, V Y; Sei, C; Yasothornsrikul, S; Toneff, T; Kang, Y H; Efthimiopoulos, S; Robakis, N K; Van Nostrand, W

1999-01-29

Proteolytic processing of proenkephalin and proneuropeptides is required for the production of active neurotransmitters and peptide hormones. Variations in the extent of proenkephalin processing in vivo suggest involvement of endogenous protease inhibitors. This study demonstrates that "protease nexin 2 (PN2)," the secreted form of the kunitz protease inhibitor (KPI) of the amyloid precursor protein (APP), potently inhibited the proenkephalin processing enzyme known as prohormone thiol protease (PTP), with a Ki,app of 400 nM. Moreover, PTP and PN2 formed SDS-stable complexes that are typical of kunitz protease inhibitor interactions with target proteases. In vivo, KPI/APP (120 kDa), as well as a truncated form of KPI/APP that resembles PN2 in apparent molecular mass (110 kDa), were colocalized with PTP and (Met)enkephalin in secretory vesicles of adrenal medulla (chromaffin granules). KPI/APP (110-120 kDa) was also detected in pituitary secretory vesicles that contain PTP. In chromaffin cells, calcium-dependent secretion of KPI/APP with PTP and (Met)enkephalin demonstrated the colocalization of these components in functional secretory vesicles. These results suggest a role for KPI/APP inhibition of PTP in regulated secretory vesicles. In addition, these results are the first to identify an endogenous protease target of KPI/APP, which is developmentally regulated in aging and Alzheimer's disease.

Rab3 proteins involved in vesicle biogenesis and priming in embryonic mouse chromaffin cells

DEFF Research Database (Denmark)

Schonn, Jean-Sébastien; van Weering, Jan R T; Mohrmann, Ralf

2010-01-01

The four Rab3 paralogs A-D are involved in exocytosis, but their mechanisms of action are hard to study due to functional redundancy. Here we used a quadruple Rab3 knock-out (rab3a, rab3b, rab3c, rab3d null, here denoted ABCD(-/-)) mouse line to investigate Rab3 function in embryonic mouse adrena...

Feocromocitoma em criança Pheochromocytoma in childhood

Directory of Open Access Journals (Sweden)

Gesmar Volga Haddad Herdy

2005-03-01

Full Text Available Feocromocitoma é uma neoplasia de células cromafins que causa sintomas e sinais decorrentes da libertação de catecolminas e é muito rara em crianças. Relatamos um caso feocromocitoma em criança, com difícil manejo clínico, confirmado também pela anatomia-patológica e curado após a ressecção cirúrgica.Pheochromocytoma is a neoplasia of chromaffin cells that is very rare in children. Its signs and symptoms result from the release of catecholamines. We report the case of a child with pheochromocytoma of difficult clinical management, confirmed on the anatomicopathological study, and cured after surgical resection.

Biological changes of APA-BCC analgesic microcapsule in cerebrospinal fluid of patients with carcinomatous pain

International Nuclear Information System (INIS)

Luo Yun; Li Yanling; Xue Yilong; Guo Shulong; Gao Yuhong; Cui Xin

2005-01-01

To explore the changes of alginate-polylysine-alginate microcapsulated bovine adrenal medullary chromaffin cells (APA-BCC microcapsules) in morphology, survival rate and leucine- enkephalin secretion after they were transplanted into CSF of cancerpain patients, the APA- BCC microcapsules were Implanted into cavitas subarachnoidealis of cancer-pain patients by conventional lumbar puncture. After 7 or 8 days, cerebrospinal fluid was collected and the morphology of the APA-BCC microcapsule, the survival rate of cells were observed and secretory volume of leucine-enkephalin was assayed by radioimmunity method. Seven days after trans- plantation, the mean VAS decreased from 8.8 to 2.4, the survival rate of cells averagely reduced from 91.2% to 89.1%, morphology of APA-BCC microcapsules did not change obviously and secretory volume of leucine-enkephalin went up 1.65 times compared with that at pretrans- plantation. In conclusion, APA-BCC can survive, secret leucine-enkephalin and produce analgesic effect after transplanted into CSF of cancer-patients. (authors)

The SNARE protein vti1a functions in dense-core vesicle biogenesis

DEFF Research Database (Denmark)

Walter, Alexander M; Kurps, Julia; de Wit, Heidi

2014-01-01

overlapping with syntaxin-6. Exocytosis is impaired in vti1a null cells, partly due to fewer Ca(2+)-channels at the plasma membrane, partly due to fewer vesicles of reduced size and synaptobrevin-2 content. In contrast, release kinetics and Ca(2+)-sensitivity remain unchanged, indicating that the final fusion......The SNARE protein vti1a is proposed to drive fusion of intracellular organelles, but recent data also implicated vti1a in exocytosis. Here we show that vti1a is absent from mature secretory vesicles in adrenal chromaffin cells, but localizes to a compartment near the trans-Golgi network, partially...... reaction leading to transmitter release is unperturbed. Additional deletion of the closest related SNARE, vti1b, does not exacerbate the vti1a phenotype, and vti1b null cells show no secretion defects, indicating that vti1b does not participate in exocytosis. Long-term re-expression of vti1a (days...

Paraganglioma of the larynx. A case report and clinical review.

Science.gov (United States)

Shipton, E A; van der Linde, J C

1984-02-04

An 8-year-old Black boy recently underwent surgery for the excision of a non-chromaffin laryngeal paraganglioma. The peri-operative management of this patient is presented. The specific problems involved and their importance to the anaesthesiologist and surgeon alike are discussed.

Phosphatidylinositol 4,5-bisphosphate optical uncaging potentiates exocytosis

DEFF Research Database (Denmark)

Walter, Alexander M; Müller, Rainer; Tawfik, Bassam

2017-01-01

Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] is essential for exocytosis. Classical ways of manipulating PI(4,5)P2 levels are slower than metabolism, making it difficult to distinguish effects of PI(4,5)P2 from those of its metabolites. We developed a membrane-permeant, photoactivatable PI(4......,5)P2, which is loaded into cells in an inactive form and activated by light, allowing sub-second increases in PI(4,5)P2 levels. By combining this compound with electrophysiological measurements in mouse adrenal chromaffin cells, we show that PI(4,5)P2 uncaging potentiates exocytosis and identify...... synaptotagmin-1 (the Ca(2+) sensor for exocytosis) and Munc13-2 (a vesicle priming protein) as the relevant effector proteins. PI(4,5)P2 activation of exocytosis did not depend on the PI(4,5)P2-binding CAPS-proteins, suggesting that PI(4,5)P2 uncaging bypasses CAPS-function. Finally, PI(4,5)P2 uncaging...

Functional paraganglioma extra-adrenal

International Nuclear Information System (INIS)

Arroyo-Martinez, Laura; Alvarez-Pertuz, Humberto; Acuna-Calvo, Jorge; Montoya-Calles, Juan Diego

2006-01-01

Functioning paragangliomas are rare tumours that produce catecholamines.They originate from extra-adrenal chromaffin cells. They are frequently malignant and are associated with high incidence of persistent or recurrent disease after their primary treatment. They are known as glomus, chemodectomas, chromaffin paragangliomas and glomerulocytomas. The location is diverse and reflects the paragangliomar distribution in the body from the base of the skull to the pelvic floor. The paragangliomas are found where there are nodes of the autonomous system, however, approximately 90% of these tumours appear in the adrenal glands (and they constitute the pheochromocytomas) and the remaining 10% is a location extra adrenal, but it has been said that its impact can be underestimated, ranging from 18% to 22% in adults and children up to 30%. The extra-adrenal are originated more frequently in the abdomen (85%), other in the chest (12%) and more rarely in the head and neck (3%). Imaging studies and measurement of non-physiological production of catecholamines may aid in the diagnosis of this entity. Surgery is the treatment of choice. It is presented the case of a primigravidas patient aged 32 with HTAIE requiring caesarean section, who had a postpartum torpid and despite to multiple antihypertensive treatments their pathology was difficult to deal, with ophthalmic complications. Some time later, the patient is studied by hyperhidrosis, laboratory tests and images are requested and it is documented incidentally, a left retroperitoneal tumour, the studies are expanded and reach the correct diagnosis. The tumour required surgical resection. The patient had a satisfactory postoperative period and she discharged with control in the external consultation. (author) [es

Retroperitoneal extraadrenal paraganglioma report of two cases.

Science.gov (United States)

Becheanu, G; Laky, D

1997-01-01

Two cases with retroperitoneal tumours affecting young women who cannot undergo surgery were investigated by biopsy and needle sampling. One of these cases presented a mainly alveolar histologic pattern and was easily diagnosed by immunohistochemical means, as well as a chromaffin paraganglioma. We discussed different diagnoses and approaches in the literature.

Protein kinase C regulates the activity of voltage-sensitive calcium channels of the rat chromaffin cells

International Nuclear Information System (INIS)

Wakade, A.R.; Malhotra, R.K.; Wakade, T.D.

1986-01-01

Phorbol dibutyrate (PB), an activator of protein kinase C was used as a tool to study the role of protein kinase C in the secretion of catecholamines (CA) from the perfused adrenal gland of rat. Secretion of CA evoked by splanchnic nerve stimulation, nicotine (N), carbamylcholine (C) and 35 mM K (K) was enhanced (about 2-fold) by 30 nM PB, but that evoked by muscarine (M) was not. In Ca-free and 1 mM EGTA Krebs solution, N and M did not evoke secretion, and PB also had no effect. If Ca concentration of the perfusion medium was maintained at 0.1 mM, N-evoked secretion was reduced over 80% but M-evoked secretion was still about 60% of the control value. Addition of PB to this medium did not modify secretion evoked by M, but N-evoked secretion was facilitated by 3-fold. Ca 45 flux data showed that N-, C-, and K-evoked secretion of CA was associated with 2- to 3-fold increase in Ca 45 uptake. However, M-evoked secretion did not cause Ca 45 uptake. These results suggest that N utilizes extracellular whereas M utilizes mostly intracellular Ca ions for the secretion of CA. PB alone did not affect Ca 45 uptake, but after stimulation with N, C and K, Ca 45 uptake was further enhanced by PB. It is concluded that protein kinase C phosphorylates membrane proteins that control opening and closing of Ca channels regulated by nicotine receptors and changes in membrane potentials

Glomus Tumor of the Larynx: A Rare Synchronous Paraganglioma in a Patient with Bilateral Carotid Body Tumor Detected on 68Ga-DOTANOC PET/CT.

Science.gov (United States)

Tripathy, Sarthak; Mukherjee, Anirban; Singh, Chirom A; Jat, Bhinyaram; Bal, Chandrasekhar; Shamim, Shamim Ahmed

2017-01-01

Paragangliomas are neoplasms arising from extra-adrenal chromaffin tissue. They frequently cause symptoms by overproduction of catecholamines with known predilection to multicentricity. We describe the case of a patient with bilateral carotid body tumor who underwent a baseline 68 Gallium labeled [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-1-NaI3-Octreotide.

Primary meningeal pheochromocytoma: a case report

Energy Technology Data Exchange (ETDEWEB)

Yoon, Il Ju; Suh, Hyoung Sim [Myung-Ji St. Mary' s Hospital, Seoul (Korea, Republic of); Kim, Sung Nam [Green Cross Reference Lab, Seoul (Korea, Republic of)

2007-04-15

Pheochromocytoma is a rare endocrine tumor arising from the chromaffin tissue, and it is able to produce and secrete catecholamines. Lymph nodes, liver, lung and bone are the most frequent sites of metastasis. We report here on a case of pheochromocytoma arising from the dura in a patient who was surgically treated for bilateral pheochromocytoma five years previously.

v-SNAREs control exocytosis of vesicles from priming to fusion.

Science.gov (United States)

Borisovska, Maria; Zhao, Ying; Tsytsyura, Yaroslav; Glyvuk, Nataliya; Takamori, Shigeo; Matti, Ulf; Rettig, Jens; Südhof, Thomas; Bruns, Dieter

2005-06-15

SNARE proteins (soluble NSF-attachment protein receptors) are thought to be central components of the exocytotic mechanism in neurosecretory cells, but their precise function remained unclear. Here, we show that each of the vesicle-associated SNARE proteins (v-SNARE) of a chromaffin granule, synaptobrevin II or cellubrevin, is sufficient to support Ca(2+)-dependent exocytosis and to establish a pool of primed, readily releasable vesicles. In the absence of both proteins, secretion is abolished, without affecting biogenesis or docking of granules indicating that v-SNAREs are absolutely required for granule exocytosis. We find that synaptobrevin II and cellubrevin differentially control the pool of readily releasable vesicles and show that the v-SNARE's amino terminus regulates the vesicle's primed state. We demonstrate that dynamics of fusion pore dilation are regulated by v-SNAREs, indicating their action throughout exocytosis from priming to fusion of vesicles.

Synthesis, biological assessment and molecular modeling of new dihydroquinoline-3-carboxamides and dihydroquinoline-3-carbohydrazide derivatives as cholinesterase inhibitors, and Ca channel antagonists.

Science.gov (United States)

Tomassoli, Isabelle; Ismaili, Lhassane; Pudlo, Marc; de Los Ríos, Cristóbal; Soriano, Elena; Colmena, Inés; Gandía, Luis; Rivas, Luis; Samadi, Abdelouahid; Marco-Contelles, José; Refouvelet, Bernard

2011-01-01

The synthesis, biological evaluation, and molecular modeling of new 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamides(4), 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbohydrazide (6), and some hexahydropyrimido[5,4-c]quinoline-2,5-diones (9) produced earlier by our laboratory, as AChE/BuChE inhibitors, is described. From these analyses compound 4c resulted equipotent regarding the inhibition of cholinesterases'; inhibitors 6k, 9a, 9b were selective for AChE, whereas product 4d proved selective for BuChE. Docking analysis has been carry out in order to identify the binding mode in the active site, and to explain the observed selectivities. Only compound 9a has been shown to decrease K(+)-induced calcium signals in bovine chromaffin cells. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Characterization and metabolic synthetic lethal testing in a new model of SDH-loss familial pheochromocytoma and paraganglioma.

Science.gov (United States)

Smestad, John; Hamidi, Oksana; Wang, Lin; Holte, Molly Nelson; Khazal, Fatimah Al; Erber, Luke; Chen, Yue; Maher, L James

2018-01-19

Succinate dehydrogenase (SDH)-loss pheochromocytoma and paraganglioma (PPGL) are tumors driven by metabolic derangement. SDH loss leads to accumulation of intracellular succinate, which competitively inhibits dioxygenase enzymes, causing activation of pseudohypoxic signaling and hypermethylation of histones and DNA. The mechanisms by which these alterations lead to tumorigenesis are unclear, however. In an effort to fundamentally understand how SDH loss reprograms cell biology, we developed an immortalized mouse embryonic fibroblast cell line with conditional disruption of Sdhc and characterize the kinetics of Sdhc gene rearrangement, SDHC protein loss, succinate accumulation, and the resultant hypoproliferative phenotype. We further perform global transcriptomic, epigenomic, and proteomic characterization of changes resulting from SDHC loss, identifying specific perturbations at each biological level. We compare the observed patterns of epigenomic derangement to another previously-described immortalized mouse chromaffin cell model of SDHB loss, and compare both models to human SDH-loss tumors. Finally, we perform analysis of SDHC synthetic lethality with lactate dehydrogenase A (LDHA) and pyruvate carboxylase (PCX), which are important for regeneration of NAD+ and aspartate biosynthesis, respectively. Our data show that SDH-loss cells are selectively vulnerable to LDH genetic knock-down or chemical inhibition, suggesting that LDH inhibition may be an effective therapeutic strategy for SDH-loss PPGL.

Optical modulation of neurotransmission using calcium photocurrents through the ion channel LiGluR

Directory of Open Access Journals (Sweden)

Mercè eIzquierdo-Serra

2013-03-01

Full Text Available A wide range of light-activated molecules (photoswitches and phototriggers have been used to the study of computational properties of an isolated neuron by acting pre and postsynaptically. However, new tools are being pursued to elicit a presynaptic calcium influx that triggers the release of neurotransmitters, most of them based in calcium-permeable Channelrhodopsin-2 mutants. Here we describe a method to control exocytosis of synaptic vesicles through the use of a light-gated glutamate receptor (LiGluR, which has recently been demonstrated that supports secretion by means of calcium influx in chromaffin cells. Expression of LiGluR in hippocampal neurons enables reversible control of neurotransmission with light, and allows modulating the firing rate of the postsynaptic neuron with the wavelength of illumination. This method may be useful for the determination of the complex transfer function of individual synapses.

Pheochromocytoma: a rare cause of childhood hypertensive encephalopathy

International Nuclear Information System (INIS)

Aftab, S.; Yasmeen, T.; Hamid, M.H.; Sarwar, M.; Sipra, H.; Sheikh, A.; Haider, N.; Hanif, G.

2012-01-01

Pheochromocytomas are rare neuroendocrine tumours of chromaffin tissues. They are catecholamine secreting tumours which cause severe hypertension and other systemic disturbances. Of all the causes of childhood hypertension, pheochromocytoma constitutes less than 1%. We report the case of a 12 years old child who presented with hypertensive encephalopathy, confirmed histologically to be secondary to pheochromocytoma, and cured with meticulous critical care and surgical resection. (author)

Phosphopeptidomics Reveals Differential Phosphorylation States and Novel SxE Phosphosite Motifs of Neuropeptides in Dense Core Secretory Vesicles

Science.gov (United States)

Lietz, Christopher B.; Toneff, Thomas; Mosier, Charles; Podvin, Sonia; O'Donoghue, Anthony J.; Hook, Vivian

2018-03-01

Neuropeptides are vital for cell-cell communication and function in the regulation of the nervous and endocrine systems. They are generated by post-translational modification (PTM) steps resulting in small active peptides generated from prohormone precursors. Phosphorylation is a significant PTM for the bioactivity of neuropeptides. From the known diversity of distinct neuropeptide functions, it is hypothesized that the extent of phosphorylation varies among different neuropeptides. To assess this hypothesis, neuropeptide-containing dense core secretory vesicles from bovine adrenal medullary chromaffin cells were subjected to global phosphopeptidomics analyses by liquid chromatography (LC)-mass spectrometry (MS/MS). Phosphopeptides were identified directly by LC-MS/MS and indirectly by phosphatase treatment followed by LC-MS/MS. The data identified numerous phosphorylated peptides derived from neuropeptide precursors such as chromogranins, secretogranins, proenkephalin and pro-NPY. Phosphosite occupancies were observed at high and low levels among identified peptides and many of the high occupancy phosphopeptides represent prohormone-derived peptides with currently unknown bioactivities. Peptide sequence analyses demonstrated SxE as the most prevalent phosphorylation site motif, corresponding to phosphorylation sites of the Fam20C protein kinase known to be present in the secretory pathway. The range of high to low phosphosite occupancies for neuropeptides demonstrates cellular regulation of neuropeptide phosphorylation. [Figure not available: see fulltext.

Immunogold staining procedure for the localisation of regulatory peptides.

Science.gov (United States)

Varndell, I M; Tapia, F J; Probert, L; Buchan, A M; Gu, J; De Mey, J; Bloom, S R; Polak, J M

1982-01-01

The use of protein A- and IgG-conjugated colloidal gold staining methods for the immuno-localisation of peptide hormones and neurotransmitters at light- and electron microscope level are described and discussed. Bright-field and dark-ground illumination modes have been used to visualise the gold-labelled antigenic sites at the light microscope level. Immunogold staining procedures at the ultrastructural level using region-specific antisera have been adopted to localise specific molecular forms of peptides including gastrin (G17 and G34), glucagon and pro-glucagon, insulin and pro-insulin, in normal tissue and in tumours of the gastroenteropancreatic system. Similar methods have been used to demonstrate the heterogeneity of p-type nerves in the enteric nervous system. Vasoactive intestinal polypeptide (VIP) has been localised to granular sites (mean +/- S.D. granule diameter = 98 +/- 19 nm) in nerve terminals of the enteric plexuses and in tumour cells of diarrhoeogenic VIP-producing neoplasias (mean +/- S.D. granule diameter = 126 +/- 37 nm) using immunogold procedures applied to ultraviolet-cured ultrathin sections. Co-localisation of amines and peptides in carotid body type I cells and in chromaffin cells of normal adrenal medulla and phaeochromocytomas has also been demonstrated. Advantages of the immunogold procedures over alternative immunocytochemical techniques are discussed.

Phosphopeptidomics Reveals Differential Phosphorylation States and Novel SxE Phosphosite Motifs of Neuropeptides in Dense Core Secretory Vesicles

Science.gov (United States)

Lietz, Christopher B.; Toneff, Thomas; Mosier, Charles; Podvin, Sonia; O'Donoghue, Anthony J.; Hook, Vivian

2018-05-01

Neuropeptides are vital for cell-cell communication and function in the regulation of the nervous and endocrine systems. They are generated by post-translational modification (PTM) steps resulting in small active peptides generated from prohormone precursors. Phosphorylation is a significant PTM for the bioactivity of neuropeptides. From the known diversity of distinct neuropeptide functions, it is hypothesized that the extent of phosphorylation varies among different neuropeptides. To assess this hypothesis, neuropeptide-containing dense core secretory vesicles from bovine adrenal medullary chromaffin cells were subjected to global phosphopeptidomics analyses by liquid chromatography (LC)-mass spectrometry (MS/MS). Phosphopeptides were identified directly by LC-MS/MS and indirectly by phosphatase treatment followed by LC-MS/MS. The data identified numerous phosphorylated peptides derived from neuropeptide precursors such as chromogranins, secretogranins, proenkephalin and pro-NPY. Phosphosite occupancies were observed at high and low levels among identified peptides and many of the high occupancy phosphopeptides represent prohormone-derived peptides with currently unknown bioactivities. Peptide sequence analyses demonstrated SxE as the most prevalent phosphorylation site motif, corresponding to phosphorylation sites of the Fam20C protein kinase known to be present in the secretory pathway. The range of high to low phosphosite occupancies for neuropeptides demonstrates cellular regulation of neuropeptide phosphorylation. [Figure not available: see fulltext.

Relative mitochondrial membrane potential and [Ca2+]i in type I cells isolated from the rabbit carotid body.

Science.gov (United States)

Duchen, M R; Biscoe, T J

1992-05-01

psi m closely followed changes in PO2 with time. 5. The rate of rise of [Ca2+]i in response to anoxia is strongly temperature sensitive. The rate of depolarization of delta psi m with anoxia similarly increased at least two- to fivefold on warming from 22 to 36 degrees C. The change with FCCP was not significantly altered by temperature. 6. These data show that the mitochondrial membrane potential changes over a physiological range of PO2 values in type I cells. This contrasts with the behaviour in dissociated chromaffin cells and sensory neurons, in which no change was measurable until the PO2 fell close to zero.(ABSTRACT TRUNCATED AT 400 WORDS)

Doc2B acts as a calcium sensor for vesicle priming requiring synaptotagmin-1, Munc13-2 and SNAREs

DEFF Research Database (Denmark)

Houy, Sébastien; Groffen, Alexander J; Ziomkiewicz, Iwona

2017-01-01

Doc2B is a cytosolic protein with binding sites for Munc13 and Tctex-1 (dynein light chain), and two C2-domains that bind to phospholipids, Ca2+ and SNAREs. Whether Doc2B functions as a calcium sensor akin to synaptotagmins, or in other calcium-independent or calcium-dependent capacities is debated....... We here show by mutation and overexpression that Doc2B plays distinct roles in two sequential priming steps in mouse adrenal chromaffin cells. Mutating Ca2+-coordinating aspartates in the C2A-domain localizes Doc2B permanently at the plasma membrane, and renders an upstream priming step Ca2......+-independent, whereas a separate function in downstream priming depends on SNARE-binding, Ca2+-binding to the C2B-domain of Doc2B, interaction with ubMunc13-2 and the presence of synaptotagmin-1. Another function of Doc2B - inhibition of release during sustained calcium elevations - depends on an overlapping...

Developmental programming of O2 sensing by neonatal intermittent hypoxia via epigenetic mechanisms

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Nanduri, Jayasri; Prabhakar, Nanduri R.

2014-01-01

Recurrent apnea with intermittent hypoxia (IH) is a major clinical problem in infants born preterm. Carotid body chemo-reflex and catecholamine secretion from adrenal medullary chromaffin cells (AMC) are important for maintenance of cardio-respiratory homeostasis during hypoxia. This article highlights studies on the effects of IH on O2 sensing by the carotid body and AMC in neonatal rodents. Neonatal IH augments hypoxia-evoked carotid body sensory excitation and catecholamine secretion from AMC which are mediated by reactive oxygen species (ROS)-dependent recruitment of endothelin-1 and Ca2+ signaling, respectively. The effects of neonatal IH persist into adulthood. Evidence is emerging that neonatal IH initiates epigenetic mechanisms involving DNA hypermethylation contributing to long-lasting increase in ROS levels. Since adult human subjects born preterm exhibit higher incidence of sleep-disordered breathing and hypertension, DNA hypomethylating agents might offer a novel therapeutic intervention to decrease long-term cardio-respiratory morbidity caused by neonatal IH. PMID:22846496

Developmental programming of O(2) sensing by neonatal intermittent hypoxia via epigenetic mechanisms.

Science.gov (United States)

Nanduri, Jayasri; Prabhakar, Nanduri R

2013-01-01

Recurrent apnea with intermittent hypoxia (IH) is a major clinical problem in infants born preterm. Carotid body chemo-reflex and catecholamine secretion from adrenal medullary chromaffin cells (AMC) are important for maintenance of cardio-respiratory homeostasis during hypoxia. This article highlights studies on the effects of IH on O(2) sensing by the carotid body and AMC in neonatal rodents. Neonatal IH augments hypoxia-evoked carotid body sensory excitation and catecholamine secretion from AMC which are mediated by reactive oxygen species (ROS)-dependent recruitment of endothelin-1 and Ca(2+) signaling, respectively. The effects of neonatal IH persist into adulthood. Evidence is emerging that neonatal IH initiates epigenetic mechanisms involving DNA hypermethylation contributing to long-lasting increase in ROS levels. Since adult human subjects born preterm exhibit higher incidence of sleep-disordered breathing and hypertension, DNA hypomethylating agents might offer a novel therapeutic intervention to decrease long-term cardio-respiratory morbidity caused by neonatal IH. Copyright © 2012. Published by Elsevier B.V.

Identification of a membrane-bound, glycol-stimulated phospholipase A2 located in the secretory granules of the adrenal medulla

International Nuclear Information System (INIS)

Hildebrandt, E.; Albanesi, J.P.

1991-01-01

Chromaffin granule membranes prepared from bovine adrenal medullae showed Ca 2+ -stimulated phospholipase A 2 (PLA 2 ) activity when assayed at pH 9.0 with phosphatidylcholine containing an [ 14 C]-arachidonyl group in the 2-position. However, the activity occurred in both soluble and particulate subcellular fractions, and did not codistribute with markers for the secretory granule. PLA 2 activity in the granule membrane preparation was stimulated dramatically by addition of glycerol, ethylene glycole, or poly(ethylene glycol). This glycol-stimulated PLA 2 activity codistributed with membrane-bound dopamine β-hydroxylase, a marker for the granule membranes, through the sequence of differential centrifugation steps employed to prepare the granule membrane fraction, as well as on a sucrose density gradient which resolved the granules from mitochondria, lysosomes, and plasma membrane. The glycol-stimulated PLA 2 of the chromaffin granule was membrane-bound, exhibited a pH optimum of 7.8, retained activity in the presence of EDTA, and was inactivated by p-bromophenacyl bromide. When different 14 C-labeled phospholipids were incorporated into diarachidonylphosphatidylcholine liposomes, 1-palmitoyl-2-arachidonylphosphatidylcholine was a better substrate for this enzyme than 1-palmitoyl-2-oleylphosphatidylcholine or 1-acyl-2-arachidonyl-phosphatidylethhanolamine, and distearoylphosphatidylcholine was not hydrolyzed

The surging role of Chromogranin A in cardiovascular homeostasis

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Tota, Bruno; Angelone, Tommaso; Cerra, Maria

2014-08-01

Together with Chromogranin B and Secretogranins, Chromogranin A (CGA) is stored in secretory (chromaffin) granules of the diffuse neuroendocrine system and released with noradrenalin and adrenalin. Co-stored within the granule together with neuropeptideY, cardiac natriuretic peptide hormones, several prohormones and their proteolytic enzymes, CGA is a multifunctional protein and a major marker of the sympatho-adrenal neuroendocrine activity. Due to its partial processing to several biologically active peptides, CGA appears an important pro-hormone implicated in relevant modulatory actions on endocrine, cardiovascular, metabolic, and immune systems through both direct and indirect sympatho-adrenergic interactions. As a part of this scenario, we here illustrate the emerging role exerted by the full-length CGA and its three derived fragments, i.e. Vasostatin 1, catestatin and serpinin, in the control of circulatory homeostasis with particular emphasis on their cardio-vascular actions under both physiological and physio-pathological conditions. The Vasostatin 1- and catestatin-induced cardiodepressive influences are achieved through anti-beta-adrenergic-NO-cGMP signalling, while serpinin acts like beta1-adrenergic agonist through AD-cAMP-independent NO signalling. On the whole, these actions contribute to wide our knowledge regarding the sympatho-chromaffin control of the cardiovascular system and its highly integrated “whip-brake” networks.

[Alfred Kohn, professor of histology at German University in Prague].

Science.gov (United States)

Nanka, O; Grim, M

2008-01-01

Prof. Kohn (1867-1959) was the head of the Institute of Histology at the Medical Faculty of German University in Prague for 26 years. In 2007 we commemorated his 140th birthday, and 2009 we will remember the 50th anniversary of his death. He entered the history of medicine by discovery of nature and origin of parathyroid glands and by pioneer research into chromaffin cells and sympathetic paraganglia. Kohn's papers on the pituitary, interstitial cells of testes, and ovaries are also related to endocrinology. All his studies are based on descriptive and comparative histological and embryological observations. Kohn was twice the dean of German Medical Faculty, and a member or honorary member of many important scientific societies. He was repeatedly nominated for Nobel Prize for physiology and medicine. For his Jewish origin he was expelled from Deutsche Gesellschaft der Wissenschaften und Künste für die Tschechoslowakische Republik in 1939 and transported to Terezin ghetto in 1943. After the war he lived in Prague. On the occasion of his 90th birthday he was elected honorary president of Anatomische Gesellschaft and awarded by the Czechoslovak Order of Labour. Alfred Kohn died in 1959. He was one of the outstanding personalities that Prague gave to the world of science.

The V-ATPase membrane domain is a sensor of granular pH that controls the exocytotic machinery.

Science.gov (United States)

Poëa-Guyon, Sandrine; Ammar, Mohamed Raafet; Erard, Marie; Amar, Muriel; Moreau, Alexandre W; Fossier, Philippe; Gleize, Vincent; Vitale, Nicolas; Morel, Nicolas

2013-10-28

Several studies have suggested that the V0 domain of the vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) is directly implicated in secretory vesicle exocytosis through a role in membrane fusion. We report in this paper that there was a rapid decrease in neurotransmitter release after acute photoinactivation of the V0 a1-I subunit in neuronal pairs. Likewise, inactivation of the V0 a1-I subunit in chromaffin cells resulted in a decreased frequency and prolonged kinetics of amperometric spikes induced by depolarization, with shortening of the fusion pore open time. Dissipation of the granular pH gradient was associated with an inhibition of exocytosis and correlated with the V1-V0 association status in secretory granules. We thus conclude that V0 serves as a sensor of intragranular pH that controls exocytosis and synaptic transmission via the reversible dissociation of V1 at acidic pH. Hence, the V-ATPase membrane domain would allow the exocytotic machinery to discriminate fully loaded and acidified vesicles from vesicles undergoing neurotransmitter reloading.

Plasma metanephrines: a novel and cost-effective test for pheochromocytoma

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G. Eisenhofer

2000-10-01

Full Text Available Pheochromocytomas are rare chromaffin cell tumors that nevertheless must be excluded in large numbers of patients who develop sustained or episodic hypertension as well as in many others with suggestive symptoms or with a familial history of pheochromocytoma. Diagnosis of pheochromocytoma depends importantly on biochemical evidence of excess catecholamine production by a tumor. Imperfect sensitivity and specificity of commonly available biochemical tests and the low incidence of the tumor among the tested population mean that considerable time and effort can be expended in confirming or ruling out pheochromocytoma in patients where the tumor is suspected. Measurements of plasma free metanephrines provide a superior test compared to other available tests for diagnosis of pheochromocytoma. In particular, the high sensitivity of plasma free metanephrines means that a normal test result reliably excludes all but the smallest of pheochromocytomas so that no other tests are necessary. Measurements of plasma free metanephrines, when systematically combined with other diagnostic procedures outlined in this review, provide a more efficient, reliable and cost-effective approach for diagnosis of pheochromocytoma than offered by previously available approaches.

Mitochondrial function in type I cells isolated from rabbit arterial chemoreceptors.

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Duchen, M R; Biscoe, T J

1992-05-01

activation could last several minutes following only 100 ms depolarization, while the changes accompanying hypoxia closely followed the time course of the change in PO2. 7. In similarly isolated rat or mouse chromaffin cells and mouse dorsal root ganglion neurons under identical conditions, no measurable change in autofluorescence or in [Ca2+]i was seen until the PO2 fell below about 5 mmHg. 8. Carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) increases O2 consumption, oxidizing mitochondrial NADH and hence decreasing autofluorescence, (delta FFCCP). Blockade of electron transport by anoxia or CN- decreases O2 consumption, increasing mitochondrial NADH/NAD and autofluorescence (delta FCN). The fractional change in autofluorescence with FCCP, delta FFCCP/delta FFCCP+FCN), is thus a measure of resting O2 consumption.(ABSTRACT TRUNCATED AT 400 WORDS)

Reversible catecholamine-induced cardiomyopathy due to pheochromocytoma: case report.

Science.gov (United States)

Satendra, Milan; de Jesus, Cláudia; Bordalo e Sá, Armando L; Rosário, Luís; Rocha, José; Bicha Castelo, Henrique; Correia, Maria José; Nunes Diogo, António

2014-03-01

Pheochromocytoma is a tumor originating from chromaffin tissue. It commonly presents with symptoms and signs of catecholamine excess, such as hypertension, tachycardia, headache and sweating. Cardiovascular manifestations include catecholamine-induced cardiomyopathy, which may present as severe left ventricular dysfunction and congestive heart failure. We report a case of pheochromocytoma which was diagnosed following investigation of dilated cardiomyopathy. We highlight the dramatic symptomatic improvement and reversal of cardiomyopathy, with recovery of left ventricular function after treatment. Copyright © 2013 Sociedade Portuguesa de Cardiologia. Published by Elsevier España. All rights reserved.

Biological indicators of stress in pacu (Piaractus mesopotamicus after capture

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JS. Abreu

Full Text Available The effects of capture (chasing, netting and air exposure on cortisol, glucose, chloride, sodium, potassium and calcium concentrations, osmolality, hematocrit, hemoglobin concentration, red blood cells count (RBC and mean corpuscular volume (MCV were investigated in pacu (Piaractus mesopotamicus. A total of 132 fish (49.7 ± 11.7 g were subjected to capture and 3 minutes air exposure and capture and 5 minutes air exposure. Nine fish at each treatment were sampled at 5, 15, 30, 60 minutes and 24 hours after the procedure. Nine undisturbed fish were sacrificed before the handling and used as controls. Capture resulted in a rise in blood cortisol and glucose 30 and 5 minutes, respectively, after both air exposures. Both indicators returned to resting levels 24 hours after capture. In both fish groups, plasma chloride decreased 60 minutes after capture, not recovering the resting levels within 24 hours after, and serum sodium rose at 15 and 30 minutes and recovered the resting levels 24 hours later. There were no significant changes neither in potassium, calcium and osmolality nor in hematocrit, hemoglobin, RBC and MCV as a consequence of capture. The sequential stressors imposed to pacu during capture activated the brain-pituitary-interrenal axis (cortisol and glucose responses but the activation of the brain-sympathetic-chromaffin cell axis was apparently moderate (ionic and hematological responses.

Epigenetic regulation of hypoxic sensing disrupts cardiorespiratory homeostasis.

Science.gov (United States)

Nanduri, Jayasri; Makarenko, Vladislav; Reddy, Vaddi Damodara; Yuan, Guoxiang; Pawar, Anita; Wang, Ning; Khan, Shakil A; Zhang, Xin; Kinsman, Brian; Peng, Ying-Jie; Kumar, Ganesh K; Fox, Aaron P; Godley, Lucy A; Semenza, Gregg L; Prabhakar, Nanduri R

2012-02-14

Recurrent apnea with intermittent hypoxia is a major clinical problem in preterm infants. Recent studies, although limited, showed that adults who were born preterm exhibit increased incidence of sleep-disordered breathing and hypertension, suggesting that apnea of prematurity predisposes to autonomic dysfunction in adulthood. Here, we demonstrate that adult rats that were exposed to intermittent hypoxia as neonates exhibit exaggerated responses to hypoxia by the carotid body and adrenal chromaffin cells, which regulate cardio-respiratory function, resulting in irregular breathing with apneas and hypertension. The enhanced hypoxic sensitivity was associated with elevated oxidative stress, decreased expression of genes encoding antioxidant enzymes, and increased expression of pro-oxidant enzymes. Decreased expression of the Sod2 gene, which encodes the antioxidant enzyme superoxide dismutase 2, was associated with DNA hypermethylation of a single CpG dinucleotide close to the transcription start site. Treating neonatal rats with decitabine, an inhibitor of DNA methylation, during intermittent hypoxia exposure prevented oxidative stress, enhanced hypoxic sensitivity, and autonomic dysfunction. These findings implicate a hitherto uncharacterized role for DNA methylation in mediating neonatal programming of hypoxic sensitivity and the ensuing autonomic dysfunction in adulthood.

Pheochromocytoma/Paraganglioma: A Poster Child for Cancer Metabolism.

Science.gov (United States)

Tevosian, Sergei G; Ghayee, Hans K

2018-05-01

Pheochromocytomas (PCCs) are tumors that are derived from the chromaffin cells of the adrenal medulla. Extra-adrenal PCCs called paragangliomas (PGLs) are derived from the sympathetic and parasympathetic chain ganglia. PCCs secrete catecholamines, which cause hypertension and have adverse cardiovascular consequences as a result of catecholamine excess. PGLs may or may not produce catecholamines depending on their genetic type and anatomical location. The most worrisome aspect of these tumors is their ability to become aggressive and metastasize; there are no known cures for metastasized PGLs. Original articles and reviews indexed in PubMed were identified by querying with specific PCC/PGL- and Krebs cycle pathway-related terms. Additional references were selected through the in-depth analysis of the relevant publications. We primarily discuss Krebs cycle mutations that can be instrumental in helping investigators identify key biological pathways and molecules that may serve as biomarkers of or treatment targets for PCC/PGL. The mainstay of treatment of patients with PCC/PGLs is surgical. However, the tide may be turning with the discovery of new genes associated with PCC/PGLs that may shed light on oncometabolites used by these tumors.

Calpain activation by ROS mediates human ether-a-go-go-related gene protein degradation by intermittent hypoxia.

Science.gov (United States)

Wang, N; Kang, H S; Ahmmed, G; Khan, S A; Makarenko, V V; Prabhakar, N R; Nanduri, J

2016-03-01

Human ether-a-go-go-related gene (hERG) channels conduct delayed rectifier K(+) current. However, little information is available on physiological situations affecting hERG channel protein and function. In the present study we examined the effects of intermittent hypoxia (IH), which is a hallmark manifestation of sleep apnea, on hERG channel protein and function. Experiments were performed on SH-SY5Y neuroblastoma cells, which express hERG protein. Cells were exposed to IH consisting of alternating cycles of 30 s of hypoxia (1.5% O2) and 5 min of 20% O2. IH decreased hERG protein expression in a stimulus-dependent manner. A similar reduction in hERG protein was also seen in adrenal medullary chromaffin cells from IH-exposed neonatal rats. The decreased hERG protein was associated with attenuated hERG K(+) current. IH-evoked hERG protein degradation was not due to reduced transcription or increased proteosome/lysomal degradation. Rather it was mediated by calcium-activated calpain proteases. Both COOH- and NH2-terminal sequences of the hERG protein were the targets of calpain-dependent degradation. IH increased reactive oxygen species (ROS) levels, intracellular Ca(2+) concentration ([Ca(2+)]i), calpain enzyme activity, and hERG protein degradation, and all these effects were prevented by manganese-(111)-tetrakis-(1-methyl-4-pyridyl)-porphyrin pentachloride, a membrane-permeable ROS scavenger. These results demonstrate that activation of calpains by ROS-dependent elevation of [Ca(2+)]i mediates hERG protein degradation by IH. Copyright © 2016 the American Physiological Society.

Stretch-induced Ca2+ independent ATP release in hippocampal astrocytes.

Science.gov (United States)

Xiong, Yingfei; Teng, Sasa; Zheng, Lianghong; Sun, Suhua; Li, Jie; Guo, Ning; Li, Mingli; Wang, Li; Zhu, Feipeng; Wang, Changhe; Rao, Zhiren; Zhou, Zhuan

2018-02-28

Similar to neurons, astrocytes actively participate in synaptic transmission via releasing gliotransmitters. The Ca 2+ -dependent release of gliotransmitters includes glutamate and ATP. Following an 'on-cell-like' mechanical stimulus to a single astrocyte, Ca 2+ independent single, large, non-quantal, ATP release occurs. Astrocytic ATP release is inhibited by either selective antagonist treatment or genetic knockdown of P2X7 receptor channels. Our work suggests that ATP can be released from astrocytes via two independent pathways in hippocampal astrocytes; in addition to the known Ca 2+ -dependent vesicular release, larger non-quantal ATP release depends on P2X7 channels following mechanical stretch. Astrocytic ATP release is essential for brain functions such as synaptic long-term potentiation for learning and memory. However, whether and how ATP is released via exocytosis remains hotly debated. All previous studies of non-vesicular ATP release have used indirect assays. By contrast, two recent studies report vesicular ATP release using more direct assays. In the present study, using patch clamped 'ATP-sniffer cells', we re-investigated astrocytic ATP release at single-vesicle resolution in hippocampal astrocytes. Following an 'on-cell-like' mechanical stimulus of a single astrocyte, a Ca 2+ independent single large non-quantal ATP release occurred, in contrast to the Ca 2+ -dependent multiple small quantal ATP release in a chromaffin cell. The mechanical stimulation-induced ATP release from an astrocyte was inhibited by either exposure to a selective antagonist or genetic knockdown of P2X7 receptor channels. Functional P2X7 channels were expressed in astrocytes in hippocampal brain slices. Thus, in addition to small quantal ATP release, larger non-quantal ATP release depends on P2X7 channels in astrocytes. © 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.

"Slow" Voltage-Dependent Inactivation of CaV2.2 Calcium Channels Is Modulated by the PKC Activator Phorbol 12-Myristate 13-Acetate (PMA.

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Lei Zhu

Full Text Available CaV2.2 (N-type voltage-gated calcium channels (Ca2+ channels play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. "Fast" voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of "slow" voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate "slow" inactivation of sodium channels, but little is known about if/how second messengers control "slow" inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA dramatically prolonged recovery from "slow" inactivation, but an inactive control (4α-PMA had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel β-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-β-S reduced the effect of PMA suggesting a role for G proteins in modulating "slow" inactivation. We postulate that the kinetics of recovery from "slow" inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.

Inclusões intracitoplasmáticas hialinas na medular da adrenal de bovinos

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L.P Mesquita

2011-02-01

Full Text Available Cytoplasmic inclusion bodies in adrenal medullary chromaffin cells have been described in various species including humans. These inclusions are believed to be related to certain infectious, toxic and neurodegenerative diseases. No reports concerning such adrenal inclusions have been described in bovines. Adrenal glands from twenty bovines were evaluated in a retrospective study. Seven of these exhibited inclusions - three cases of rabies, two cases of chronic suppurative bronchopneumonia, one case of chronic suppurative peritonitis, and one case of gangrenous mastitis. The inclusions were present in higher numbers especially in cases of rabies and also in one case of chronic suppurative bronchopneumonia. The inclusions were intracytoplasmic, eosinophilic, rounded, single or multiple, of various sizes, strongly stained by PAS and were present in higher numbers in the external layer of the adrenal medulla. The inclusions were negative when subjected to immunohistochemistry for detection of viral antigens in the cases of rabies. Although inclusion bodies were present in adrenal glands devoid of other histological alterations, they were more abundant in cases in which the adrenal gland had other alterations. The correlation between certain diseases and the development of inclusion bodies is not known, which highlights the importance of further studies on these inclusions in adrenal glands of bovines.

Epigenetic Regulation of Carotid Body Oxygen Sensing: Clinical Implications.

Science.gov (United States)

Nanduri, Jayasri; Prabhakar, Nanduri R

2015-01-01

Recurrent apnea with intermittent hypoxia (IH) is a major clinical problem in infants born preterm. Recent epidemiological studies showed that adults who were born preterm exhibit increased incidence of sleep-disordered breathing and hypertension. Thus, apnea of prematurity predisposes individuals to autonomic dysfunction in adulthood. Experimental studies showed that adult rats exposed to IH as neonates exhibit augmented carotid body and adrenal chromaffin cells (AMC) response to hypoxia and irregular breathing with apneas and hypertension. The enhanced hypoxic sensitivity of the carotid body and AMC in adult rats exposed to neonatal IH was associated with increased oxidative stress, decreased expression of genes encoding anti-oxidant enzymes, and increased expression of pro-oxidant enzymes. Epigenetic mechanisms including DNA methylation leads to long-term changes in gene expression. The decreased expression of the Sod2 gene, which encodes the anti-oxidant enzyme, superoxide dismutase 2, was associated with DNA hypermethylation of a single CpG dinucleotide close to the transcription start site. Treating neonatal rats with decitabine, an inhibitor of DNA methylation, during IH exposure prevented the oxidative stress, enhanced hypoxic sensitivity, and autonomic dysfunction in adult rats. These findings suggest that epigenetic mechanisms, especially DNA methylation contributes to neonatal programming of hypoxic sensitivity and the ensuing autonomic dysfunction in adulthood.

Anesthesia Management for Pheochromocytoma Removal in a 17-Year-Old Girl: Case Report

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Gulsen Keskin

2018-03-01

Full Text Available A rare tumor of the chromaffin tissue, pheochromocytoma is characterized by increased secretion of catecholamines. Pediatric cases represent only 5% of all pheochromocytomas. In this report, we presented anesthesia management of a 17-year-old girl who would undergo right suprarenal mass excision due to pheochromocytoma accompanied with familial Mediterranean fever (FMF. Preoperative blood pressure control was achieved with phenoxybenzamine. General anesthesia was established with thiopental, vecuronium, fentanyl, and sevoflurane. Sodium nitroprusside and phentolamine was used for perioperative blood pressure control. Tramadol and diclofenac were administered for postoperative pain. The patient was discharged after a good preoperative preparation and perioperative management with uneventful treatment period. [J Contemp Med 2018; 8(1.000: 70-73

An inhibitory effect of extracellular Ca2+ on Ca2+-dependent exocytosis.

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Wei Xiong

Full Text Available AIM: Neurotransmitter release is elicited by an elevation of intracellular Ca(2+ concentration ([Ca(2+](i. The action potential triggers Ca(2+ influx through Ca(2+ channels which causes local changes of [Ca(2+](i for vesicle release. However, any direct role of extracellular Ca(2+ (besides Ca(2+ influx on Ca(2+-dependent exocytosis remains elusive. Here we set out to investigate this possibility on rat dorsal root ganglion (DRG neurons and chromaffin cells, widely used models for studying vesicle exocytosis. RESULTS: Using photolysis of caged Ca(2+ and caffeine-induced release of stored Ca(2+, we found that extracellular Ca(2+ inhibited exocytosis following moderate [Ca(2+](i rises (2-3 µM. The IC(50 for extracellular Ca(2+ inhibition of exocytosis (ECIE was 1.38 mM and a physiological reduction (∼30% of extracellular Ca(2+ concentration ([Ca(2+](o significantly increased the evoked exocytosis. At the single vesicle level, quantal size and release frequency were also altered by physiological [Ca(2+](o. The calcimimetics Mg(2+, Cd(2+, G418, and neomycin all inhibited exocytosis. The extracellular Ca(2+-sensing receptor (CaSR was not involved because specific drugs and knockdown of CaSR in DRG neurons did not affect ECIE. CONCLUSION/SIGNIFICANCE: As an extension of the classic Ca(2+ hypothesis of synaptic release, physiological levels of extracellular Ca(2+ play dual roles in evoked exocytosis by providing a source of Ca(2+ influx, and by directly regulating quantal size and release probability in neuronal cells.

Molecular and therapeutic advances in the diagnosis and management of malignant pheochromocytomas and paragangliomas.

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Lowery, Aoife J

2013-01-01

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare catecholamine-secreting tumors derived from chromaffin cells originating in the neural crest. These tumors represent a significant diagnostic and therapeutic challenge because the diagnosis of malignancy is frequently made in retrospect by the development of metastatic or recurrent disease. Complete surgical resection offers the only potential for cure; however, recurrence can occur even after apparently successful resection of the primary tumor. The prognosis for malignant disease is poor because traditional treatment modalities have been limited. The last decade has witnessed exciting discoveries in the study of PCCs and PGLs; advances in molecular genetics have uncovered hereditary and germline mutations of at least 10 genes that contribute to the development of these tumors, and increasing knowledge of genotype-phenotype interactions has facilitated more accurate determination of malignant potential. Elucidating the molecular mechanisms responsible for malignant transformation in these tumors has opened avenues of investigation into targeted therapeutics that show promising results. There have also been significant advances in functional and radiological imaging and in the surgical approach to adrenalectomy, which remains the mainstay of treatment for PCC. In this review, we discuss the currently available diagnostic and therapeutic options for patients with malignant PCCs and PGLs and detail the molecular rationale and clinical evidence for novel and emerging diagnostic and therapeutic strategies.

Conotoxins as Tools to Understand the Physiological Function of Voltage-Gated Calcium (CaV Channels

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David Ramírez

2017-10-01

Full Text Available Voltage-gated calcium (CaV channels are widely expressed and are essential for the completion of multiple physiological processes. Close regulation of their activity by specific inhibitors and agonists become fundamental to understand their role in cellular homeostasis as well as in human tissues and organs. CaV channels are divided into two groups depending on the membrane potential required to activate them: High-voltage activated (HVA, CaV1.1–1.4; CaV2.1–2.3 and Low-voltage activated (LVA, CaV3.1–3.3. HVA channels are highly expressed in brain (neurons, heart, and adrenal medulla (chromaffin cells, among others, and are also classified into subtypes which can be distinguished using pharmacological approaches. Cone snails are marine gastropods that capture their prey by injecting venom, “conopeptides”, which cause paralysis in a few seconds. A subset of conopeptides called conotoxins are relatively small polypeptides, rich in disulfide bonds, that target ion channels, transporters and receptors localized at the neuromuscular system of the animal target. In this review, we describe the structure and properties of conotoxins that selectively block HVA calcium channels. We compare their potency on several HVA channel subtypes, emphasizing neuronal calcium channels. Lastly, we analyze recent advances in the therapeutic use of conotoxins for medical treatments.

Sequelae and survivorship in patients treated with (131)I-MIBG therapy.

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Sze, W C C; Grossman, A B; Goddard, I; Amendra, D; Shieh, S C C; Plowman, P N; Drake, W M; Akker, S A; Druce, M R

2013-08-06

(131)I-meta-iodobenzylguanidine ((131)I-MIBG) has been in therapeutic use since 1980s. Newer treatment modalities are emerging for neuroendocrine tumours (NETs) and chromaffin cell tumours (CCTs), but many of these do not yet have adequate long-term follow-up to determine their longer term efficacy and sequelae. Fifty-eight patients with metastatic NETs and CCTs who had received (131)I-MIBG therapy between 2000 and 2011 were analysed. Survival and any long-term haematological or renal sequelae were investigated. In the NET group, the overall median survival and median survival following the diagnosis of metastatic disease was 124 months. The median survival following the commencement of (131)I-MIBG was 66 months. For the CCT group, median survival had not been reached. The 5-year survival from diagnosis and following the diagnosis of metastatic disease was 67% and 67.5% for NETs and CCTs, respectively. The 5-year survival following the commencement of (131)I-MIBG therapy was 68%. Thirty-two patients had long-term haematological sequelae: 5 of these 32 patients developed haematological malignancies. Two patients developed a mild deterioration in renal function. Long follow up of (131)I-MIBG therapy reveals a noteable rate of bone marrow toxicities and malignancy and long term review of all patients receiving radionuclide therapies is recommended.

Epigenetic programming of autonomic functions in an experimental model of apnea of prematurity

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Jayasri Nanduri

2017-01-01

Full Text Available Intermittent hypoxia (IH is a hallmark manifestation of recurrent apneas, which is a major clinical problem in infants born preterm. Carotid body (CB chemoreflex and catecholamine (CA secretion from adrenal medullary chromaffin cells (AMCs are two major mechanisms contributing to the maintenance of cardiorespiratory homeostasis during hypoxia. The purpose of this article is to highlight recent studies showing how neonates experiencing IH affect the CB and AMC function and their consequences in adult life. To simulate apneas, rat pups were treated with IH consisting of alternating cycles of hypoxia (1.5% O2 for 15 s and room air for 5 min, 8 h/day from ages P0–P10. Rats treated neonatal IH displayed augmented CB response to hypoxia and augmented CA secretion from AMC. Rats treated for 10 days of IH in the neonatal period were allowed to grow into adulthood. Remarkably, the effects of neonatal IH on CB and AMC persisted in the adulthood. Moreover, adult rats that were exposed to IH in neonatal period exhibited hypertension, increased incidence apnea. Analysis of the underlying molecular mechanisms revealed re-programming of the redox state by epigenetic mechanisms involving suppression of transcription of antioxidant enzyme genes by DNA hypermethylation. DNA hypomethylating agents might offer a novel therapeutic intervention to prevent early onset of cardiorespiratory morbidities caused by neonatal IH.

Regulation of cell cycle progression by cell-cell and cell-matrix forces

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Uroz, Marina; Wistorf, Sabrina; Serra-Picamal, Xavier; Conte, Vito; Sales-Pardo, Marta; Roca-Cusachs, Pere; Guimerà, Roger; Trepat, Xavier

2018-01-01

It has long been proposed that the cell cycle is regulated by physical forces at the cell-cell and cell-extracellular matrix (ECM) interfaces 1-12 . However, the evolution of these forces during the cycle has never been measured in a tissue, and whether this evolution affects cell cycle progression

Salinity-dependent in vitro effects of homologous natriuretic peptides on the pituitary-interrenal axis in eels.

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Ventura, Albert; Kusakabe, Makoto; Takei, Yoshio

2011-08-01

We examined the effects of atrial, B-type, ventricular and C-type natriuretic peptides (ANP, BNP, VNP and CNP1, 3, 4) on cortisol secretion from interrenal tissue in vitro in both freshwater (FW) and seawater (SW)-acclimated eels. We first localized the interrenal and chromaffin cells in the eel head kidney using cell specific markers (cholesterol side-chain cleavage enzyme (P450ssc) and tyrosine hydroxylase (TH), respectively) and established the in vitro incubation system for eel interrenal tissue. Unexpectedly, none of the NPs given alone to the interrenal tissue of FW and SW eels stimulated cortisol secretion. However, ANP and VNP, but not BNP and three CNPs, enhanced the steroidogenic action of ACTH in SW interrenal preparations, while CNP1 and CNP4, but not ANP, BNP, VNP and CNP3, potentiated the ACTH action in FW preparations. These salinity dependent effects of NPs are consistent with the previous in vivo study in the eel where endogenous ACTH can act with the injected NPs. 8-Br-cGMP also enhanced the ACTH action in both FW and SW eel preparations, suggesting that the NP actions were mediated by the guanylyl cyclase-coupled NP receptors (GC-A and B) that were localized in the eel interrenal. Further, ANP and CNP1 stimulated ACTH secretion from isolated pituitary glands of SW and/or FW eels. In summary, the present study revealed complex mechanisms of NP action on corticosteroidogenesis through the pituitary-interrenal axis in eels, thereby providing a deeper insight into the role of the NP family in the acclimation of this euryhaline teleost to diverse salinity environments. Copyright © 2011 Elsevier Inc. All rights reserved.

Well-Controlled Cell-Trapping Systems for Investigating Heterogeneous Cell-Cell Interactions.

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Kamiya, Koki; Abe, Yuta; Inoue, Kosuke; Osaki, Toshihisa; Kawano, Ryuji; Miki, Norihisa; Takeuchi, Shoji

2018-03-01

Microfluidic systems have been developed for patterning single cells to study cell-cell interactions. However, patterning multiple types of cells to understand heterogeneous cell-cell interactions remains difficult. Here, it is aimed to develop a cell-trapping device to assemble multiple types of cells in the well-controlled order and morphology. This device mainly comprises a parylene sheet for assembling cells and a microcomb for controlling the cell-trapping area. The cell-trapping area is controlled by moving the parylene sheet on an SU-8 microcomb using tweezers. Gentle downward flow is used as a driving force for the cell-trapping. The assembly of cells on a parylene sheet with round and line-shaped apertures is demonstrated. The cell-cell contacts of the trapped cells are then investigated by direct cell-cell transfer of calcein via connexin nanopores. Finally, using the device with a system for controlling the cell-trapping area, three different types of cells in the well-controlled order are assembled. The correct cell order rate obtained using the device is 27.9%, which is higher than that obtained without the sliding parylene system (0.74%). Furthermore, the occurrence of cell-cell contact between the three cell types assembled is verified. This cell-patterning device will be a useful tool for investigating heterogeneous cell-cell interactions. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cell Adhesions: Actin-Based Modules that Mediate Cell-Extracellular Matrix and Cell-Cell Interactions

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Bachir, Alexia; Horwitz, Alan Rick; Nelson, W. James; Bianchini, Julie M.

2018-01-01

Cell adhesions link cells to the extracellular matrix (ECM) and to each other, and depend on interactions with the actin cytoskeleton. Both cell-ECM and cell-cell adhesion sites contain discrete, yet overlapping functional modules. These modules establish physical association with the actin cytoskeleton, locally modulate actin organization and dynamics, and trigger intracellular signaling pathways. Interplay between these modules generates distinct actin architectures that underlie different stages, types, and functions of cell-ECM and cell-cell adhesions. Actomyosin contractility is required to generate mature, stable adhesions, as well as sense and translate the mechanical properties of the cellular environment to changes in cell organization and behavior. In this chapter we discuss the organization and function of different adhesion modules and how they interact with the actin cytoskeleton. We highlight the molecular mechanisms of mechanotransduction in adhesions, and how adhesion molecules mediate crosstalk between cell-ECM and cell-cell adhesion sites. PMID:28679638

Meningeal mast cell-T cell crosstalk regulates T cell encephalitogenicity.

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Russi, Abigail E; Walker-Caulfield, Margaret E; Guo, Yong; Lucchinetti, Claudia F; Brown, Melissa A

2016-09-01

GM-CSF is a cytokine produced by T helper (Th) cells that plays an essential role in orchestrating neuroinflammation in experimental autoimmune encephalomyelitis, a rodent model of multiple sclerosis. Yet where and how Th cells acquire GM-CSF expression is unknown. In this study we identify mast cells in the meninges, tripartite tissues surrounding the brain and spinal cord, as important contributors to antigen-specific Th cell accumulation and GM-CSF expression. In the absence of mast cells, Th cells do not accumulate in the meninges nor produce GM-CSF. Mast cell-T cell co-culture experiments and selective mast cell reconstitution of the meninges of mast cell-deficient mice reveal that resident meningeal mast cells are an early source of caspase-1-dependent IL-1β that licenses Th cells to produce GM-CSF and become encephalitogenic. We also provide evidence of mast cell-T cell co-localization in the meninges and CNS of recently diagnosed acute MS patients indicating similar interactions may occur in human demyelinating disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mast cells enhance T cell activation: Importance of mast cell-derived TNF

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Nakae, Susumu; Suto, Hajime; Kakurai, Maki; Sedgwick, Jonathon D.; Tsai, Mindy; Galli, Stephen J.

2005-05-01

Mast cells are not only important effector cells in immediate hypersensitivity reactions and immune responses to pathogens but also can contribute to T cell-mediated disorders. However, the mechanisms by which mast cells might influence T cells in such settings are not fully understood. We find that mast cells can enhance proliferation and cytokine production in multiple T cell subsets. Mast cell-dependent enhancement of T cell activation can be promoted by FcRI-dependent mast cell activation, TNF production by both mast cells and T cells, and mast cell-T cell contact. However, at high concentrations of cells, mast cells can promote T cell activation independent of IgE or TNF. Finally, mast cells also can promote T cell activation by means of soluble factors. These findings identify multiple mechanisms by which mast cells can influence T cell proliferation and cytokine production. allergy | asthma | autoimmunity | cytokines | immune response

Induction of Functional Hair-Cell-Like Cells from Mouse Cochlear Multipotent Cells

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Quanwen Liu

2016-01-01

Full Text Available In this paper, we developed a two-step-induction method of generating functional hair cells from inner ear multipotent cells. Multipotent cells from the inner ear were established and induced initially into progenitor cells committed to the inner ear cell lineage on the poly-L-lysine substratum. Subsequently, the committed progenitor cells were cultured on the mitotically inactivated chicken utricle stromal cells and induced into hair-cell-like cells containing characteristic stereocilia bundles. The hair-cell-like cells exhibited rapid permeation of FM1-43FX. The whole-cell patch-clamp technique was used to measure the membrane currents of cells differentiated for 7 days on chicken utricle stromal cells and analyze the biophysical properties of the hair-cell-like cells by recording membrane properties of cells. The results suggested that the hair-cell-like cells derived from inner ear multipotent cells were functional following differentiation in an enabling environment.

Management of pediatric pheochromocytoma. A review of the literature

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Mauriello C

2016-06-01

Full Text Available Introduction Pheochromocytoma is a tumor originated from the chromaffin tissue of the adrenal medulla or from extra-adrenal paraganglionic tissue. Pheochromocytomasare extremely rare in the pediatric population, accounting for 1% of pediatric hypertension. Material and Methods The Authors conduced a systematic review of the pediatric PCC focusing on the indications and surgical technique. Results Surgery remains the mainstay of treatment of pheochromocytomain children. Prior to surgery all children must be prepared with alpha-blockade with adequate fluid and salt replacement in order to reduce surgical complications. Discussion and Conclusions Minimally invasive adrenalectomy is the gold standard for benign lesions of the adrenal gland. The lateral transperitoneal adrenalectomy is the standard approach. Laparoscopic bilateral partial adrenalectomies should be considered in children with bilateral PCC in order to avoid lifelong glucocorticoid and mineralocorticoid replacement.

Cell Cycle Related Differentiation of Bone Marrow Cells into Lung Cells

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Dooner, Mark; Aliotta, Jason M.; Pimental, Jeffrey; Dooner, Gerri J.; Abedi, Mehrdad; Colvin, Gerald; Liu, Qin; Weier, Heinz-Ulli; Dooner, Mark S.; Quesenberry, Peter J.

2007-12-31

Green-fluorescent protein (GFP) labeled marrow cells transplanted into lethally irradiated mice can be detected in the lungs of transplanted mice and have been shown to express lung specific proteins while lacking the expression of hematopoietic markers. We have studied marrow cells induced to transit cell cycle by exposure to IL-3, IL-6, IL-11 and steel factor at different times of culture corresponding to different phases of cell cycle. We have found that marrow cells at the G1/S interface have a 3-fold increase in cells which assume a lung phenotype and that this increase is no longer seen in late S/G2. These cells have been characterized as GFP{sup +} CD45{sup -} and GFP{sup +} cytokeratin{sup +}. Thus marrow cells with the capacity to convert into cells with a lung phenotype after transplantation show a reversible increase with cytokine induced cell cycle transit. Previous studies have shown the phenotype of bone marrow stem cells fluctuates reversibly as these cells traverse cell cycle, leading to a continuum model of stem cell regulation. The present studies indicate that marrow stem cell production of nonhematopoietic cells also fluctuates on a continuum.

Deletion of Notch1 converts pro-T cells to dendritic cells and promotes thymic B cells by cell-extrinsic and cell-intrinsic mechanisms.

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Feyerabend, Thorsten B; Terszowski, Grzegorz; Tietz, Annette; Blum, Carmen; Luche, Hervé; Gossler, Achim; Gale, Nicholas W; Radtke, Freddy; Fehling, Hans Jörg; Rodewald, Hans-Reimer

2009-01-16

Notch1 signaling is required for T cell development and has been implicated in fate decisions in the thymus. We showed that Notch1 deletion in progenitor T cells (pro-T cells) revealed their latent developmental potential toward becoming conventional and plasmacytoid dendritic cells. In addition, Notch1 deletion in pro-T cells resulted in large numbers of thymic B cells, previously explained by T-to-B cell fate conversion. Single-cell genotyping showed, however, that the majority of these thymic B cells arose from Notch1-sufficient cells by a cell-extrinsic pathway. Fate switching nevertheless exists for a subset of thymic B cells originating from Notch1-deleted pro-T cells. Chimeric mice lacking the Notch ligand delta-like 4 (Dll4) in thymus epithelium revealed an essential role for Dll4 in T cell development. Thus, Notch1-Dll4 signaling fortifies T cell commitment by suppressing non-T cell lineage potential in pro-T cells, and normal Notch1-driven T cell development repels excessive B cells in the thymus.

Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

International Nuclear Information System (INIS)

Varga, Nóra; Veréb, Zoltán; Rajnavölgyi, Éva; Német, Katalin; Uher, Ferenc; Sarkadi, Balázs; Apáti, Ágota

2011-01-01

Highlights: ► MSC like cells were derived from hESC by a simple and reproducible method. ► Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. ► MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

Involvement of plant stem cells or stem cell-like cells in dedifferentiation

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Fangwei eJiang

2015-11-01

Full Text Available Dedifferentiation is the transformation of cells from a given differentiated state to a less differentiated or stem cell-like state. Stem cell-related genes play important roles in dedifferentiation, which exhibits similar histone modification and DNA methylation features to stem cell maintenance. Hence, stem cell-related factors possibly synergistically function to provide a specific niche beneficial to dedifferentiation. During callus formation in Arabidopsis petioles, cells adjacent to procambium cells (stem cell-like cells are dedifferentiated and survive more easily than other cell types. This finding indicates that stem cells or stem cell-like cells may influence the dedifferentiating niche. In this paper, we provide a brief overview of stem cell maintenance and dedifferentiation regulation. We also summarize current knowledge of genetic and epigenetic mechanisms underlying the balance between differentiation and dedifferentiation. Furthermore, we discuss the correlation of stem cells or stem cell-like cells with dedifferentiation.

Cell-cell interactions mediate cytoskeleton organization and collective endothelial cell chemotaxis.

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Shamloo, Amir

2014-09-01

This study investigates the role of cell-cell and cell-ligand interactions in cytoskeleton organization of endothelial cells (ECs) and their directional migration within a microfluidic device. The migration of ECs in response to a biochemical factor was studied. Mathematical analysis of the cell migration pathways and cellular cytoskeleton revealed that directional migration, migration persistence length, migration speed, and cytoskeletal stress fiber alignment can be mediated by the level of cell contacts as well as the presence or absence of a biochemical polarizing factor. It was shown that in the presence of a biochemical polarizing factor, higher cell density and more frequent cell contacts has a reinforcing effect on collective cell chemotaxis. In contrast, in the absence of a polarizing factor, high cell density can decrease or suppress the ability of the cells to migrate. Also, the correlation of actin stress fiber organization and alignment with directional migration of ECs was investigated. It was shown that in the presence of a biochemical polarizing factor, stress fibers within the cytoskeleton of ECs can be significantly aligned parallel to the gradient direction when the cells have higher level of contacts. The results also show that the organization and alignment of actin stress fibers is mediated by cell adhesion junctions during collective cell migration and introduce cell-cell interactions as a key factor during collective cell chemotaxis. © 2014 Wiley Periodicals, Inc.

Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

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Varga, Nora [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Vereb, Zoltan; Rajnavoelgyi, Eva [Department of Immunology, Medical and Health Science Centre, University of Debrecen, Debrecen (Hungary); Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Apati, Agota, E-mail: apati@kkk.org.hu [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary)

2011-10-28

Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

Computational cell model based on autonomous cell movement regulated by cell-cell signalling successfully recapitulates the "inside and outside" pattern of cell sorting

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Ajioka Itsuki

2007-09-01

Full Text Available Abstract Background Development of multicellular organisms proceeds from a single fertilized egg as the combined effect of countless numbers of cellular interactions among highly dynamic cells. Since at least a reminiscent pattern of morphogenesis can be recapitulated in a reproducible manner in reaggregation cultures of dissociated embryonic cells, which is known as cell sorting, the cells themselves must possess some autonomous cell behaviors that assure specific and reproducible self-organization. Understanding of this self-organized dynamics of heterogeneous cell population seems to require some novel approaches so that the approaches bridge a gap between molecular events and morphogenesis in developmental and cell biology. A conceptual cell model in a computer may answer that purpose. We constructed a dynamical cell model based on autonomous cell behaviors, including cell shape, growth, division, adhesion, transformation, and motility as well as cell-cell signaling. The model gives some insights about what cellular behaviors make an appropriate global pattern of the cell population. Results We applied the model to "inside and outside" pattern of cell-sorting, in which two different embryonic cell types within a randomly mixed aggregate are sorted so that one cell type tends to gather in the central region of the aggregate and the other cell type surrounds the first cell type. Our model can modify the above cell behaviors by varying parameters related to them. We explored various parameter sets with which the "inside and outside" pattern could be achieved. The simulation results suggested that direction of cell movement responding to its neighborhood and the cell's mobility are important for this specific rearrangement. Conclusion We constructed an in silico cell model that mimics autonomous cell behaviors and applied it to cell sorting, which is a simple and appropriate phenomenon exhibiting self-organization of cell population. The model

Endothelial cell subpopulations in vitro: cell volume, cell cycle, and radiosensitivity

International Nuclear Information System (INIS)

Rubin, D.B.; Drab, E.A.; Bauer, K.D.

1989-01-01

Vascular endothelial cells (EC) are important clinical targets of radiation and other forms of free radical/oxidant stresses. In this study, we found that the extent of endothelial damage may be determined by the different cytotoxic responses of EC subpopulations. The following characteristics of EC subpopulations were examined: (1) cell volume; (2) cell cycle position; and (3) cytotoxic indexes for both acute cell survival and proliferative capacity after irradiation (137Cs, gamma, 0-10 Gy). EC cultured from bovine aortas were separated by centrifugal elutriation into subpopulations of different cell volumes. Through flow cytometry, we found that cell volume was related to the cell cycle phase distribution. The smallest EC were distributed in G1 phase and the larger cells were distributed in either early S, middle S, or late S + G2M phases. Cell cycle phase at the time of irradiation was not associated with acute cell loss. However, distribution in the cell cycle did relate to cell survival based on proliferative capacity (P less than 0.01). The order of increasing radioresistance was cells in G1 (D0 = 110 cGy), early S (135 cGy), middle S (145 cGy), and late S + G2M phases (180 cGy). These findings (1) suggest an age-related response to radiation in a nonmalignant differentiated cell type and (2) demonstrate EC subpopulations in culture

Quantitative imaging of epithelial cell scattering identifies specific inhibitors of cell motility and cell-cell dissociation

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Loerke, D.; le Duc, Q.; Blonk, I.; Kerstens, A.; Spanjaard, E.; Machacek, M.; Danuser, G.; de Rooij, J.

2012-01-01

The scattering of cultured epithelial cells in response to hepatocyte growth factor (HGF) is a model system that recapitulates key features of metastatic cell behavior in vitro, including disruption of cell-cell adhesions and induction of cell migration. We have developed image analysis tools that

The cell cycle as a brake for β-cell regeneration from embryonic stem cells.

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El-Badawy, Ahmed; El-Badri, Nagwa

2016-01-13

The generation of insulin-producing β cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic β cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle machinery. Both β cells and ES cells possess unique cell cycle machinery yet with significant contrasts. In this review, we compare the cell cycle control mechanisms in both ES cells and β cells, and highlight the fundamental differences between pluripotent cells of embryonic origin and differentiated β cells. Through critical analysis of the differences of the cell cycle between these two cell types, we propose that the cell cycle of ES cells may act as a brake for β-cell regeneration. Based on these differences, we discuss the potential of modulating the cell cycle of ES cells for the large-scale generation of functionally mature β cells in vitro. Further understanding of the factors that modulate the ES cell cycle will lead to new approaches to enhance the production of functional mature insulin-producing cells, and yield a reliable system to generate bona fide β cells in vitro.

{sup 68}Ga-labelled peptides in the management of neuroectodermal tumours

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Naji, Meeran [Maidstone and Tunbridge Wells NHS Trust, Departments of Nuclear Medicine and Radiology, Maidstone (United Kingdom); Al-Nahhas, Adil [Hammersmith Hospital, Imperial College NHS Trust, Department of Nuclear Medicine, London (United Kingdom)

2012-02-15

Neuroectodermal tumours arise from chromaffin cells and possess the ability to secrete catecholamines. They are generally rare and may occur in association with a variety of hereditary syndromes such as MEN-2A and 2B, neurofibromatosis type 1 and von Hippel-Lindau disease. The most common types are phaeochromocytoma arising from the adrenal medulla and paraganglioma of extra-adrenal origin. Phaeochromocytomas tend to be benign and are often associated with a gene mutation if the disease is bilateral, while paragangliomas are often malignant, have a more aggressive nature and tend to metastasize. There are no specific histological or immunohistochemical features that indicate the malignant potential and the diagnosis of malignancy can only be established by the presence of distant metastases. Therefore, imaging can play a vital role in the diagnosis, localization, staging and assessment of spread. Traditionally, this is achieved with a combination of cross-sectional (CT and MRI) and functional ({sup 123}I-MIBG or {sup 111}In-octreotide) imaging. However, these modalities are not adequate and achieve moderate sensitivity. The introduction of {sup 68}Ga-DOTA peptide in PET/CT imaging has led to improved receptor targeting and superb PET resolution, as well as accurate localization of lesions. The use of this technique in neuroectodermal tumours has been shown to be superior to all available modalities, but the available data are limited and larger studies are awaited to establish its role in the management of these tumours. (orig.)

Adrenal Gland Microenvironment and Its Involvement in the Regulation of Stress-induced Hormone Secretion during Sepsis.

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Waldemar Kanczkowski

2016-12-01

Full Text Available Survival of all living organisms depends on maintenance of a steady state of homeostasis, which process relies on its ability to react and adapt to various physical and emotional threats. The defense against stress is executed by the hypothalamic-pituitary-adrenal axis and the sympathetic-adrenal medullary system. Adrenal gland is a major effector organ of stress system. During stress adrenal gland rapidly respond with increased secretion of glucocorticoids and catecholamines into circulation, which hormones, in turn, affect metabolism, to provide acutely energy, vasculature to increase blood pressure and the immune system to prevent it from extensive activation. Sepsis resulting from microbial infections is a sustained and extreme example of stress situation. In many critical ill patients levels of both corticotropin-releasing hormone and adrenocorticotropin, two major regulators of adrenal hormone production, are suppressed. Levels of glucocorticoids however, remain normal or are elevated in these patients, suggesting a shift from central to local intraadrenal regulation of adrenal stress response. Among many mechanisms potentially involved in this process, reduced glucocorticoid metabolism and local intraadrenal activation of hormone production mediated by adrenocortical and chromaffin cell interactions, the adrenal vascular system and the immune-adrenal crosstalk play a key role. Consequently, any impairment in function of these systems, can ultimately affect adrenal stress response. The purpose of this mini review is to present and discuss recent advances in our understanding of the adrenal gland microenvironment, and its role in regulation of stress-induced hormone secretion.

Pheochromocytomas and secreting paragangliomas

Directory of Open Access Journals (Sweden)

Gimenez-Roqueplo Anne-Paule

2006-12-01

Full Text Available Abstract Catecholamine-producing tumors may arise in the adrenal medulla (pheochromocytomas or in extraadrenal chromaffin cells (secreting paragangliomas. Their prevalence is about 0.1% in patients with hypertension and 4% in patients with a fortuitously discovered adrenal mass. An increase in the production of catecholamines causes symptoms (mainly headaches, palpitations and excess sweating and signs (mainly hypertension, weight loss and diabetes reflecting the effects of epinephrine and norepinephrine on α- and β-adrenergic receptors. Catecholamine-producing tumors mimic paroxysmal conditions with hypertension and/or cardiac rhythm disorders, including panic attacks, in which sympathetic activation linked to anxiety reproduces the same signs and symptoms. These tumors may be sporadic or part of any of several genetic diseases: familial pheochromocytoma-paraganglioma syndromes, multiple endocrine neoplasia type 2, neurofibromatosis 1 and von Hippel-Lindau disease. Familial cases are diagnosed earlier and are more frequently bilateral and recurring than sporadic cases. The most specific and sensitive diagnostic test for the tumor is the determination of plasma or urinary metanephrines. The tumor can be located by computed tomography, magnetic resonance imaging and metaiodobenzylguanidine scintigraphy. Treatment requires resection of the tumor, generally by laparoscopic surgery. About 10% of tumors are malignant either at first operation or during follow-up, malignancy being diagnosed by the presence of lymph node, visceral or bone metastases. Recurrences and malignancy are more frequent in cases with large or extraadrenal tumors. Patients, especially those with familial or extraadrenal tumors, should be followed-up indefinitely.

Pancreatic stellate cells enhance stem cell-like phenotypes in pancreatic cancer cells

International Nuclear Information System (INIS)

Hamada, Shin; Masamune, Atsushi; Takikawa, Tetsuya; Suzuki, Noriaki; Kikuta, Kazuhiro; Hirota, Morihisa; Hamada, Hirofumi; Kobune, Masayoshi; Satoh, Kennichi; Shimosegawa, Tooru

2012-01-01

Highlights: ► Pancreatic stellate cells (PSCs) promote the progression of pancreatic cancer. ► Pancreatic cancer cells co-cultured with PSCs showed enhanced spheroid formation. ► Expression of stem cell-related genes ABCG2, Nestin and LIN28 was increased. ► Co-injection of PSCs enhanced tumorigenicity of pancreatic cancer cells in vivo. ► This study suggested a novel role of PSCs as a part of the cancer stem cell niche. -- Abstract: The interaction between pancreatic cancer cells and pancreatic stellate cells (PSCs), a major profibrogenic cell type in the pancreas, is receiving increasing attention. There is accumulating evidence that PSCs promote the progression of pancreatic cancer by increasing cancer cell proliferation and invasion as well as by protecting them from radiation- and gemcitabine-induced apoptosis. Recent studies have identified that a portion of cancer cells, called “cancer stem cells”, within the entire cancer tissue harbor highly tumorigenic and chemo-resistant phenotypes, which lead to the recurrence after surgery or re-growth of the tumor. The mechanisms that maintain the “stemness” of these cells remain largely unknown. We hypothesized that PSCs might enhance the cancer stem cell-like phenotypes in pancreatic cancer cells. Indirect co-culture of pancreatic cancer cells with PSCs enhanced the spheroid-forming ability of cancer cells and induced the expression of cancer stem cell-related genes ABCG2, Nestin and LIN28. In addition, co-injection of PSCs enhanced tumorigenicity of pancreatic cancer cells in vivo. These results suggested a novel role of PSCs as a part of the cancer stem cell niche.

In silico characterization of cell-cell interactions using a cellular automata model of cell culture.

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Kihara, Takanori; Kashitani, Kosuke; Miyake, Jun

2017-07-14

Cell proliferation is a key characteristic of eukaryotic cells. During cell proliferation, cells interact with each other. In this study, we developed a cellular automata model to estimate cell-cell interactions using experimentally obtained images of cultured cells. We used four types of cells; HeLa cells, human osteosarcoma (HOS) cells, rat mesenchymal stem cells (MSCs), and rat smooth muscle A7r5 cells. These cells were cultured and stained daily. The obtained cell images were binarized and clipped into squares containing about 10 4 cells. These cells showed characteristic cell proliferation patterns. The growth curves of these cells were generated from the cell proliferation images and we determined the doubling time of these cells from the growth curves. We developed a simple cellular automata system with an easily accessible graphical user interface. This system has five variable parameters, namely, initial cell number, doubling time, motility, cell-cell adhesion, and cell-cell contact inhibition (of proliferation). Within these parameters, we obtained initial cell numbers and doubling times experimentally. We set the motility at a constant value because the effect of the parameter for our simulation was restricted. Therefore, we simulated cell proliferation behavior with cell-cell adhesion and cell-cell contact inhibition as variables. By comparing growth curves and proliferation cell images, we succeeded in determining the cell-cell interaction properties of each cell. Simulated HeLa and HOS cells exhibited low cell-cell adhesion and weak cell-cell contact inhibition. Simulated MSCs exhibited high cell-cell adhesion and positive cell-cell contact inhibition. Simulated A7r5 cells exhibited low cell-cell adhesion and strong cell-cell contact inhibition. These simulated results correlated with the experimental growth curves and proliferation images. Our simulation approach is an easy method for evaluating the cell-cell interaction properties of cells.

Host cell reactivation in mammalian cells

International Nuclear Information System (INIS)

Lytle, C.D.; Benane, S.G.; Stafford, J.E.

1976-01-01

The survival of UV-irradiated herpes simplex virus was determined in cultured Potoroo (a marsupial) and human cells under lighting conditions which promoted photereactivation. Photoreactivation was readily demonstrated for herpes virus in two lines of Potoroo cells with dose reduction factors of 0.7 to 0.8 for ovary cells and 0.5 to 0.7 for kidney cells. Light from Blacklite (near UV) lamps was more effective than from Daylight (mostly visible) lamps, suggesting that near UV radiation was more effecient for photoreactivation in Potoroo cells. The quantitative and qualitative aspects of this photoreactivation were similar to those reported for a similar virus infecting chick embryo cells. UV-survival curves of herpes virus in Potoroo cells indicated a high level of 'dark' host cell reactivation. No photoreactivation was found for UV-irradiated vaccinia virus in Potoroo cells. A similar photoreactivation study was done using special control lighting (lambda>600 nm) and human cells with normal repair and with cells deficient in excision repair (XP). No photoreactivation was found for UV-irradiated herpes virus in either human cell with either Blacklite or Daylight lamps as the sources of photoreactivating light. This result contrasts with a report of photoreactivation for a herpes virus in the same XP cells using incandescent lamps. (author)

Local cell metrics: a novel method for analysis of cell-cell interactions

Directory of Open Access Journals (Sweden)

Chen Chien-Chiang

2009-10-01

Full Text Available Abstract Background The regulation of many cell functions is inherently linked to cell-cell contact interactions. However, effects of contact interactions among adherent cells can be difficult to detect with global summary statistics due to the localized nature and noise inherent to cell-cell interactions. The lack of informatics approaches specific for detecting cell-cell interactions is a limitation in the analysis of large sets of cell image data, including traditional and combinatorial or high-throughput studies. Here we introduce a novel histogram-based data analysis strategy, termed local cell metrics (LCMs, which addresses this shortcoming. Results The new LCM method is demonstrated via a study of contact inhibition of proliferation of MC3T3-E1 osteoblasts. We describe how LCMs can be used to quantify the local environment of cells and how LCMs are decomposed mathematically into metrics specific to each cell type in a culture, e.g., differently-labelled cells in fluorescence imaging. Using this approach, a quantitative, probabilistic description of the contact inhibition effects in MC3T3-E1 cultures has been achieved. We also show how LCMs are related to the naïve Bayes model. Namely, LCMs are Bayes class-conditional probability functions, suggesting their use for data mining and classification. Conclusion LCMs are successful in robust detection of cell contact inhibition in situations where conventional global statistics fail to do so. The noise due to the random features of cell behavior was suppressed significantly as a result of the focus on local distances, providing sensitive detection of cell-cell contact effects. The methodology can be extended to any quantifiable feature that can be obtained from imaging of cell cultures or tissue samples, including optical, fluorescent, and confocal microscopy. This approach may prove useful in interpreting culture and histological data in fields where cell-cell interactions play a critical

Local cell metrics: a novel method for analysis of cell-cell interactions.

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Su, Jing; Zapata, Pedro J; Chen, Chien-Chiang; Meredith, J Carson

2009-10-23

The regulation of many cell functions is inherently linked to cell-cell contact interactions. However, effects of contact interactions among adherent cells can be difficult to detect with global summary statistics due to the localized nature and noise inherent to cell-cell interactions. The lack of informatics approaches specific for detecting cell-cell interactions is a limitation in the analysis of large sets of cell image data, including traditional and combinatorial or high-throughput studies. Here we introduce a novel histogram-based data analysis strategy, termed local cell metrics (LCMs), which addresses this shortcoming. The new LCM method is demonstrated via a study of contact inhibition of proliferation of MC3T3-E1 osteoblasts. We describe how LCMs can be used to quantify the local environment of cells and how LCMs are decomposed mathematically into metrics specific to each cell type in a culture, e.g., differently-labelled cells in fluorescence imaging. Using this approach, a quantitative, probabilistic description of the contact inhibition effects in MC3T3-E1 cultures has been achieved. We also show how LCMs are related to the naïve Bayes model. Namely, LCMs are Bayes class-conditional probability functions, suggesting their use for data mining and classification. LCMs are successful in robust detection of cell contact inhibition in situations where conventional global statistics fail to do so. The noise due to the random features of cell behavior was suppressed significantly as a result of the focus on local distances, providing sensitive detection of cell-cell contact effects. The methodology can be extended to any quantifiable feature that can be obtained from imaging of cell cultures or tissue samples, including optical, fluorescent, and confocal microscopy. This approach may prove useful in interpreting culture and histological data in fields where cell-cell interactions play a critical role in determining cell fate, e.g., cancer, developmental

Single-cell sequencing in stem cell biology.

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Wen, Lu; Tang, Fuchou

2016-04-15

Cell-to-cell variation and heterogeneity are fundamental and intrinsic characteristics of stem cell populations, but these differences are masked when bulk cells are used for omic analysis. Single-cell sequencing technologies serve as powerful tools to dissect cellular heterogeneity comprehensively and to identify distinct phenotypic cell types, even within a 'homogeneous' stem cell population. These technologies, including single-cell genome, epigenome, and transcriptome sequencing technologies, have been developing rapidly in recent years. The application of these methods to different types of stem cells, including pluripotent stem cells and tissue-specific stem cells, has led to exciting new findings in the stem cell field. In this review, we discuss the recent progress as well as future perspectives in the methodologies and applications of single-cell omic sequencing technologies.

NKT Cell Responses to B Cell Lymphoma.

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Li, Junxin; Sun, Wenji; Subrahmanyam, Priyanka B; Page, Carly; Younger, Kenisha M; Tiper, Irina V; Frieman, Matthew; Kimball, Amy S; Webb, Tonya J

2014-06-01

Natural killer T (NKT) cells are a unique subset of CD1d-restricted T lymphocytes that express characteristics of both T cells and natural killer cells. NKT cells mediate tumor immune-surveillance; however, NKT cells are numerically reduced and functionally impaired in lymphoma patients. Many hematologic malignancies express CD1d molecules and co-stimulatory proteins needed to induce anti-tumor immunity by NKT cells, yet most tumors are poorly immunogenic. In this study, we sought to investigate NKT cell responses to B cell lymphoma. In the presence of exogenous antigen, both mouse and human NKT cell lines produce cytokines following stimulation by B cell lymphoma lines. NKT cell populations were examined ex vivo in mouse models of spontaneous B cell lymphoma, and it was found that during early stages, NKT cell responses were enhanced in lymphoma-bearing animals compared to disease-free animals. In contrast, in lymphoma-bearing animals with splenomegaly and lymphadenopathy, NKT cells were functionally impaired. In a mouse model of blastoid variant mantle cell lymphoma, treatment of tumor-bearing mice with a potent NKT cell agonist, α-galactosylceramide (α-GalCer), resulted in a significant decrease in disease pathology. Ex vivo studies demonstrated that NKT cells from α-GalCer treated mice produced IFN-γ following α-GalCer restimulation, unlike NKT cells from vehicle-control treated mice. These data demonstrate an important role for NKT cells in the immune response to an aggressive hematologic malignancy like mantle cell lymphoma.

Automated Cell-Cutting for Cell Cloning

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Ichikawa, Akihiko; Tanikawa, Tamio; Matsukawa, Kazutsugu; Takahashi, Seiya; Ohba, Kohtaro

We develop an automated cell-cutting technique for cell cloning. Animal cells softened by the cytochalasin treatment are injected into a microfluidic chip. The microfluidic chip contains two orthogonal channels: one microchannel is wide, used to transport cells, and generates the cutting flow; the other is thin and used for aspiration, fixing, and stretching of the cell. The injected cell is aspirated and stretched in the thin microchannel. Simultaneously, the volumes of the cell before and after aspiration are calculated; the volumes are used to calculate the fluid flow required to aspirate half the volume of the cell into the thin microchannel. Finally, we apply a high-speed flow in the orthogonal microchannel to bisect the cell. This paper reports the cutting process, the cutting system, and the results of the experiment.

Huntingtin-associated protein-1 (HAP1) regulates endocytosis and interacts with multiple trafficking-related proteins.

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Mackenzie, Kimberly D; Lim, Yoon; Duffield, Michael D; Chataway, Timothy; Zhou, Xin-Fu; Keating, Damien J

2017-07-01

Huntingtin-associated protein 1 (HAP1) was initially identified as a binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking, cell signalling and receptor internalization. In this study, a proteomics approach was used for the identification of novel HAP1-interacting partners to attempt to shed light on the physiological function of HAP1. Using affinity chromatography with HAP1-GST protein fragments bound to Sepharose columns, this study identified a number of trafficking-related proteins that bind to HAP1. Interestingly, many of the proteins that were identified by mass spectrometry have trafficking-related functions and include the clathrin light chain B and Sec23A, an ER to Golgi trafficking vesicle coat component. Using co-immunoprecipitation and GST-binding assays the association between HAP1 and clathrin light chain B has been validated in vitro. This study also finds that HAP1 co-localizes with clathrin light chain B. In line with a physiological function of the HAP1-clathrin interaction this study detected a dramatic reduction in vesicle retrieval and endocytosis in adrenal chromaffin cells. Furthermore, through examination of transferrin endocytosis in HAP1 -/- cortical neurons, this study has determined that HAP1 regulates neuronal endocytosis. In this study, the interaction between HAP1 and Sec23A was also validated through endogenous co-immunoprecipitation in rat brain homogenate. Through the identification of novel HAP1 binding partners, many of which have putative trafficking roles, this study provides us with new insights into the mechanisms underlying the important physiological function of HAP1 as an intracellular trafficking protein through its protein-protein interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

Different cell fates from cell-cell interactions: core architectures of two-cell bistable networks.

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Rouault, Hervé; Hakim, Vincent

2012-02-08

The acquisition of different fates by cells that are initially in the same state is central to development. Here, we investigate the possible structures of bistable genetic networks that can allow two identical cells to acquire different fates through cell-cell interactions. Cell-autonomous bistable networks have been previously sampled using an evolutionary algorithm. We extend this evolutionary procedure to take into account interactions between cells. We obtain a variety of simple bistable networks that we classify into major subtypes. Some have long been proposed in the context of lateral inhibition through the Notch-Delta pathway, some have been more recently considered and others appear to be new and based on mechanisms not previously considered. The results highlight the role of posttranscriptional interactions and particularly of protein complexation and sequestration, which can replace cooperativity in transcriptional interactions. Some bistable networks are entirely based on posttranscriptional interactions and the simplest of these is found to lead, upon a single parameter change, to oscillations in the two cells with opposite phases. We provide qualitative explanations as well as mathematical analyses of the dynamical behaviors of various created networks. The results should help to identify and understand genetic structures implicated in cell-cell interactions and differentiation. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Low Doses of Curcuma longa Modulates Cell Migration and Cell-Cell Adhesion.

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de Campos, Paloma Santos; Matte, Bibiana Franzen; Diel, Leonardo Francisco; Jesus, Luciano Henrique; Bernardi, Lisiane; Alves, Alessandro Menna; Rados, Pantelis Varvaki; Lamers, Marcelo Lazzaron

2017-09-01

Cell invasion and metastasis are involved in clinical failures in cancer treatment, and both events require the acquisition of a migratory behavior by tumor cells. Curcumin is a promising natural product with anti-proliferative activity, but its effects on cell migration are still unclear. We evaluated the effects of curcumin on the proliferation, apoptosis, migration, and cell-cell adhesion of keratinocyte, oral squamous cell carcinoma (OSCC), and fibroblast cell lines, as well as in a xenograft model of OSCC. Curcumin (2 μM) decreased cell proliferation in cell lines with mesenchymal characteristics, while cell death was detected only at 50 μM. We observed that highly migratory cells showed a decrease on migration speed and directionality when treated with 2 or 5 μM of curcumin (50% and 40%, respectively, p curcumin dose dependently decreased cell-cell adhesion, especially on tumor-derived spheroids. Also, in a xenograft model with patient-derived OSCC cells, the administration of curcumin decreased tumor growth and aggressiveness when compared with untreated tumors, indicating the potential antitumor effect in oral cancer. These results suggest that lower doses of curcumin can influence several steps involved in tumorigenesis, including migration properties, suggesting a possible use in cancer therapy. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Nonimmune cells equipped with T-cell-receptor-like signaling for cancer cell ablation.

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Kojima, Ryosuke; Scheller, Leo; Fussenegger, Martin

2018-01-01

The ability to engineer custom cell-contact-sensing output devices into human nonimmune cells would be useful for extending the applicability of cell-based cancer therapies and for avoiding risks associated with engineered immune cells. Here we have developed a new class of synthetic T-cell receptor-like signal-transduction device that functions efficiently in human nonimmune cells and triggers release of output molecules specifically upon sensing contact with a target cell. This device employs an interleukin signaling cascade, whose OFF/ON switching is controlled by biophysical segregation of a transmembrane signal-inhibitory protein from the sensor cell-target cell interface. We further show that designer nonimmune cells equipped with this device driving expression of a membrane-penetrator/prodrug-activating enzyme construct could specifically kill target cells in the presence of the prodrug, indicating its potential usefulness for target-cell-specific, cell-based enzyme-prodrug cancer therapy. Our study also contributes to the advancement of synthetic biology by extending available design principles to transmit extracellular information to cells.

Proliferating cells in psoriatic dermis are comprised primarily of T cells, endothelial cells, and factor XIIIa+ perivascular dendritic cells

International Nuclear Information System (INIS)

Morganroth, G.S.; Chan, L.S.; Weinstein, G.D.; Voorhees, J.J.; Cooper, K.D.

1991-01-01

Determination of the cell types proliferating in the dermis of patients with psoriasis should identify those cells experiencing activation or responding to growth factors in the psoriatic dermal milieu. Toward that end, sections of formalin-fixed biopsies obtained from 3H-deoxyuridine (3H-dU)-injected skin of eight psoriatic patients were immunostained, followed by autoradiography. Proliferating dermal cells exhibit silver grains from tritium emissions. The identity of the proliferating cells could then be determined by simultaneous visualization with antibodies specific for various cell types. UCHL1+ (CD45RO+) T cells (recall antigen-reactive helper T-cell subset) constituted 36.6 +/- 3.1% (mean +/- SEM, n = 6) of the proliferating dermal cells in involved skin, whereas Leu 18+ (CD45RA+) T cells (recall antigen naive T-cell subsets) comprised only 8.7 +/- 1.5% (n = 6). The Factor XIIIa+ dermal perivascular dendritic cell subset (24.9 +/- 1.5% of proliferating dermal cells, n = 6) and Factor VIII+ endothelial cells represented the two other major proliferating populations in lesional psoriatic dermis. Differentiated tissue macrophages, identified by phase microscopy as melanophages or by immunostaining with antibodies to Leu M1 (CD15) or myeloid histiocyte antigen, comprised less than 5% of the proliferating population in either skin type. In addition to calculating the relative proportions of these cells to each other as percent, we also determined the density of cells, in cells/mm2 of tissue. The density of proliferating cells within these populations was increased in involved versus uninvolved skin: UCHL1+, 9.0 +/- 1.7 cells/mm2 versus 1.8 +/- 0.6 cells/mm2, p less than 0.01; Factor XIIIa+, 6.0 +/- 0.7 cells/mm2 versus 1.5 +/- 0.5 cells/mm2, p less than 0.01; Factor VIII+, 5.5 +/- 1.4 cells/mm2 versus 0.0 cells/mm2, p less than 0.05

Imaging the primate adrenal medulla with [123I] and [131I] metaiodobenzylguanidine: concise communication

International Nuclear Information System (INIS)

Wieland, D.M.; Brown, L.E.; Tobes, M.C.; Rogers, W.L.; Marsh, D.D.; Mangner, T.J.; Swanson, D.P.; Beierwaltes, W.H.

1981-01-01

An evaluation of radioiodinated meta-iodobenzylguanidine (m-IBG) as an adrenomedullary imaging agent is reported in 15 rhesus monkeys. Scintiscans of the monkey adrenal medulla have been obtained with [ 123 I]- and [m- 131 ]IBG at 2 to 6 days after injection. The imaging superiority of m-IBG over its positional isomer, para-iodobenzylguanidine (p-IBG), is documented in both dogs and monkeys. Administration of reserpine, a depletor of catecholamine stores, markedly lowers the [m- 131 I]-IBG content of the dog adrenal medulla, but the adrenergic blocking agents phenoxybenzamine and propanolol have no effect. Subcellular fractionation of the dog's adrenal medullae reveals that m-IBG is sequestered mainly in the chromaffin storage granules. The results of this study suggest that radioiodinated m-IBG, previously reported to image the primate myocardium, also merits evaluation as a clinical radiopharmaceutical for the adrenal medulla

Pathological significance and prognostic roles of densities of CD57+ cells, CD68+ cells, and mast cells, and their ratios in clear cell renal cell carcinoma.

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Nakanishi, Hiromi; Miyata, Yasuyoshi; Mochizuki, Yasushi; Yasuda, Takuji; Nakamura, Yuichiro; Araki, Kyohei; Sagara, Yuji; Matsuo, Tomohiro; Ohba, Kojiro; Sakai, Hideki

2018-05-19

The immune system is closely associated with malignant behavior in renal cell carcinoma (RCC). Therefore, understanding the pathological roles of immune cells in tumor stroma is essential to discuss the pathological characteristics of RCC. In this study, the clinical significance of densities of CD57+ cells, CD68+ cells, and mast cells, and their ratios were investigated in patients with clear cell RCC. The densities of CD57+, CD68+, and mast cells were evaluated by immunohistochemical techniques in 179 patients. Proliferation index (PI), apoptotic index (AI), and microvessel density (MVD) were evaluated by using anti-Ki-67, anti-cleaved caspase-3, and anti-CD31 antibodies, respectively. The density of CD57+ cell was negatively correlated with grade, pT stage, and metastasis, although densities of CD68+ cell and mast cell were positively correlated. Ratios of CD68+ cell/CD57+ cell and mast cell/CD57+ cell were significantly correlated with grade, pT stage, and metastasis. Survival analyses showed that the CD68+ cell/CD57+ cell ratio was a significant predictor for cause-specific survival by multi-variate analyses (hazard ratio=1.41, 95% confidential interval=1.03-1.93, P=.031), and was significantly correlated with PI, AI, and MVD (r=.47; P <. 001, r=-.31, P<.001, and r=.40, P<.001, respectively). In conclusion, CD57+ cell, CD68+ cell, and mast cell played important roles in malignancy in clear cell RCC. The CD68+ cell/CD57+ cell ratio was strongly correlated with pathological features and prognosis in these patients because this ratio reflected the status of cancer cell proliferation, apoptosis, and angiogenesis. Copyright © 2018. Published by Elsevier Inc.

Cell-extracellular matrix and cell-cell adhesion are linked by syndecan-4

DEFF Research Database (Denmark)

Pakideeri Karat, Sandeep Gopal; Multhaupt, Hinke A B; Pocock, Roger

2017-01-01

Cell-extracellular matrix (ECM) and cell-cell junctions that employ microfilaments are sites of tension. They are important for tissue repair, morphogenetic movements and can be emblematic of matrix contraction in fibrotic disease and the stroma of solid tumors. One cell surface receptor, syndecan...... calcium. While it is known that cell-ECM and cell-cell junctions may be linked, possible roles for syndecans in this process are not understood. Here we show that wild type primary fibroblasts and those lacking syndecan-4 utilize different cadherins in their adherens junctions and that tension is a major...... factor in this differential response. This corresponds to the reduced ability of fibroblasts lacking syndecan-4 to exert tension on the ECM and we now show that this may extend to reduced tension in cell-cell adhesion....

MARCKS-related protein regulates cytoskeletal organization at cell-cell and cell-substrate contacts in epithelial cells.

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Van Itallie, Christina M; Tietgens, Amber Jean; Aponte, Angel; Gucek, Marjan; Cartagena-Rivera, Alexander X; Chadwick, Richard S; Anderson, James M

2018-02-02

Treatment of epithelial cells with interferon-γ and TNF-α (IFN/TNF) results in increased paracellular permeability. To identify relevant proteins mediating barrier disruption, we performed proximity-dependent biotinylation (BioID) of occludin and found that tagging of MARCKS-related protein (MRP; also known as MARCKSL1) increased ∼20-fold following IFN/TNF administration. GFP-MRP was focused at the lateral cell membrane and its overexpression potentiated the physiological response of the tight junction barrier to cytokines. However, deletion of MRP did not abrogate the cytokine responses, suggesting that MRP is not required in the occludin-dependent IFN/TNF response. Instead, our results reveal a key role for MRP in epithelial cells in control of multiple actin-based structures, likely by regulation of integrin signaling. Changes in focal adhesion organization and basal actin stress fibers in MRP-knockout (KO) cells were reminiscent of those seen in FAK-KO cells. In addition, we found alterations in cell-cell interactions in MRP-KO cells associated with increased junctional tension, suggesting that MRP may play a role in focal adhesion-adherens junction cross talk. Together, our results are consistent with a key role for MRP in cytoskeletal organization of cell contacts in epithelial cells. © 2018. Published by The Company of Biologists Ltd.

Stem cells engineering for cell-based therapy.

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Taupin, Philippe

2007-09-01

Stem cells carry the promise to cure a broad range of diseases and injuries, from diabetes, heart and muscular diseases, to neurological diseases, disorders and injuries. Significant progresses have been made in stem cell research over the past decade; the derivation of embryonic stem cells (ESCs) from human tissues, the development of cloning technology by somatic cell nuclear transfer (SCNT) and the confirmation that neurogenesis occurs in the adult mammalian brain and that neural stem cells (NSCs) reside in the adult central nervous system (CNS), including that of humans. Despite these advances, there may be decades before stem cell research will translate into therapy. Stem cell research is also subject to ethical and political debates, controversies and legislation, which slow its progress. Cell engineering has proven successful in bringing genetic research to therapy. In this review, I will review, in two examples, how investigators are applying cell engineering to stem cell biology to circumvent stem cells' ethical and political constraints and bolster stem cell research and therapy.

Dissecting engineered cell types and enhancing cell fate conversion via CellNet

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Morris, Samantha A.; Cahan, Patrick; Li, Hu; Zhao, Anna M.; San Roman, Adrianna K.; Shivdasani, Ramesh A.; Collins, James J.; Daley, George Q.

2014-01-01

SUMMARY Engineering clinically relevant cells in vitro holds promise for regenerative medicine, but most protocols fail to faithfully recapitulate target cell properties. To address this, we developed CellNet, a network biology platform that determines whether engineered cells are equivalent to their target tissues, diagnoses aberrant gene regulatory networks, and prioritizes candidate transcriptional regulators to enhance engineered conversions. Using CellNet, we improved B cell to macrophage conversion, transcriptionally and functionally, by knocking down predicted B cell regulators. Analyzing conversion of fibroblasts to induced hepatocytes (iHeps), CellNet revealed an unexpected intestinal program regulated by the master regulator Cdx2. We observed long-term functional engraftment of mouse colon by iHeps, thereby establishing their broader potential as endoderm progenitors and demonstrating direct conversion of fibroblasts into intestinal epithelium. Our studies illustrate how CellNet can be employed to improve direct conversion and to uncover unappreciated properties of engineered cells. PMID:25126792

cgCorrect: a method to correct for confounding cell-cell variation due to cell growth in single-cell transcriptomics

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Blasi, Thomas; Buettner, Florian; Strasser, Michael K.; Marr, Carsten; Theis, Fabian J.

2017-06-01

Accessing gene expression at a single-cell level has unraveled often large heterogeneity among seemingly homogeneous cells, which remains obscured when using traditional population-based approaches. The computational analysis of single-cell transcriptomics data, however, still imposes unresolved challenges with respect to normalization, visualization and modeling the data. One such issue is differences in cell size, which introduce additional variability into the data and for which appropriate normalization techniques are needed. Otherwise, these differences in cell size may obscure genuine heterogeneities among cell populations and lead to overdispersed steady-state distributions of mRNA transcript numbers. We present cgCorrect, a statistical framework to correct for differences in cell size that are due to cell growth in single-cell transcriptomics data. We derive the probability for the cell-growth-corrected mRNA transcript number given the measured, cell size-dependent mRNA transcript number, based on the assumption that the average number of transcripts in a cell increases proportionally to the cell’s volume during the cell cycle. cgCorrect can be used for both data normalization and to analyze the steady-state distributions used to infer the gene expression mechanism. We demonstrate its applicability on both simulated data and single-cell quantitative real-time polymerase chain reaction (PCR) data from mouse blood stem and progenitor cells (and to quantitative single-cell RNA-sequencing data obtained from mouse embryonic stem cells). We show that correcting for differences in cell size affects the interpretation of the data obtained by typically performed computational analysis.

Single cell time-lapse analysis reveals that podoplanin enhances cell survival and colony formation capacity of squamous cell carcinoma cells.

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Miyashita, Tomoyuki; Higuchi, Youichi; Kojima, Motohiro; Ochiai, Atsushi; Ishii, Genichiro

2017-01-06

Tumor initiating cells (TICs) are characterized by high clonal expansion capacity. We previously reported that podoplanin is a TIC-specific marker for the human squamous cell carcinoma cell line A431. The aim of this study is to explore the molecular mechanism underlying the high clonal expansion potential of podoplanin-positive A431cells using Fucci imaging. Single podoplanin-positive cells created large colonies at a significantly higher frequency than single podoplanin-negative cells, whereas no difference was observed between the two types of cells with respect to cell cycle status. Conversely, the cell death ratio of progenies derived from podoplanin-positive single cell was significantly lower than that of cells derived from podoplanin-negative cells. Single A431 cells, whose podoplanin expression was suppressed by RNA interference, exhibited increased cell death ratios and decreased frequency of large colony forming. Moreover, the frequency of large colony forming decreased significantly when podoplanin-positive single cells was treated with a ROCK (Rho-associated coiled-coil kinase) inhibitor, whereas no difference was observed in single podoplanin-negative cells. Our current study cleared that high clonal expansion capacity of podoplanin-positive TICs populations was the result of reduced cell death by podoplanin-mediated signaling. Therefore, podoplanin activity may be a therapeutic target in the treatment of squamous cell carcinomas.

Modeling cell-in-cell structure into its biological significance

OpenAIRE

He, M-f; Wang, S; Wang, Y; Wang, X-n

2013-01-01

Although cell-in-cell structure was noted 100 years ago, the molecular mechanisms of ?entering' and the destination of cell-in-cell remain largely unclear. It takes place among the same type of cells (homotypic cell-in-cell) or different types of cells (heterotypic cell-in-cell). Cell-in-cell formation affects both effector cells and their host cells in multiple aspects, while cell-in-cell death is under more intensive investigation. Given that cell-in-cell has an important role in maintainin...

Cell volume change through water efflux impacts cell stiffness and stem cell fate

NARCIS (Netherlands)

Guo, Ming; Pegoraro, Adrian F.; Mao, Angelo; Zhou, Enhua H.; Arany, Praveen R.; Han, Yulong; Burnette, Dylan T.; Jensen, Mikkel H.; Kasza, Karen E.; Moore, Jeffrey R.; Mackintosh, Frederick C.; Fredberg, Jeffrey J.; Mooney, David J.; Lippincott-Schwartz, Jennifer; Weitz, David A.

2017-01-01

Cells alter their mechanical properties in response to their local microenvironment; this plays a role in determining cell function and can even influence stem cell fate. Here, we identify a robust and unified relationship between cell stiffness and cell volume. As a cell spreads on a substrate, its

Retinal stem cells and potential cell transplantation treatments

Directory of Open Access Journals (Sweden)

Tai-Chi Lin

2014-11-01

Full Text Available The retina, histologically composed of ten delicate layers, is responsible for light perception and relaying electrochemical signals to the secondary neurons and visual cortex. Retinal disease is one of the leading clinical causes of severe vision loss, including age-related macular degeneration, Stargardt's disease, and retinitis pigmentosa. As a result of the discovery of various somatic stem cells, advances in exploring the identities of embryonic stem cells, and the development of induced pluripotent stem cells, cell transplantation treatment for retinal diseases is currently attracting much attention. The sources of stem cells for retinal regeneration include endogenous retinal stem cells (e.g., neuronal stem cells, Müller cells, and retinal stem cells from the ciliary marginal zone and exogenous stem cells (e.g., bone mesenchymal stem cells, adipose-derived stem cells, embryonic stem cells, and induced pluripotent stem cells. The success of cell transplantation treatment depends mainly on the cell source, the timing of cell harvesting, the protocol of cell induction/transplantation, and the microenvironment of the recipient's retina. This review summarizes the different sources of stem cells for regeneration treatment in retinal diseases and surveys the more recent achievements in animal studies and clinical trials. Future directions and challenges in stem cell transplantation are also discussed.

Hybrid cell adhesive material for instant dielectrophoretic cell trapping and long-term cell function assessment.

Science.gov (United States)

Reyes, Darwin R; Hong, Jennifer S; Elliott, John T; Gaitan, Michael

2011-08-16

Dielectrophoresis (DEP) for cell manipulation has focused, for the most part, on approaches for separation/enrichment of cells of interest. Advancements in cell positioning and immobilization onto substrates for cell culture, either as single cells or as cell aggregates, has benefited from the intensified research efforts in DEP (electrokinetic) manipulation. However, there has yet to be a DEP approach that provides the conditions for cell manipulation while promoting cell function processes such as cell differentiation. Here we present the first demonstration of a system that combines DEP with a hybrid cell adhesive material (hCAM) to allow for cell entrapment and cell function, as demonstrated by cell differentiation into neuronlike cells (NLCs). The hCAM, comprised of polyelectrolytes and fibronectin, was engineered to function as an instantaneous cell adhesive surface after DEP manipulation and to support long-term cell function (cell proliferation, induction, and differentiation). Pluripotent P19 mouse embryonal carcinoma cells flowing within a microchannel were attracted to the DEP electrode surface and remained adhered onto the hCAM coating under a fluid flow field after the DEP forces were removed. Cells remained viable after DEP manipulation for up to 8 d, during which time the P19 cells were induced to differentiate into NLCs. This approach could have further applications in areas such as cell-cell communication, three-dimensional cell aggregates to create cell microenvironments, and cell cocultures.

Cell cycle regulation in human embryonic stem cells: links to adaptation to cell culture.

Science.gov (United States)

Barta, Tomas; Dolezalova, Dasa; Holubcova, Zuzana; Hampl, Ales

2013-03-01

Cell cycle represents not only a tightly orchestrated mechanism of cell replication and cell division but it also plays an important role in regulation of cell fate decision. Particularly in the context of pluripotent stem cells or multipotent progenitor cells, regulation of cell fate decision is of paramount importance. It has been shown that human embryonic stem cells (hESCs) show unique cell cycle characteristics, such as short doubling time due to abbreviated G1 phase; these properties change with the onset of differentiation. This review summarizes the current understanding of cell cycle regulation in hESCs. We discuss cell cycle properties as well as regulatory machinery governing cell cycle progression of undifferentiated hESCs. Additionally, we provide evidence that long-term culture of hESCs is accompanied by changes in cell cycle properties as well as configuration of several cell cycle regulatory molecules.

Cell patterning without chemical surface modification: Cell cell interactions between printed bovine aortic endothelial cells (BAEC) on a homogeneous cell-adherent hydrogel

Science.gov (United States)

Chen, C. Y.; Barron, J. A.; Ringeisen, B. R.

2006-10-01

Cell printing offers the unique ability to directly deposit one or multiple cell types directly onto a surface without the need to chemically pre-treat the surface with lithographic methods. We utilize biological laser printing (BioLP ™) to form patterns of bovine aortic endothelial cells (BAECs) onto a homogeneous cell adherent hydrogel surface. These normal cells are shown to retain near-100% viability post-printing. In order to determine whether BAECs encountered shear and/or heat stress during printing, immunocytochemical staining experiments were performed to detect potential expression of heat shock proteins (HSP) by the deposited cells. Printed BAECs expressed HSP at levels similar to negative control cells, indicating that the BioLP process does not expose cells to damaging levels of stress. However, HSP expression was slightly higher at the highest laser energy studied, suggesting more stress was present under these extreme conditions. Printed BAECs also showed preferential asymmetric growth and migration towards each other and away from the originally printed pattern, demonstrating a retained ability for the cells to communicate post-printing.

Llgl1 Connects Cell Polarity with Cell-Cell Adhesion in Embryonic Neural Stem Cells.

Science.gov (United States)

Jossin, Yves; Lee, Minhui; Klezovitch, Olga; Kon, Elif; Cossard, Alexia; Lien, Wen-Hui; Fernandez, Tania E; Cooper, Jonathan A; Vasioukhin, Valera

2017-06-05

Malformations of the cerebral cortex (MCCs) are devastating developmental disorders. We report here that mice with embryonic neural stem-cell-specific deletion of Llgl1 (Nestin-Cre/Llgl1 fl/fl ), a mammalian ortholog of the Drosophila cell polarity gene lgl, exhibit MCCs resembling severe periventricular heterotopia (PH). Immunohistochemical analyses and live cortical imaging of PH formation revealed that disruption of apical junctional complexes (AJCs) was responsible for PH in Nestin-Cre/Llgl1 fl/fl brains. While it is well known that cell polarity proteins govern the formation of AJCs, the exact mechanisms remain unclear. We show that LLGL1 directly binds to and promotes internalization of N-cadherin, and N-cadherin/LLGL1 interaction is inhibited by atypical protein kinase C-mediated phosphorylation of LLGL1, restricting the accumulation of AJCs to the basolateral-apical boundary. Disruption of the N-cadherin-LLGL1 interaction during cortical development in vivo is sufficient for PH. These findings reveal a mechanism responsible for the physical and functional connection between cell polarity and cell-cell adhesion machineries in mammalian cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Snail modulates cell metabolism in MDCK cells

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Haraguchi, Misako, E-mail: haraguci@m3.kufm.kagoshima-u.ac.jp [Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Indo, Hiroko P. [Department of Maxillofacial Radiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Iwasaki, Yasumasa [Health Care Center, Kochi University, Kochi 780-8520 (Japan); Iwashita, Yoichiro [Department of Maxillofacial Radiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Fukushige, Tomoko [Department of Dermatology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Majima, Hideyuki J. [Department of Maxillofacial Radiology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Izumo, Kimiko; Horiuchi, Masahisa [Department of Environmental Medicine, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Kanekura, Takuro [Department of Dermatology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Furukawa, Tatsuhiko [Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan); Ozawa, Masayuki [Department of Biochemistry and Molecular Biology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544 (Japan)

2013-03-22

Highlights: ► MDCK/snail cells were more sensitive to glucose deprivation than MDCK/neo cells. ► MDCK/snail cells had decreased oxidative phosphorylation, O{sub 2} consumption and ATP content. ► TCA cycle enzyme activity, but not expression, was lower in MDCK/snail cells. ► MDCK/snail cells showed reduced PDH activity and increased PDK1 expression. ► MDCK/snail cells showed reduced expression of GLS2 and ACLY. -- Abstract: Snail, a repressor of E-cadherin gene transcription, induces epithelial-to-mesenchymal transition and is involved in tumor progression. Snail also mediates resistance to cell death induced by serum depletion. By contrast, we observed that snail-expressing MDCK (MDCK/snail) cells undergo cell death at a higher rate than control (MDCK/neo) cells in low-glucose medium. Therefore, we investigated whether snail expression influences cell metabolism in MDCK cells. Although gylcolysis was not affected in MDCK/snail cells, they did exhibit reduced pyruvate dehydrogenase (PDH) activity, which controls pyruvate entry into the tricarboxylic acid (TCA) cycle. Indeed, the activity of multiple enzymes involved in the TCA cycle was decreased in MDCK/snail cells, including that of mitochondrial NADP{sup +}-dependent isocitrate dehydrogenase (IDH2), succinate dehydrogenase (SDH), and electron transport Complex II and Complex IV. Consequently, lower ATP content, lower oxygen consumption and increased survival under hypoxic conditions was also observed in MDCK/snail cells compared to MDCK/neo cells. In addition, the expression and promoter activity of pyruvate dehydrogenase kinase 1 (PDK1), which phosphorylates and inhibits the activity of PDH, was increased in MDCK/snail cells, while expression levels of glutaminase 2 (GLS2) and ATP-citrate lyase (ACLY), which are involved in glutaminolysis and fatty acid synthesis, were decreased in MDCK/snail cells. These results suggest that snail modulates cell metabolism by altering the expression and activity of

nduced pluripotent stem cells and cell therapy

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Banu İskender

2013-12-01

Full Text Available Human embryonic stem cells are derived from the inner cell mass of a blastocyst-stage embryo. They hold a huge promise for cell therapy with their self-renewing ability and pluripotency, which is known as the potential to differentiate into all cell types originating from three embryonic germ layers. However, their unique pluripotent feature could not be utilised for therapeutic purposes due to the ethical and legal problems during derivation. Recently, it was shown that the cells from adult tissues could be reverted into embryonic state, thereby restoring their pluripotent feature. This has strenghtened the possiblity of directed differentition of the reprogrammed somatic cells into the desired cell types in vitro and their use in regenerative medicine. Although these cells were termed as induced pluripotent cells, the mechanism of pluripotency has yet to be understood. Still, induced pluripotent stem cell technology is considered to be significant by proposing novel approaches in disease modelling, drug screening and cell therapy. Besides their self-renewing ability and their potential to differentiate into all cell types in a human body, they arouse a great interest in scientific world by being far from the ethical concerns regarding their embryonic counterparts and their unique feature of being patient-specific in prospective cell therapies. In this review, induced pluripotent stem cell technology and its role in cell-based therapies from past to present will be discussed. J Clin Exp Invest 2013; 4 (4: 550-561

Cell-mediated mutagenesis and cell transformation of mammalian cells by chemical carcinogens

International Nuclear Information System (INIS)

Huberman, E.; Langenbach, R.

1977-01-01

We have developed a cell-mediated mutagenesis assay in which cells with the appropriate markers for mutagenesis are co-cultivated with either lethally irradiated rodent embryonic cells that can metabolize carcinogenic hydrocarbons or with primary rat liver cells that can metabolize chemicals carcinogenic to the liver. During co-cultivation, the reactive metabolites of the procarcinogen appear to be transmitted to the mutable cells and induce mutations in them. Assays of this type make it possible to demonstrate a relationship between carcinogenic potency of the chemicals and their ability to induce mutations in mammalian cells. In addition, by simultaneously comparing the frequencies of transformation and mutation induced in normal diploid hamster cells by benzo(a)pyrene (BP) and one of its metabolites, it is possible to estimate the genetic target size for cell transformation in vitro

Gastric stem cells and gastric cancer stem cells

OpenAIRE

Han, Myoung-Eun; Oh, Sae-Ock

2013-01-01

The gastric epithelium is continuously regenerated by gastric stem cells, which give rise to various kinds of daughter cells, including parietal cells, chief cells, surface mucous cells, mucous neck cells, and enteroendocrine cells. The self-renewal and differentiation of gastric stem cells need delicate regulation to maintain the normal physiology of the stomach. Recently, it was hypothesized that cancer stem cells drive the cancer growth and metastasis. In contrast to conventional clonal ev...

Hybrid clone cells derived from human breast epithelial cells and human breast cancer cells exhibit properties of cancer stem/initiating cells.

Science.gov (United States)

Gauck, Daria; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S; Dittmar, Thomas

2017-08-02

The biological phenomenon of cell fusion has been associated with cancer progression since it was determined that normal cell × tumor cell fusion-derived hybrid cells could exhibit novel properties, such as enhanced metastatogenic capacity or increased drug resistance, and even as a mechanism that could give rise to cancer stem/initiating cells (CS/ICs). CS/ICs have been proposed as cancer cells that exhibit stem cell properties, including the ability to (re)initiate tumor growth. Five M13HS hybrid clone cells, which originated from spontaneous cell fusion events between M13SV1-EGFP-Neo human breast epithelial cells and HS578T-Hyg human breast cancer cells, and their parental cells were analyzed for expression of stemness and EMT-related marker proteins by Western blot analysis and confocal laser scanning microscopy. The frequency of ALDH1-positive cells was determined by flow cytometry using AldeRed fluorescent dye. Concurrently, the cells' colony forming capabilities as well as the cells' abilities to form mammospheres were investigated. The migratory activity of the cells was analyzed using a 3D collagen matrix migration assay. M13HS hybrid clone cells co-expressed SOX9, SLUG, CK8 and CK14, which were differently expressed in parental cells. A variation in the ALDH1-positive putative stem cell population was observed among the five hybrids ranging from 1.44% (M13HS-7) to 13.68% (M13HS-2). In comparison to the parental cells, all five hybrid clone cells possessed increased but also unique colony formation and mammosphere formation capabilities. M13HS-4 hybrid clone cells exhibited the highest colony formation capacity and second highest mammosphere formation capacity of all hybrids, whereby the mean diameter of the mammospheres was comparable to the parental cells. In contrast, the largest mammospheres originated from the M13HS-2 hybrid clone cells, whereas these cells' mammosphere formation capacity was comparable to the parental breast cancer cells. All M13HS

PINCH1 regulates cell-matrix and cell-cell adhesions, cell polarity and cell survival during the peri-implantation stage

DEFF Research Database (Denmark)

Li, Shaohua; Bordoy, Randi; Stanchi, Fabio

2005-01-01

PINCH1 is composed of 5 LIM domains, binds integrin-linked kinase (ILK) and locates to integrin-mediated adhesion sites. In order to investigate PINCH1 function we generated mice and embryonic stem (ES) cell-derived embryoid bodies (EBs) lacking the PINCH1 gene. Similar to mice lacking beta1...... integrin or Ilk, loss of PINCH1 arrested development at the peri-implantation stage. In contrast to beta1 integrin or Ilk mutants, however, disruption of the PINCH1 gene produced implantation chambers with visible cell clumps even at embryonic day 9.5. In order to define the phenotype leading to the peri...... not observed in beta1 integrin- or ILK-deficient mice or EBs, included abnormal cell-cell adhesion of endoderm and epiblast as well as the presence of apoptotic cells in the endodermal cell layer. Although ILK and PINCH1 were shown to be involved in the phosphorylation of serine-473 of PKB/Akt, immunostaining...

In vitro differentiation of primordial germ cells and oocyte-like cells from stem cells.

Science.gov (United States)

Costa, José J N; Souza, Glaucinete B; Soares, Maria A A; Ribeiro, Regislane P; van den Hurk, Robert; Silva, José R V

2018-02-01

Infertility is the result of failure due to an organic disorder of the reproductive organs, especially their gametes. Recently, much progress has been made on generating germ cells, including oocytes, from various types of stem cells. This review focuses on advances in female germ cell differentiation from different kinds of stem cells, with emphasis on embryonic stem cells, adult stem cells, and induced pluripotent stem cells. The advantages and disadvantages of the derivation of female germ cells from several types of stem cells are also highlighted, as well as the ability of stem cells to generate mature and functional female gametes. This review shows that stem cell therapies have opened new frontiers in medicine, especially in the reproductive area, with the possibility of regenerating fertility.

Cell cycle control by components of cell anchorage

OpenAIRE

Gad, Annica

2005-01-01

Extracellular factors, such as growth factors and cell anchorage to the extracellular matrix, control when and where cells may proliferate. This control is abolished when a normal cell transforms into a tumour cell. The control of cell proliferation by cell anchorage was elusive and less well studied than the control by growth factors. Therefore, we aimed to clarify at what points in the cell cycle and through which molecular mechanisms cell anchorage controls cell cycle pro...

Casticin impairs cell growth and induces cell apoptosis via cell cycle arrest in human oral cancer SCC-4 cells.

Science.gov (United States)

Chou, Guan-Ling; Peng, Shu-Fen; Liao, Ching-Lung; Ho, Heng-Chien; Lu, Kung-Wen; Lien, Jin-Cherng; Fan, Ming-Jen; La, Kuang-Chi; Chung, Jing-Gung

2018-02-01

Casticin, a polymethoxyflavone, present in natural plants, has been shown to have biological activities including anti-cancer activities. Herein, we investigated the anti-oral cancer activity of casticin on SCC-4 cells in vitro. Viable cells, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS) production, and Ca 2+ production, levels of ΔΨ m and caspase activity were measured by flow cytometric assay. Cell apoptosis associated protein expressions were examined by Western blotting and confocal laser microscopy. Results indicated that casticin induced cell morphological changes, DNA condensation and damage, decreased the total viable cells, induced G 2 /M phase arrest in SCC-4 cells. Casticin promoted ROS and Ca 2+ productions, decreases the levels of ΔΨ m , promoted caspase-3, -8, and -9 activities in SCC-4 cells. Western blotting assay demonstrated that casticin affect protein level associated with G2/M phase arrest and apoptosis. Confocal laser microscopy also confirmed that casticin increased the translocation of AIF and cytochrome c in SCC-4 cells. In conclusion, casticin decreased cell number through G 2 /M phase arrest and the induction of cell apoptosis through caspase- and mitochondria-dependent pathways in SCC-4 cells. © 2017 Wiley Periodicals, Inc.

Skin Stem Cells in Skin Cell Therapy

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Mollapour Sisakht

2015-12-01

Full Text Available Context Preclinical and clinical research has shown that stem cell therapy is a promising therapeutic option for many diseases. This article describes skin stem cells sources and their therapeutic applications. Evidence Acquisition Compared with conventional methods, cell therapy reduces the surgical burden for patients because it is simple and less time-consuming. Skin cell therapy has been developed for variety of diseases. By isolation of the skin stem cell from the niche, in vitro expansion and transplantation of cells offers a surprising healing capacity profile. Results Stem cells located in skin cells have shown interesting properties such as plasticity, transdifferentiation, and specificity. Mesenchymal cells of the dermis, hypodermis, and other sources are currently being investigated to promote regeneration. Conclusions Because skin stem cells are highly accessible from autologous sources and their immunological profile is unique, they are ideal for therapeutic approaches. Optimization of administrative routes requires more investigation own to the lack of a standard protocol.

Cell biology of mesangial cells: the third cell that maintains the glomerular capillary.

Science.gov (United States)

Kurihara, Hidetake; Sakai, Tatsuo

2017-03-01

The renal glomerulus consists of glomerular endothelial cells, podocytes, and mesangial cells, which cooperate with each other for glomerular filtration. We have produced monoclonal antibodies against glomerular cells in order to identify different types of glomerular cells. Among these antibodies, the E30 clone specifically recognizes the Thy1.1 molecule expressed on mesangial cells. An injection of this antibody into rats resulted in mesangial cell-specific injury within 15 min, and induced mesangial proliferative glomerulonephritis in a reproducible manner. We examined the role of mesangial cells in glomerular function using several experimental tools, including an E30-induced nephritis model, mesangial cell culture, and the deletion of specific genes. Herein, we describe the characterization of E30-induced nephritis, formation of the glomerular capillary network, mesangial matrix turnover, and intercellular signaling between glomerular cells. New molecules that are involved in a wide variety of mesangial cell functions are also introduced.

Natural killer cells for immunotherapy – Advantages of cell lines over blood NK cells

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Hans eKlingemann

2016-03-01

Full Text Available Natural killer cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells form a patient’s blood since they represent only 10% of the lymphocytes. Especially, cancer patients are known to have dysfunctional NK cells. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T-cells. Establishing cell lines from donor blood NK cells have not been successful, in contrast to blood NK cells obtained from patients with a clonal NK cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. However, except for the NK-92 cell line none of the other six known cell lines has consistent and reproducibly high anti-tumor cytotoxicity, nor can they be easily genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through ADCC. NK-92 is also the only cell line product that has been widely given to patients with advanced cancer with demonstrated efficiency and minimal side effects.

Cell sheet technology and cell patterning for biofabrication

Energy Technology Data Exchange (ETDEWEB)

Hannachi, Imen Elloumi; Yamato, Masayuki; Okano, Teruo [Institute of Advanced Biomedical Engineering and Science, Tokyo Women' s Medical University, 8-1 Kawada-cho, Shinjuku, Tokyo (Japan)

2009-06-01

We have developed cell sheet technology as a modern method for the fabrication of functional tissue-like and organ-like structures. This technology allows for a sheet of interconnected cells and cells in full contact with their natural extracellular environment to be obtained. A cell sheet can be patterned and composed according to more than one cell type. The key technology of cell sheet engineering is that a fabricated cell sheet can be harvested and transplanted utilizing temperature-responsive surfaces. In this review, we summarize different aspects of cell sheet engineering and provide a survey of the application of cell sheets as a suitable material for biofabrication and clinics. Moreover, since cell micropatterning is a key tool for cell sheet engineering, in this review we focus on the introduction of our approaches to cell micropatterning and cell co-culture to the principles of automation and how they can be subjected to easy robotics programming. Finally, efforts towards making cell sheet technology suitable for biofabrication and robotic biofabrication are also summarized. (topical review)

Free-zone electrophoresis of animal cells. 1: Experiments on cell-cell interactions

Science.gov (United States)

Todd, P. W.; Hjerten, S.

1985-01-01

The electrophoretically migrating zones wasa monitored. The absence of fluid flows in the direction of migration permits direct measurement of electrophoretic velocities of any material. Sedimentation is orthogonal to electrokinetic motion and the effects of particle-particle interaction on electrophoretic mobility is studied by free zone electrophoresis. Fixed erythrocytes at high concentrations, mixtures of fixed erythrocytes from different animal species, and mixtures of cultured human cells were studied in low ionic strength buffers. The electrophoretic velocity of fixed erythrocytes was not altered by increasing cell concentration or by the mixing of erythrocytes from different species. When zones containing cultured human glial cells and neuroblastoma cells are permitted to interact during electrophoresis, altered migration patterns occur. It is found that cell-cell interactions depends upon cell type.

Characterization of a Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Cell Line CVG-1.

Science.gov (United States)

Velásquez, Celestino; Amako, Yutaka; Harold, Alexis; Toptan, Tuna; Chang, Yuan; Shuda, Masahiro

2018-01-01

Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation.

c-Myc-Dependent Cell Competition in Human Cancer Cells.

Science.gov (United States)

Patel, Manish S; Shah, Heta S; Shrivastava, Neeta

2017-07-01

Cell Competition is an interaction between cells for existence in heterogeneous cell populations of multicellular organisms. This phenomenon is involved in initiation and progression of cancer where heterogeneous cell populations compete directly or indirectly for the survival of the fittest based on differential gene expression. In Drosophila, cells having lower dMyc expression are eliminated by cell competition through apoptosis when present in the milieu of cells having higher dMyc expression. Thus, we designed a study to develop c-Myc (human homolog) dependent in vitro cell competition model of human cancer cells. Cells with higher c-Myc were transfected with c-myc shRNA to prepare cells with lower c-Myc and then co-cultured with the same type of cells having a higher c-Myc in equal ratio. Cells with lower c-Myc showed a significant decrease in numbers when compared with higher c-Myc cells, suggesting "loser" and "winner" status of cells, respectively. During microscopy, engulfment of loser cells by winner cells was observed with higher expression of JNK in loser cells. Furthermore, elimination of loser cells was prevented significantly, when co-cultured cells were treated with the JNK (apoptosis) inhibitor. Above results indicate elimination of loser cells in the presence of winner cells by c-Myc-dependent mechanisms of cell competition in human cancer cells. This could be an important mechanism in human tumors where normal cells are eliminated by c-Myc-overexpressed tumor cells. J. Cell. Biochem. 118: 1782-1791, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

β-Cell regeneration through the transdifferentiation of pancreatic cells: Pancreatic progenitor cells in the pancreas.

Science.gov (United States)

Kim, Hyo-Sup; Lee, Moon-Kyu

2016-05-01

Pancreatic progenitor cell research has been in the spotlight, as these cells have the potential to replace pancreatic β-cells for the treatment of type 1 and 2 diabetic patients with the absence or reduction of pancreatic β-cells. During the past few decades, the successful treatment of diabetes through transplantation of the whole pancreas or isolated islets has nearly been achieved. However, novel sources of pancreatic islets or insulin-producing cells are required to provide sufficient amounts of donor tissues. To overcome this limitation, the use of pancreatic progenitor cells is gaining more attention. In particular, pancreatic exocrine cells, such as duct epithelial cells and acinar cells, are attractive candidates for β-cell regeneration because of their differentiation potential and pancreatic lineage characteristics. It has been assumed that β-cell neogenesis from pancreatic progenitor cells could occur in pancreatic ducts in the postnatal stage. Several studies have shown that insulin-producing cells can arise in the duct tissue of the adult pancreas. Acinar cells also might have the potential to differentiate into insulin-producing cells. The present review summarizes recent progress in research on the transdifferentiation of pancreatic exocrine cells into insulin-producing cells, especially duct and acinar cells.

Intrinsic Plasma Cell Differentiation Defects in B Cell Expansion with NF-κB and T Cell Anergy Patient B Cells

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Swadhinya Arjunaraja

2017-08-01

Full Text Available B cell Expansion with NF-κB and T cell Anergy (BENTA disease is a novel B cell lymphoproliferative disorder caused by germline, gain-of-function mutations in the lymphocyte scaffolding protein CARD11, which drives constitutive NF-κB signaling. Despite dramatic polyclonal expansion of naive and immature B cells, BENTA patients also present with signs of primary immunodeficiency, including markedly reduced percentages of class-switched/memory B cells and poor humoral responses to certain vaccines. Using purified naive B cells from our BENTA patient cohort, here we show that BENTA B cells exhibit intrinsic defects in B cell differentiation. Despite a profound in vitro survival advantage relative to normal donor B cells, BENTA patient B cells were severely impaired in their ability to differentiate into short-lived IgDloCD38hi plasmablasts or CD138+ long-lived plasma cells in response to various stimuli. These defects corresponded with diminished IgG antibody production and correlated with poor induction of specific genes required for plasma cell commitment. These findings provide important mechanistic clues that help explain both B cell lymphocytosis and humoral immunodeficiency in BENTA disease.

Activated Allogeneic NK Cells Preferentially Kill Poor Prognosis B-Cell Chronic Lymphocytic Leukemia Cells.

Science.gov (United States)

Sánchez-Martínez, Diego; Lanuza, Pilar M; Gómez, Natalia; Muntasell, Aura; Cisneros, Elisa; Moraru, Manuela; Azaceta, Gemma; Anel, Alberto; Martínez-Lostao, Luis; Villalba, Martin; Palomera, Luis; Vilches, Carlos; García Marco, José A; Pardo, Julián

2016-01-01

Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV ) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.

Lung cells support osteosarcoma cell migration and survival.

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Yu, Shibing; Fourman, Mitchell Stephen; Mahjoub, Adel; Mandell, Jonathan Brendan; Crasto, Jared Anthony; Greco, Nicholas Giuseppe; Weiss, Kurt Richard

2017-01-25

Osteosarcoma (OS) is the most common primary bone tumor, with a propensity to metastasize to the lungs. Five-year survival for metastatic OS is below 30%, and has not improved for several decades despite the introduction of multi-agent chemotherapy. Understanding OS cell migration to the lungs requires an evaluation of the lung microenvironment. Here we utilized an in vitro lung cell and OS cell co-culture model to explore the interactions between OS and lung cells, hypothesizing that lung cells would promote OS cell migration and survival. The impact of a novel anti-OS chemotherapy on OS migration and survival in the lung microenvironment was also examined. Three human OS cell lines (SJSA-1, Saos-2, U-2) and two human lung cell lines (HULEC-5a, MRC-5) were cultured according to American Type Culture Collection recommendations. Human lung cell lines were cultured in growth medium for 72 h to create conditioned media. OS proliferation was evaluated in lung co-culture and conditioned media microenvironment, with a murine fibroblast cell line (NIH-3 T3) in fresh growth medium as controls. Migration and invasion were measured using a real-time cell analysis system. Real-time PCR was utilized to probe for Aldehyde Dehydrogenase (ALDH1) expression. Osteosarcoma cells were also transduced with a lentivirus encoding for GFP to permit morphologic analysis with fluorescence microscopy. The anti-OS efficacy of Disulfiram, an ALDH-inhibitor previously shown to inhibit OS cell proliferation and metastasis in vitro, was evaluated in each microenvironment. Lung-cell conditioned medium promoted osteosarcoma cell migration, with a significantly higher attractive effect on all three osteosarcoma cell lines compared to basic growth medium, 10% serum containing medium, and NIH-3 T3 conditioned medium (p cell conditioned medium induced cell morphologic changes, as demonstrated with GFP-labeled cells. OS cells cultured in lung cell conditioned medium had increased alkaline

Human regulatory B cells control the TFH cell response.

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Achour, Achouak; Simon, Quentin; Mohr, Audrey; Séité, Jean-François; Youinou, Pierre; Bendaoud, Boutahar; Ghedira, Ibtissem; Pers, Jacques-Olivier; Jamin, Christophe

2017-07-01

Follicular helper T (T FH ) cells support terminal B-cell differentiation. Human regulatory B (Breg) cells modulate cellular responses, but their control of T FH cell-dependent humoral immune responses is unknown. We sought to assess the role of Breg cells on T FH cell development and function. Human T cells were polyclonally stimulated in the presence of IL-12 and IL-21 to generate T FH cells. They were cocultured with B cells to induce their terminal differentiation. Breg cells were included in these cultures, and their effects were evaluated by using flow cytometry and ELISA. B-cell lymphoma 6, IL-21, inducible costimulator, CXCR5, and programmed cell death protein 1 (PD-1) expressions increased on stimulated human T cells, characterizing T FH cell maturation. In cocultures they differentiated B cells into CD138 + plasma and IgD - CD27 + memory cells and triggered immunoglobulin secretions. Breg cells obtained by Toll-like receptor 9 and CD40 activation of B cells prevented T FH cell development. Added to T FH cell and B-cell cocultures, they inhibited B-cell differentiation, impeded immunoglobulin secretions, and expanded Foxp3 + CXCR5 + PD-1 + follicular regulatory T cells. Breg cells modulated IL-21 receptor expressions on T FH cells and B cells, and their suppressive activities involved CD40, CD80, CD86, and intercellular adhesion molecule interactions and required production of IL-10 and TGF-β. Human Breg cells control T FH cell maturation, expand follicular regulatory T cells, and inhibit the T FH cell-mediated antibody secretion. These novel observations demonstrate a role for the Breg cell in germinal center reactions and suggest that deficient activities might impair the T FH cell-dependent control of humoral immunity and might lead to the development of aberrant autoimmune responses. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Single-cell protein secretomic signatures as potential correlates to tumor cell lineage evolution and cell-cell interaction

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Minsuk eKwak

2013-02-01

Full Text Available Secreted proteins including cytokines, chemokines and growth factors represent important functional regulators mediating a range of cellular behavior and cell-cell paracrine/autocrine signaling, e.g. in the immunological system, tumor microenvironment or stem cell niche. Detection of these proteins is of great value not only in basic cell biology but also for diagnosis and therapeutic monitoring of human diseases such as cancer. However, due to co-production of multiple effector proteins from a single cell, referred to as polyfunctionality, it is biologically informative to measure a panel of secreted proteins, or secretomic signature, at the level of single cells. Recent evidence further indicates that a genetically-identical cell population can give rise to diverse phenotypic differences. It is known that cytokines, for example, in the immune system define the effector functions and lineage differentiation of immune cells. In this Perspective Article, we hypothesize that protein secretion profile may represent a universal measure to identify the definitive correlate in the larger context of cellular functions to dissect cellular heterogeneity and evolutionary lineage relationship in human cancer.

Mesenchymal Stem Cells Induce Epithelial to Mesenchymal Transition in Colon Cancer Cells through Direct Cell-to-Cell Contact

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Hidehiko Takigawa

2017-05-01

Full Text Available We previously reported that in an orthotopic nude mouse model of human colon cancer, bone marrow–derived mesenchymal stem cells (MSCs migrated to the tumor stroma and promoted tumor growth and metastasis. Here, we evaluated the proliferation and migration ability of cancer cells cocultured with MSCs to elucidate the mechanism of interaction between cancer cells and MSCs. Proliferation and migration of cancer cells increased following direct coculture with MSCs but not following indirect coculture. Thus, we hypothesized that direct contact between cancer cells and MSCs was important. We performed a microarray analysis of gene expression in KM12SM colon cancer cells directly cocultured with MSCs. Expression of epithelial-mesenchymal transition (EMT–related genes such as fibronectin (FN, SPARC, and galectin 1 was increased by direct coculture with MSCs. We also confirmed the upregulation of these genes with real-time polymerase chain reaction. Gene expression was not elevated in cancer cells indirectly cocultured with MSCs. Among the EMT-related genes upregulated by direct coculture with MSCs, we examined the immune localization of FN, a well-known EMT marker. In coculture assay in chamber slides, expression of FN was seen only at the edges of cancer clusters where cancer cells directly contacted MSCs. FN expression in cancer cells increased at the tumor periphery and invasive edge in orthotopic nude mouse tumors and human colon cancer tissues. These results suggest that MSCs induce EMT in colon cancer cells via direct cell-to-cell contact and may play an important role in colon cancer metastasis.

Activated allogeneic NK cells preferentially kill poor prognosis B-cell chronic lymphocytic leukemia cells

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Diego Sanchez-Martinez

2016-10-01

Full Text Available Mutational status of TP53 together with expression of wild type (wt IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL patients. Adoptive cell therapy using allogeneic HLA mismatched Natural Killer (NK cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs the most effective stimulus to activate NK cells. Here we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell activating receptors (NKG2D and NCRs and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments.□

Mast cells dysregulate apoptotic and cell cycle genes in mucosal squamous cell carcinoma

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Davis Paul

2006-12-01

Full Text Available Abstract Background Mucosal squamous cell carcinoma of the head and neck is a disease of high mortality and morbidity. Interactions between the squamous cell carcinoma and the host's local immunity, and how the latter contributes to the biological behavior of the tumor are unclear. In vivo studies have demonstrated sequential mast cell infiltration and degranulation during squamous cell carcinogenesis. The degree of mast cell activation correlates closely with distinct phases of hyperkeratosis, dysplasia, carcinoma in-situ and invasive carcinoma. However, the role of mast cells in carcinogenesis is unclear. Aim This study explores the effects of mast cells on the proliferation and gene expression profile of mucosal squamous cell carcinoma using human mast cell line (HMC-1 and human glossal squamous cell carcinoma cell line (SCC25. Methods HMC-1 and SCC25 were co-cultured in a two-compartment chamber, separated by a polycarbonate membrane. HMC-1 was stimulated to degranulate with calcium ionophore A23187. The experiments were done in quadruplicate. Negative controls were established where SCC25 were cultured alone without HMC-1. At 12, 24, 48 and 72 hours, proliferation and viability of SCC25 were assessed with MTT colorimetric assay. cDNA microarray was employed to study differential gene expression between co-cultured and control SCC25. Results HMC-1/SCC25 co-culture resulted in suppression of growth rate for SCC-25 (34% compared with 110% for the control by 72 hours, p Conclusion We show that mast cells have a direct inhibitory effect on the proliferation of mucosal squamous cell carcinoma in vitro by dysregulating key genes in apoptosis and cell cycle control.

Plant cell wall polysaccharide analysis during cell elongation

DEFF Research Database (Denmark)

Guo, Xiaoyuan

Plant cell walls are complex structures whose composition and architecture are important to various cellular activities. Plant cell elongation requires a high level of rearrangement of the cell wall polymers to enable cell expansion. However, the cell wall polysaccharides dynamics during plant cell...... elongation is poorly understood. This PhD project aims to elucidate the cell wall compositional and structural change during cell elongation by using Comprehensive Microarray Polymer Profiling (CoMPP), microscopic techniques and molecular modifications of cell wall polysaccharide. Developing cotton fibre......, pea and Arabidopsis thaliana were selected as research models to investigate different types of cell elongation, developmental elongation and tropism elongation. A set of comprehensive analysis covering 4 cotton species and 11 time points suggests that non-cellulosic polysaccharides contribute...

Estrogen enhanced cell-cell signalling in breast cancer cells exposed to targeted irradiation

International Nuclear Information System (INIS)

Shao, Chunlin; Folkard, Melvyn; Held, Kathryn D; Prise, Kevin M

2008-01-01

Radiation-induced bystander responses, where cells respond to their neighbours being irradiated are being extensively studied. Although evidence shows that bystander responses can be induced in many types of cells, it is not known whether there is a radiation-induced bystander effect in breast cancer cells, where the radiosensitivity may be dependent on the role of the cellular estrogen receptor (ER). This study investigated radiation-induced bystander responses in estrogen receptor-positive MCF-7 and estrogen receptor-negative MDA-MB-231 breast cancer cells. The influence of estrogen and anti-estrogen treatments on the bystander response was determined by individually irradiating a fraction of cells within the population with a precise number of helium-3 using a charged particle microbeam. Damage was scored as chromosomal damage measured as micronucleus formation. A bystander response measured as increased yield of micronucleated cells was triggered in both MCF-7 and MDA-MB-231 cells. The contribution of the bystander response to total cell damage in MCF-7 cells was higher than that in MDA-MB-231 cells although the radiosensitivity of MDA-MB-231 was higher than MCF-7. Treatment of cells with 17β-estradiol (E2) increased the radiosensitivity and the bystander response in MCF-7 cells, and the effect was diminished by anti-estrogen tamoxifen (TAM). E2 also increased the level of intracellular reactive oxygen species (ROS) in MCF-7 cells in the absence of radiation. In contrast, E2 and TAM had no influence on the bystander response and ROS levels in MDA-MB-231 cells. Moreover, the treatment of MCF-7 cells with antioxidants eliminated both the E2-induced ROS increase and E2-enhanced bystander response triggered by the microbeam irradiation, which indicates that ROS are involved in the E2-enhanced bystander micronuclei formation after microbeam irradiation. The observation of bystander responses in breast tumour cells may offer new potential targets for radiation

Follow-the-leader cell migration requires biased cell-cell contact and local microenvironmental signals

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Wynn, Michelle L.; Rupp, Paul; Trainor, Paul A.; Schnell, Santiago; Kulesa, Paul M.

2013-06-01

Directed cell migration often involves at least two types of cell motility that include multicellular streaming and chain migration. However, what is unclear is how cell contact dynamics and the distinct microenvironments through which cells travel influence the selection of one migratory mode or the other. The embryonic and highly invasive neural crest (NC) are an excellent model system to study this question since NC cells have been observed in vivo to display both of these types of cell motility. Here, we present data from tissue transplantation experiments in chick and in silico modeling that test our hypothesis that cell contact dynamics with each other and the microenvironment promote and sustain either multicellular stream or chain migration. We show that when premigratory cranial NC cells (at the pre-otic level) are transplanted into a more caudal region in the head (at the post-otic level), cells alter their characteristic stream behavior and migrate in chains. Similarly, post-otic NC cells migrate in streams after transplantation into the pre-otic hindbrain, suggesting that local microenvironmental signals dictate the mode of NC cell migration. Simulations of an agent-based model (ABM) that integrates the NC cell behavioral data predict that chain migration critically depends on the interplay of biased cell-cell contact and local microenvironment signals. Together, this integrated modeling and experimental approach suggests new experiments and offers a powerful tool to examine mechanisms that underlie complex cell migration patterns.

Hilar mossy cell circuitry controlling dentate granule cell excitability

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Seiichiro eJinde

2013-02-01

Full Text Available Glutamatergic hilar mossy cells of the dentate gyrus can either excite or inhibit distant granule cells, depending on whether their direct excitatory projections to granule cells or their projections to local inhibitory interneurons dominate. However, it remains controversial whether the net effect of mossy cell loss is granule cell excitation or inhibition. Clarifying this controversy has particular relevance to temporal lobe epilepsy, which is marked by dentate granule cell hyperexcitability and extensive loss of dentate hilar mossy cells. Two diametrically opposed hypotheses have been advanced to explain this granule cell hyperexcitability – the dormant basket cell and the irritable mossy cell hypotheses. The dormant basket cell hypothesis proposes that mossy cells normally exert a net inhibitory effect on granule cells and therefore their loss causes dentate granule cell hyperexcitability. The irritable mossy cell hypothesis takes the opposite view that mossy cells normally excite granule cells and that the surviving mossy cells in epilepsy increase their activity, causing granule cell excitation. The inability to eliminate mossy cells selectively has made it difficult to test these two opposing hypotheses. To this end, we developed a transgenic toxin-mediated, mossy cell-ablation mouse line. Using these mutants, we demonstrated that the extensive elimination of hilar mossy cells causes granule cell hyperexcitability, although the mossy cell loss observed appeared insufficient to cause clinical epilepsy. In this review, we focus on this topic and also suggest that different interneuron populations may mediate mossy cell-induced translamellar lateral inhibition and intralamellar recurrent inhibition. These unique local circuits in the dentate hilar region may be centrally involved in the functional organization of the dentate gyrus.

Cell Biochips

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Pioufle, B. Le; Picollet-D'Hahan, N.

A cell biochip is a microsystem, equipped with electronic and microfluidic functions, designed to manipulate or analyse living cells. The first publications in this emerging area of research appeared toward the end of the 1980s. In 1989 Washizu described a biochip designed to fuse two cells by electropermeabilisation of the cytoplasmic membrane [1]. Research centers have devised a whole range of cell chip structures, for simultaneous or sequential analysis of single cells, cell groups, or cell tissues reconstituted on the chip. The cells are arranged in a square array on a parallel cell chip for parallel analysis, while they are examined and processed one by one in a microchannel in the case of a series cell chip. In contrast to these biochips for high-throughput analysis of a large number of cells, single-cell chips focus on the analysis of a single isolated cell. As in DNA microarrays, where a large number of oligonucleotides are ordered in a matrix array, parallel cell chips order living cells in a similar way. At each point of the array, the cells can be isolated, provided that the cell type allows this, e.g., blood cells, or cultivated in groups (most adhesion cells can only survive in groups). The aim is to allow massively parallel analysis or processing. Le Pioufle et al. describe a microdevice for the culture of single cells or small groups of cells in a micropit array [2]. Each pit is equipped to stimulate the cell or group of cells either electrically or fluidically. Among the applications envisaged are gene transfer, cell sorting, and screening in pharmacology. A complementary approach, combining the DNA microarray and cell biochip ideas, has been put forward by Bailey et al. [3]. Genes previously arrayed on the chip transfect the cultured cells on the substrate depending on their position in the array (see Fig. 19.1). This way of achieving differential lipofection on a chip was then taken up again by Yoshikawa et al. [4] with primary cells, more

Pharmacologic suppression of target cell recognition by engineered T cells expressing chimeric T-cell receptors.

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Alvarez-Vallina, L; Yañez, R; Blanco, B; Gil, M; Russell, S J

2000-04-01

Adoptive therapy with autologous T cells expressing chimeric T-cell receptors (chTCRs) is of potential interest for the treatment of malignancy. To limit possible T-cell-mediated damage to normal tissues that weakly express the targeted tumor antigen (Ag), we have tested a strategy for the suppression of target cell recognition by engineered T cells. Jurkat T cells were transduced with an anti-hapten chTCR tinder the control of a tetracycline-suppressible promoter and were shown to respond to Ag-positive (hapten-coated) but not to Ag-negative target cells. The engineered T cells were then reacted with hapten-coated target cells at different effector to target cell ratios before and after exposure to tetracycline. When the engineered T cells were treated with tetracycline, expression of the chTCR was greatly decreased and recognition of the hapten-coated target cells was completely suppressed. Tetracycline-mediated suppression of target cell recognition by engineered T cells may be a useful strategy to limit the toxicity of the approach to cancer gene therapy.

The usefulness of three-dimensional cell culture in induction of cancer stem cells from esophageal squamous cell carcinoma cell lines

International Nuclear Information System (INIS)

Fujiwara, Daisuke; Kato, Kazunori; Nohara, Shigeo; Iwanuma, Yoshimi; Kajiyama, Yoshiaki

2013-01-01

Highlights: •Spheroids were created from esophageal carcinoma cells using NanoCulture® Plates. •The proportion of strongly ALDH-positive cells increased in 3-D culture. •Expression of cancer stem cell-related genes was enhanced in 3-D culture. •CA-9 expression was enhanced, suggesting hypoxia had been induced in 3-D culture. •Drug resistance was increased. 3-D culture is useful for inducing cancer stem cells. -- Abstract: In recent years, research on resistance to chemotherapy and radiotherapy in cancer treatment has come under the spotlight, and researchers have also begun investigating the relationship between resistance and cancer stem cells. Cancer stem cells are assumed to be present in esophageal cancer, but experimental methods for identification and culture of these cells have not yet been established. To solve this problem, we created spheroids using a NanoCulture® Plate (NCP) for 3-dimensional (3-D) cell culture, which was designed as a means for experimentally reproducing the 3-D structures found in the body. We investigated the potential for induction of cancer stem cells from esophageal cancer cells. Using flow cytometry we analyzed the expression of surface antigen markers CD44, CD133, CD338 (ABCG2), CD318 (CDCP1), and CD326 (EpCAM), which are known cancer stem cell markers. None of these surface antigen markers showed enhanced expression in 3-D cultured cells. We then analyzed aldehyde dehydrogenase (ALDH) enzymatic activity using the ALDEFLUOR reagent, which can identify immature cells such as stem cells and precursor cells. 3-D-cultured cells were strongly positive for ALDH enzyme activity. We also analyzed the expression of the stem cell-related genes Sox-2, Nanog, Oct3/4, and Lin28 using RT-PCR. Expression of Sox-2, Nanog, and Lin28 was enhanced. Analysis of expression of the hypoxic surface antigen marker carbonic anhydrase-9 (CA-9), which is an indicator of cancer stem cell induction and maintenance, revealed that CA-9 expression

A novel cell division factor from tobacco 2B-13 cells that induced cell division in auxin-starved tobacco BY-2 cells

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Shimizu, Takashi; Eguchi, Kentaro; Nishida, Ikuo; Laukens, Kris; Witters, Erwin; van Onckelen, Harry; Nagata, Toshiyuki

2006-06-01

Effects of auxin as plant hormones are widespread; in fact in almost all aspects of plant growth and development auxin plays a pivotal role. Although auxin is required for propagating cell division in plant cells, its effect upon cell division is least understood. If auxin is depleted from the culture medium, cultured cells cease to divide. It has been demonstrated in this context that the addition of auxin to auxin-starved nondividing tobacco BY-2 cells induced semisynchronous cell division. On the other hand, there are some cell lines, named habituated cells, that can grow without auxin. The cause and reason for the habituated cells have not been clarified. A habituated cell line named 2B-13 is derived from the tobacco BY-2 cell line, which has been most intensively studied among plant cell lines. When we tried to find the difference between two cell lines of BY-2 and 2B-13 cells, we found that the addition of culture filtrated from the auxin-habituated 2B-13 cells induced semisynchronous cell division in auxin-starved BY-2 cells. The cell division factor (CDF) that is responsible for inducing cell division in auxin-starved BY-2 cells was purified to near-homogeneity by sequential passage through a hydroxyapatite column, a ConA Sepharose column and a Sephadex gel filtration column. The resulting purified fraction appeared as a single band of high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels by silver staining and was able to induce cell division in auxin-starved BY-2 cells. Identification of the protein by MALD-TOF-MS/MS revealed that it is structurally related to P-glycoprotein from Gossypioides kirkii, which belongs to ATP-binding cassette (ABC)-transporters. The significance of CDF as a possible ABC-transporter is discussed in relationship to auxin-autotrophic growth and auxin-signaling pathway.

Cell damage evaluation of mammalian cells in cell manipulation by amplified femtosecond ytterbium laser

Science.gov (United States)

Hong, Z.-Y.; Iino, T.; Hagihara, H.; Maeno, T.; Okano, K.; Yasukuni, R.; Hosokawa, Y.

2018-03-01

A micrometer-scale explosion with cavitation bubble generation is induced by focusing a femtosecond laser in an aqueous solution. We have proposed to apply the explosion as an impulsive force to manipulate mammalian cells especially in microfluidic chip. Herein, we employed an amplified femtosecond ytterbium laser as an excitation source for the explosion and evaluated cell damage in the manipulation process to clarify the application potential. The damage of C2C12 myoblast cell prepared as a representative mammalian cell was investigated as a function of distance between cell and laser focal point. Although the cell received strong damage on the direct laser irradiation condition, the damage sharply decreased with increasing distance. Since the threshold distance, above which the cell had no damage, was consistent with radius of the cavitation bubble, impact of the cavitation bubble would be a critical factor for the cell damage. The damage had strong nonlinearity in the pulse energy dependence. On the other hand, cell position shift by the impact of the cavitation bubble was almost proportional to the pulse energy. In balance between the cell viability and the cell position shift, we elucidated controllability of the cell manipulation in microfluidic chip.

Can resting B cells present antigen to T cells

International Nuclear Information System (INIS)

Ashwell, J.D.; DeFranco, A.L.; Paul, W.E.; Schwartz, R.H.

1985-01-01

Antigen stimulation of T lymphocytes can occur only in the presence of an antigen-presenting cell (APC). An ever-increasing number of cell types have been found to act as APCs; these include macrophages, splenic and lymph node dendritic cells, and Langerhans cells of the skin. Although activated B lymphocytes and B cell lymphomas are known to serve as APCs, it has been generally believed that resting B cells cannot perform this function. However, in recent studies the authors have found that resting B cells can indeed present soluble antigen to T cell clones as well as to antigen-primed T cells. The previous difficulty in demonstrating this activity can be explained by the finding that, in contrast to macrophages and dendritic cells, the antigen-presenting ability of resting B cells is very radiosensitive. Macrophages are usually irradiated with 2000-3300 rads to prevent them from incorporating [ 3 H]thymidine in the T cell proliferation assay. Resting B cells, however, begin to lose presenting function at 1500 rads and have completely lost this activity at 3300 rads. It was also possible to distinguish two distinct T cell clonal phenotypes when resting B cells were used as APCs on the basis of two different assays (T cell proliferation, and B cell proliferation resulting from T cell activation). The majority of T cell clones tested were capable of both proliferating themselves and inducing the proliferation of B cells. Some T cells clones, however, could not proliferate in the presence of antigen and B cell APCs, although they were very good at inducing the proliferation of B cells

Analysis of individual cells identifies cell-to-cell variability following induction of cellular senescence.

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Wiley, Christopher D; Flynn, James M; Morrissey, Christapher; Lebofsky, Ronald; Shuga, Joe; Dong, Xiao; Unger, Marc A; Vijg, Jan; Melov, Simon; Campisi, Judith

2017-10-01

Senescent cells play important roles in both physiological and pathological processes, including cancer and aging. In all cases, however, senescent cells comprise only a small fraction of tissues. Senescent phenotypes have been studied largely in relatively homogeneous populations of cultured cells. In vivo, senescent cells are generally identified by a small number of markers, but whether and how these markers vary among individual cells is unknown. We therefore utilized a combination of single-cell isolation and a nanofluidic PCR platform to determine the contributions of individual cells to the overall gene expression profile of senescent human fibroblast populations. Individual senescent cells were surprisingly heterogeneous in their gene expression signatures. This cell-to-cell variability resulted in a loss of correlation among the expression of several senescence-associated genes. Many genes encoding senescence-associated secretory phenotype (SASP) factors, a major contributor to the effects of senescent cells in vivo, showed marked variability with a subset of highly induced genes accounting for the increases observed at the population level. Inflammatory genes in clustered genomic loci showed a greater correlation with senescence compared to nonclustered loci, suggesting that these genes are coregulated by genomic location. Together, these data offer new insights into how genes are regulated in senescent cells and suggest that single markers are inadequate to identify senescent cells in vivo. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Dynamic imaging of cell-free and cell-associated viral capture in mature dendritic cells.

Science.gov (United States)

Izquierdo-Useros, Nuria; Esteban, Olga; Rodriguez-Plata, Maria T; Erkizia, Itziar; Prado, Julia G; Blanco, Julià; García-Parajo, Maria F; Martinez-Picado, Javier

2011-12-01

Dendritic cells (DCs) capture human immunodeficiency virus (HIV) through a non-fusogenic mechanism that enables viral transmission to CD4(+) T cells, contributing to in vivo viral dissemination. Although previous studies have provided important clues to cell-free viral capture by mature DCs (mDCs), dynamic and kinetic insight on this process is still missing. Here, we used three-dimensional video microscopy and single-particle tracking approaches to dynamically dissect both cell-free and cell-associated viral capture by living mDCs. We show that cell-free virus capture by mDCs operates through three sequential phases: virus binding through specific determinants expressed in the viral particle, polarized or directional movements toward concrete regions of the cell membrane and virus accumulation in a sac-like structure where trapped viral particles display a hindered diffusive behavior. Moreover, real-time imaging of cell-associated viral transfer to mDCs showed a similar dynamics to that exhibited by cell-free virus endocytosis leading to viral accumulation in compartments. However, cell-associated HIV type 1 transfer to mDCs was the most effective pathway, boosted throughout enhanced cellular contacts with infected CD4(+) T cells. Our results suggest that in lymphoid tissues, mDC viral uptake could occur either by encountering cell-free or cell-associated virus produced by infected cells generating the perfect scenario to promote HIV pathogenesis and impact disease progression. © 2011 John Wiley & Sons A/S.

Role of Cell-Cell bond for the viability and the function of vascular smooth muscle cells

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M. Mura

2010-01-01

Full Text Available Vascular smooth muscle cell (VSMC viability and homeostasis is regulated by cell-matrix and cell-cell contact: disruption of these interactions are responsible of a switch from a mature to a high proliferative phenotype. VSMCs migration, rate of growth and apoptosis, and the extent of their extracellular matrix (ECM deposition can be also modulated by proatherogenic peptides. Among them, ATII induces the transactivation of IGF I R, which, together with the binding protein IGFBP3, represents a determinant of cell survival, growth and proliferation. Aim of our in vitro study was to verify the role of elective cell-cell bond in moulating the response to ATII. Thus, we evaluated viability, proliferation, IGFIR, IGFBP3 expression and the long term survival and production of ECM in a provisional tissue. A7r5 cell-line was used in adherent cultures or incubated in agarose-coated culture plates to inhibit cell-matrix interactions. Cells, treated or not with ATII 100 nM, were evaluated for apoptosis rate, cell cycle, IGFIR and IGFBP3 protei expression. Fibrin provisional tissue was developed polymerizing a fibrin solution. cantaining A7r5 cells with thrombin. Histological stainings for ECM components were performed on sections of prvisional tissue. An exclusive cell-cell contact resulted to monolayer cell cultures. ATII did not affect the cell survival in both culture conditions, but promoted a 10% decrease in "S" phase and an increases IGFIR expression only in adherent cells. while suspended cell aggregates were resistant to ATII administration; IGFBP3 was reduced both in ATII treated adherent cells and in floating clustered cells, irrespective of the treatmentn. VSMC conditioning in agarose-coated plates before seeding in fibrin provisional matrix reduced, but not abolished, the cell ability to colonize the clot and to produce ECM. This study demonstrates that the elective cell-cell contact induces a quiescent status in cells lacking of cell

Semiallogenic fusions of MSI+ tumor cells and activated B cells induce MSI-specific T cell responses

International Nuclear Information System (INIS)

Garbe, Yvette; Klier, Ulrike; Linnebacher, Michael

2011-01-01

Various strategies have been developed to transfer tumor-specific antigens into antigen presenting cells in order to induce cytotoxic T cell responses against tumor cells. One approach uses cellular vaccines based on fusions of autologous antigen presenting cells and allogeneic tumor cells. The fusion cells combine antigenicity of the tumor cell with optimal immunostimulatory capacity of the antigen presenting cells. Microsatellite instability caused by mutational inactivation of DNA mismatch repair genes results in translational frameshifts when affecting coding regions. It has been shown by us and others that these mutant proteins lead to the presentation of immunogenic frameshift peptides that are - in principle - recognized by a multiplicity of effector T cells. We chose microsatellite instability-induced frameshift antigens as ideal to test for induction of tumor specific T cell responses by semiallogenic fusions of microsatellite instable carcinoma cells with CD40-activated B cells. Two fusion clones of HCT116 with activated B cells were selected for stimulation of T cells autologous to the B cell fusion partner. Outgrowing T cells were phenotyped and tested in functional assays. The fusion clones expressed frameshift antigens as well as high amounts of MHC and costimulatory molecules. Autologous T cells stimulated with these fusions were predominantly CD4 + , activated, and reacted specifically against the fusion clones and also against the tumor cell fusion partner. Interestingly, a response toward 6 frameshift-derived peptides (of 14 tested) could be observed. Cellular fusions of MSI + carcinoma cells and activated B cells combine the antigen-presenting capacity of the B cell with the antigenic repertoire of the carcinoma cell. They present frameshift-derived peptides and can induce specific and fully functional T cells recognizing not only fusion cells but also the carcinoma cells. These hybrid cells may have great potential for cellular immunotherapy and

NK Cells and Other Innate Lymphoid Cells in Hematopoietic Stem Cell Transplantation.

Science.gov (United States)

Vacca, Paola; Montaldo, Elisa; Croxatto, Daniele; Moretta, Francesca; Bertaina, Alice; Vitale, Chiara; Locatelli, Franco; Mingari, Maria Cristina; Moretta, Lorenzo

2016-01-01

Natural killer (NK) cells play a major role in the T-cell depleted haploidentical hematopoietic stem cell transplantation (haplo-HSCT) to cure high-risk leukemias. NK cells belong to the expanding family of innate lymphoid cells (ILCs). At variance with NK cells, the other ILC populations (ILC1/2/3) are non-cytolytic, while they secrete different patterns of cytokines. ILCs provide host defenses against viruses, bacteria, and parasites, drive lymphoid organogenesis, and contribute to tissue remodeling. In haplo-HSCT patients, the extensive T-cell depletion is required to prevent graft-versus-host disease (GvHD) but increases risks of developing a wide range of life-threatening infections. However, these patients may rely on innate defenses that are reconstituted more rapidly than the adaptive ones. In this context, ILCs may represent important players in the early phases following transplantation. They may contribute to tissue homeostasis/remodeling and lymphoid tissue reconstitution. While the reconstitution of NK cell repertoire and its role in haplo-HSCT have been largely investigated, little information is available on ILCs. Of note, CD34(+) cells isolated from different sources of HSC may differentiate in vitro toward various ILC subsets. Moreover, cytokines released from leukemia blasts (e.g., IL-1β) may alter the proportions of NK cells and ILC3, suggesting the possibility that leukemia may skew the ILC repertoire. Further studies are required to define the timing of ILC development and their potential protective role after HSCT.

NK cells and other innate lymphoid cells in haematopoietic stem cell transplantation

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Paola eVacca

2016-05-01

Full Text Available Natural Killer (NK cells play a major role in the T-cell depleted haploidentical haematopoietic stem cell transplantation (haplo-HSCT to cure high-risk leukemias. NK cells belong to the expanding family of innate lymphoid cells (ILC. At variance with NK cells, the other ILC populations (ILC1/2/3 are non-cytolytic, while they secrete different patterns of cytokines. ILC provide host defences against viruses, bacteria and parasites, drive lymphoid organogenesis, and contribute to tissue remodelling. In haplo-HSCT patients, the extensive T-cell depletion is required to prevent graft-versus-host disease (GvHD but increases risks of developing a wide range of life-threatening infections. However, these patients may rely on innate defences that are reconstituted more rapidly than the adaptive ones. In this context, ILC may represent important players in the early phases following transplantation. They may contribute to tissue homeostasis/remodelling and lymphoid tissue reconstitution. While the reconstitution of NK cell repertoire and its role in haplo-HSCT have been largely investigated, little information is available on ILC. Of note, CD34+ cells isolated from different sources of HSC, may differentiate in vitro towards various ILC subsets. Moreover, cytokines released from leukemia blasts (e.g. IL-1β may alter the proportions of NK cells and ILC3, suggesting the possibility that leukemia may skew the ILC repertoire. Further studies are required to define the timing of ILC development and their potential protective role after HSCT.

Cell-substrate impedance fluctuations of single amoeboid cells encode cell-shape and adhesion dynamics.

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Leonhardt, Helmar; Gerhardt, Matthias; Höppner, Nadine; Krüger, Kirsten; Tarantola, Marco; Beta, Carsten

2016-01-01

We show systematic electrical impedance measurements of single motile cells on microelectrodes. Wild-type cells and mutant strains were studied that differ in their cell-substrate adhesion strength. We recorded the projected cell area by time-lapse microscopy and observed irregular oscillations of the cell shape. These oscillations were correlated with long-term variations in the impedance signal. Superposed to these long-term trends, we observed fluctuations in the impedance signal. Their magnitude clearly correlated with the adhesion strength, suggesting that strongly adherent cells display more dynamic cell-substrate interactions.

Cell-substrate impedance fluctuations of single amoeboid cells encode cell-shape and adhesion dynamics

Science.gov (United States)

Leonhardt, Helmar; Gerhardt, Matthias; Höppner, Nadine; Krüger, Kirsten; Tarantola, Marco; Beta, Carsten

2016-01-01

We show systematic electrical impedance measurements of single motile cells on microelectrodes. Wild-type cells and mutant strains were studied that differ in their cell-substrate adhesion strength. We recorded the projected cell area by time-lapse microscopy and observed irregular oscillations of the cell shape. These oscillations were correlated with long-term variations in the impedance signal. Superposed to these long-term trends, we observed fluctuations in the impedance signal. Their magnitude clearly correlated with the adhesion strength, suggesting that strongly adherent cells display more dynamic cell-substrate interactions.

Cell division orientation is coupled to cell-cell adhesion by the E-cadherin/LGN complex

NARCIS (Netherlands)

Gloerich, Martijn; Bianchini, Julie M.; Siemers, Kathleen A.; Cohen, Daniel J.; Nelson, W. James

2017-01-01

Both cell-cell adhesion and oriented cell division play prominent roles in establishing tissue architecture, but it is unclear how they might be coordinated. Here, we demonstrate that the cell-cell adhesion protein E-cadherin functions as an instructive cue for cell division orientation. This is

Stem Cells

Science.gov (United States)

Stem cells are cells with the potential to develop into many different types of cells in the body. ... the body. There are two main types of stem cells: embryonic stem cells and adult stem cells. Stem ...

Inflammatory Cell Distribution in Primary Merkel Cell Carcinoma

Energy Technology Data Exchange (ETDEWEB)

Wheat, Rachel [School of Cancer Sciences and CR UK Centre for Cancer Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT (United Kingdom); Roberts, Claudia [School of Cancer Sciences and CR UK Centre for Cancer Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT (United Kingdom); University Hospitals Birmingham NHS Foundation Trust, New Queen Elizabeth Hospital Birmingham, Mindelsohn Way, Edgbaston, Birmingham, B15 2WB (United Kingdom); Waterboer, Tim [Infection and Cancer Program, DKFZ (German Cancer Research Centre), 69120 Heidelberg (Germany); Steele, Jane [Human Biomaterials Resource Centre, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT (United Kingdom); Marsden, Jerry [University Hospitals Birmingham NHS Foundation Trust, New Queen Elizabeth Hospital Birmingham, Mindelsohn Way, Edgbaston, Birmingham, B15 2WB (United Kingdom); Steven, Neil M., E-mail: n.m.steven@bham.ac.uk [School of Cancer Sciences and CR UK Centre for Cancer Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT (United Kingdom); University Hospitals Birmingham NHS Foundation Trust, New Queen Elizabeth Hospital Birmingham, Mindelsohn Way, Edgbaston, Birmingham, B15 2WB (United Kingdom); Blackbourn, David J., E-mail: n.m.steven@bham.ac.uk [Department of Microbial and Cellular Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH (United Kingdom)

2014-05-06

Merkel cell carcinoma (MCC) is an aggressive poorly differentiated neuroendocrine cutaneous carcinoma associated with older age, immunodeficiency and Merkel cell polyomavirus (MCPyV) integrated within malignant cells. The presence of intra-tumoural CD8+ lymphocytes reportedly predicts better MCC-specific survival. In this study, the distribution of inflammatory cells and properties of CD8+ T lymphocytes within 20 primary MCC specimens were characterised using immunohistochemistry and multicolour immunofluorescent staining coupled to confocal microscopy. CD8+ cells and CD68+ macrophages were identified in 19/20 primary MCC. CD20+ B cells were present in 5/10, CD4+ cells in 10/10 and FoxP3+ cells in 7/10 specimens. Only two specimens had almost no inflammatory cells. Within specimens, inflammatory cells followed the same patchy distribution, focused at the edge of sheets and nodules and, in some cases, more intense in trabecular areas. CD8+ cells were outside vessels on the edge of tumour. Those few within malignant sheets typically lined up in fine septa not contacting MCC cells expressing MCPyV large T antigen. The homeostatic chemokine CXCL12 was expressed outside malignant nodules whereas its receptor CXCR4 was identified within tumour but not on CD8+ cells. CD8+ cells lacked CXCR3 and granzyme B expression irrespective of location within stroma versus malignant nodules or of the intensity of the intra-tumoural infiltrate. In summary, diverse inflammatory cells were organised around the margin of malignant deposits suggesting response to aberrant signaling, but were unable to penetrate the tumour microenvironment itself to enable an immune response against malignant cells or their polyomavirus.

Inflammatory Cell Distribution in Primary Merkel Cell Carcinoma

International Nuclear Information System (INIS)

Wheat, Rachel; Roberts, Claudia; Waterboer, Tim; Steele, Jane; Marsden, Jerry; Steven, Neil M.; Blackbourn, David J.

2014-01-01

Merkel cell carcinoma (MCC) is an aggressive poorly differentiated neuroendocrine cutaneous carcinoma associated with older age, immunodeficiency and Merkel cell polyomavirus (MCPyV) integrated within malignant cells. The presence of intra-tumoural CD8+ lymphocytes reportedly predicts better MCC-specific survival. In this study, the distribution of inflammatory cells and properties of CD8+ T lymphocytes within 20 primary MCC specimens were characterised using immunohistochemistry and multicolour immunofluorescent staining coupled to confocal microscopy. CD8+ cells and CD68+ macrophages were identified in 19/20 primary MCC. CD20+ B cells were present in 5/10, CD4+ cells in 10/10 and FoxP3+ cells in 7/10 specimens. Only two specimens had almost no inflammatory cells. Within specimens, inflammatory cells followed the same patchy distribution, focused at the edge of sheets and nodules and, in some cases, more intense in trabecular areas. CD8+ cells were outside vessels on the edge of tumour. Those few within malignant sheets typically lined up in fine septa not contacting MCC cells expressing MCPyV large T antigen. The homeostatic chemokine CXCL12 was expressed outside malignant nodules whereas its receptor CXCR4 was identified within tumour but not on CD8+ cells. CD8+ cells lacked CXCR3 and granzyme B expression irrespective of location within stroma versus malignant nodules or of the intensity of the intra-tumoural infiltrate. In summary, diverse inflammatory cells were organised around the margin of malignant deposits suggesting response to aberrant signaling, but were unable to penetrate the tumour microenvironment itself to enable an immune response against malignant cells or their polyomavirus

Skeletal Muscle Cell Induction from Pluripotent Stem Cells

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Yusaku Kodaka

2017-01-01

Full Text Available Embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs have the potential to differentiate into various types of cells including skeletal muscle cells. The approach of converting ESCs/iPSCs into skeletal muscle cells offers hope for patients afflicted with the skeletal muscle diseases such as the Duchenne muscular dystrophy (DMD. Patient-derived iPSCs are an especially ideal cell source to obtain an unlimited number of myogenic cells that escape immune rejection after engraftment. Currently, there are several approaches to induce differentiation of ESCs and iPSCs to skeletal muscle. A key to the generation of skeletal muscle cells from ESCs/iPSCs is the mimicking of embryonic mesodermal induction followed by myogenic induction. Thus, current approaches of skeletal muscle cell induction of ESCs/iPSCs utilize techniques including overexpression of myogenic transcription factors such as MyoD or Pax3, using small molecules to induce mesodermal cells followed by myogenic progenitor cells, and utilizing epigenetic myogenic memory existing in muscle cell-derived iPSCs. This review summarizes the current methods used in myogenic differentiation and highlights areas of recent improvement.

Cell-cell transmission of VSV-G pseudotyped lentivector particles.

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Amy M Skinner

Full Text Available Many replicating viruses, including HIV-1 and HTLV-1, are efficiently transmitted from the cell surface of actively infected cells upon contact with bystander cells. In a previous study, we reported the prolonged cell surface retention of VSV-G replication-deficient pseudotyped lentivector prior to endocytic entry. However, the competing kinetics of cell surface versus dissociation, neutralization or direct transfer to other cells have received comparatively little attention. Here we demonstrate that the relative efficiency of cell-cell surface transmission can outpace "cell-free" transduction at limiting vector input. This coincides with the prolonged half-life of cell bound vector but occurs, unlike HTLV-1, without evidence for particle aggregation. These studies suggest that cell-surface attachment stabilizes particles and alters neutralization kinetics. Our experiments provide novel insight into the underexplored cell-cell transmission of pseudotyped particles.

Chromosome aberrations and cell survival in irradiated mammalian cells

International Nuclear Information System (INIS)

Tremp, J.

1981-01-01

A possible correlation between chromosome aberrations and reduced proliferation capacity or cell death was investigated. Synchronized Chinese hamster fibroblast cells were irradiated with 300 rad of x rays in early G 1 . Despite synchronization the cells reached the subsequent mitosis at different times. The frequency of chromosome aberrations was determined in the postirradiation division at 2-h intervals. The highest frequency occurred in cells with a first cell cycle of medium length. The colony-forming ability of mitotic cells was measured in parallel samples by following the progress of individual mitoses. The proportion of cells forming macrocolonies decreased with increasing cell cycle length, and the number of non-colony-forming cells increased. Irrespective of various first cell cycle lengths and different frequencies of chromosome aberrations, the number of cells forming microcolonies remained constant. A correlation was found between the absence of chromosome aberrations and the ability of cells to form macrocolonies. However, cells with a long first cell cycle formed fewer macrocolonies than expected

Lobatamide C: total synthesis, stereochemical assignment, preparation of simplified analogues, and V-ATPase inhibition studies.

Science.gov (United States)

Shen, Ruichao; Lin, Cheng Ting; Bowman, Emma Jean; Bowman, Barry J; Porco, John A

2003-07-02

The total synthesis and stereochemical assignment of the potent antitumor macrolide lobatamide C, as well as synthesis of simplified lobatamide analogues, is reported. Cu(I)-mediated enamide formation methodology has been developed to prepare the highly unsaturated enamide side chain of the natural product and analogues. A key fragment coupling employs base-mediated esterification of a beta-hydroxy acid and a salicylate cyanomethyl ester. Three additional stereoisomers of lobatamide C have been prepared using related synthetic routes. The stereochemistry at C8, C11, and C15 of lobatamide C was assigned by comparison of stereoisomers and X-ray analysis of a crystalline derivative. Synthetic lobatamide C, stereoisomers, and simplified analogues have been evaluated for inhibition of bovine chromaffin granule membrane V-ATPase. The salicylate phenol, enamide NH, and ortho-substitution of the salicylate ester have been shown to be important for V-ATPase inhibitory activity.

The role of Rap1 in cell-cell junction formation

NARCIS (Netherlands)

Kooistra, M.R.H.

2008-01-01

Both epithelial and endothelial cells form cell-cell junctions at the cell-cell contacts to maintain tissue integrity. Proper regulation of cell-cell junctions is required for the organisation of the tissue and to prevent leakage of blood vessels. In endothelial cells, the cell-cell junctions are

Recipient dendritic cells, but not B cells, are required antigen-presenting cells for peripheral alloreactive CD8+ T-cell tolerance.

Science.gov (United States)

Mollov, J L; Lucas, C L; Haspot, F; Gaspar, J Kurtz C; Guzman, A; Sykes, M

2010-03-01

Induction of mixed allogeneic chimerism is a promising approach for achieving donor-specific tolerance, thereby obviating the need for life-long immunosuppression for solid organ allograft acceptance. In mice receiving a low dose (3Gy) of total body irradiation, allogeneic bone marrow transplantation combined with anti-CD154 tolerizes peripheral CD4 and CD8 T cells, allowing achievement of mixed chimerism with specific tolerance to donor. With this approach, peripheral CD8 T-cell tolerance requires recipient MHC class II, CD4 T cells, B cells and DCs. Recipient-type B cells from chimeras that were tolerant to donor still promoted CD8 T-cell tolerance, but their role could not be replaced by donor-type B cells. Using recipients whose B cells or DCs specifically lack MHC class I and/or class II or lack CD80 and CD86, we demonstrate that dendritic cells (DCs) must express CD80/86 and either MHC class I or class II to promote CD8 tolerance. In contrast, B cells, though required, did not need to express MHC class I or class II or CD80/86 to promote CD8 tolerance. Moreover, recipient IDO and IL-10 were not required. Thus, antigen presentation by recipient DCs and not by B cells is critical for peripheral alloreactive CD8 T cell tolerance.

Pseudomonas aeruginosa Transmigrates at Epithelial Cell-Cell Junctions, Exploiting Sites of Cell Division and Senescent Cell Extrusion.

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Guillaume Golovkine

2016-01-01

Full Text Available To achieve systemic infection, bacterial pathogens must overcome the critical and challenging step of transmigration across epithelial barriers. This is particularly true for opportunistic pathogens such as Pseudomonas aeruginosa, an agent which causes nosocomial infections. Despite extensive study, details on the mechanisms used by this bacterium to transmigrate across epithelial tissues, as well as the entry sites it uses, remain speculative. Here, using real-time microscopy and a model epithelial barrier, we show that P. aeruginosa employs a paracellular transmigration route, taking advantage of altered cell-cell junctions at sites of cell division or when senescent cells are expelled from the cell layer. Once a bacterium transmigrates, it is followed by a cohort of bacteria using the same entry point. The basal compartment is then invaded radially from the initial penetration site. Effective transmigration and propagation require type 4 pili, the type 3 secretion system (T3SS and a flagellum, although flagellum-deficient bacteria can occasionally invade the basal compartment from wounded areas. In the basal compartment, the bacteria inject the T3SS toxins into host cells, disrupting the cytoskeleton and focal contacts to allow their progression under the cells. Thus, P. aeruginosa exploits intrinsic host cell processes to breach the epithelium and invade the subcellular compartment.

Stem Cell Therapy: Repurposing Cell-Based Regenerative Medicine Beyond Cell Replacement.

Science.gov (United States)

Napoli, Eleonora; Lippert, Trenton; Borlongan, Cesar V

2018-02-27

Stem cells exhibit simple and naive cellular features, yet their exact purpose for regenerative medicine continues to elude even the most elegantly designed research paradigms from developmental biology to clinical therapeutics. Based on their capacity to divide indefinitely and their dynamic differentiation into any type of tissue, the advent of transplantable stem cells has offered a potential treatment for aging-related and injury-mediated diseases. Recent laboratory evidence has demonstrated that transplanted human neural stem cells facilitate endogenous reparative mechanisms by initiating multiple regenerative processes in the brain neurogenic areas. Within these highly proliferative niches reside a myriad of potent regenerative molecules, including anti-inflammatory cytokines, proteomes, and neurotrophic factors, altogether representing a biochemical cocktail vital for restoring brain function in the aging and diseased brain. Here, we advance the concept of therapeutically repurposing stem cells not towards cell replacement per se, but rather exploiting the cells' intrinsic properties to serve as the host brain regenerative catalysts.

Parallel and convergent processing in grid cell, head-direction cell, boundary cell, and place cell networks.

Science.gov (United States)

Brandon, Mark P; Koenig, Julie; Leutgeb, Stefan

2014-03-01

The brain is able to construct internal representations that correspond to external spatial coordinates. Such brain maps of the external spatial topography may support a number of cognitive functions, including navigation and memory. The neuronal building block of brain maps are place cells, which are found throughout the hippocampus of rodents and, in a lower proportion, primates. Place cells typically fire in one or few restricted areas of space, and each area where a cell fires can range, along the dorsoventral axis of the hippocampus, from 30 cm to at least several meters. The sensory processing streams that give rise to hippocampal place cells are not fully understood, but substantial progress has been made in characterizing the entorhinal cortex, which is the gateway between neocortical areas and the hippocampus. Entorhinal neurons have diverse spatial firing characteristics, and the different entorhinal cell types converge in the hippocampus to give rise to a single, spatially modulated cell type-the place cell. We therefore suggest that parallel information processing in different classes of cells-as is typically observed at lower levels of sensory processing-continues up into higher level association cortices, including those that provide the inputs to hippocampus. WIREs Cogn Sci 2014, 5:207-219. doi: 10.1002/wcs.1272 Conflict of interest: The authors have declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website. © 2013 John Wiley & Sons, Ltd.

α-Mangostin Induces Apoptosis and Cell Cycle Arrest in Oral Squamous Cell Carcinoma Cell

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Hyun-Ho Kwak

2016-01-01

Full Text Available Mangosteen has long been used as a traditional medicine and is known to have antibacterial, antioxidant, and anticancer effects. Although the effects of α-mangostin, a natural compound extracted from the pericarp of mangosteen, have been investigated in many studies, there is limited data on the effects of the compound in human oral squamous cell carcinoma (OSCC. In this study, α-mangostin was assessed as a potential anticancer agent against human OSCC cells. α-Mangostin inhibited cell proliferation and induced cell death in OSCC cells in a dose- and time-dependent manner with little to no effect on normal human PDLF cells. α-Mangostin treatment clearly showed apoptotic evidences such as nuclear fragmentation and accumulation of annexin V and PI-positive cells on OSCC cells. α-Mangostin treatment also caused the collapse of mitochondrial membrane potential and the translocation of cytochrome c from the mitochondria into the cytosol. The expressions of the mitochondria-related proteins were activated by α-mangostin. Treatment with α-mangostin also induced G1 phase arrest and downregulated cell cycle-related proteins (CDK/cyclin. Hence, α-mangostin specifically induces cell death and inhibits proliferation in OSCC cells via the intrinsic apoptosis pathway and cell cycle arrest at the G1 phase, suggesting that α-mangostin may be an effective agent for the treatment of OSCC.

Two subpopulations of stem cells for T cell lineage

International Nuclear Information System (INIS)

Katsura, Y.; Amagai, T.; Kina, T.; Sado, T.; Nishikawa, S.

1985-01-01

An assay system for the stem cell that colonizes the thymus and differentiates into T cells was developed, and by using this assay system the existence of two subpopulations of stem cells for T cell lineage was clarified. Part-body-shielded and 900-R-irradiated C57BL/6 (H-2b, Thy-1.2) recipient mice, which do not require the transfer of pluripotent stem cells for their survival, were transferred with cells from B10 X Thy-1.1 (H-2b, Thy-1.1) donor mice. The reconstitution of the recipient's thymus lymphocytes was accomplished by stem cells in the donor cells and those spared in the shielded portion of the recipient that competitively colonize the thymus. Thus, the stem cell activity of donor cells can be evaluated by determining the proportion of donor-type (Thy-1.1+) cells in the recipient's thymus. Bone marrow cells were the most potent source of stem cells. By contrast, when the stem cell activity was compared between spleen and bone marrow cells of whole-body-irradiated (800 R) C57BL/6 mice reconstituted with B10 X Thy-1.1 bone marrow cells by assaying in part-body-shielded and irradiated C57BL/6 mice, the activity of these two organs showed quite a different time course of development. The results strongly suggest that the stem cells for T cell lineage in the bone marrow comprise at least two subpopulations, spleen-seeking and bone marrow-seeking cells

Nanodiamond internalization in cells and the cell uptake mechanism

Energy Technology Data Exchange (ETDEWEB)

Perevedentseva, E. [National Dong Hwa University, Department of Physics (China); Hong, S.-F.; Huang, K.-J. [National Dong Hwa University, Department of Life Sciences (China); Chiang, I.-T.; Lee, C.-Y. [National Dong Hwa University, Department of Physics (China); Tseng, Y.-T. [National Dong Hwa University, Department of Life Sciences (China); Cheng, C.-L., E-mail: clcheng@mail.ndhu.edu.tw [National Dong Hwa University, Department of Physics (China)

2013-08-15

Cell type-dependent penetration of nanodiamond in living cells is one of the important factors for using nanodiamond as cellular markers/labels, for drug delivery as well as for other biomedical applications. In this work, internalization of 100 nm nanodiamonds by A549 lung human adenocarcinoma cell, Beas-2b non-tumorigenic human bronchial epithelial cell, and HFL-1 fibroblast-like human fetal lung cell is studied and compared. The penetration of nanodiamond into the cells was observed using confocal fluorescence imaging and Raman imaging methods. Visualization of the nanodiamond in cells allows comparison of the internalization for diamond nanoparticles in cancer A549 cell, non-cancer HFL-1, and Beas-2b cells. The dose-dependent and time-dependent behavior of nanodiamond uptake is observed in both cancer as well as non-cancer cells. The mechanism of nanodiamond uptake by cancer and non-cancer cells is analyzed by blocking different pathways. The uptake of nanodiamond in both cancer and non-cancer cells was found predominantly via clathrin-dependent endocytosis. In spite of observed similarity in the uptake mechanism for cancer and non-cancer cells, the nanodiamond uptake for cancer cell quantitatively exceeds the uptake for non-cancer cells, for the studied cell lines. The observed difference in internalization of nanodiamond by cancer and non-cancer cells is discussed.

Nanodiamond internalization in cells and the cell uptake mechanism

International Nuclear Information System (INIS)

Perevedentseva, E.; Hong, S.-F.; Huang, K.-J.; Chiang, I.-T.; Lee, C.-Y.; Tseng, Y.-T.; Cheng, C.-L.

2013-01-01

Cell type-dependent penetration of nanodiamond in living cells is one of the important factors for using nanodiamond as cellular markers/labels, for drug delivery as well as for other biomedical applications. In this work, internalization of 100 nm nanodiamonds by A549 lung human adenocarcinoma cell, Beas-2b non-tumorigenic human bronchial epithelial cell, and HFL-1 fibroblast-like human fetal lung cell is studied and compared. The penetration of nanodiamond into the cells was observed using confocal fluorescence imaging and Raman imaging methods. Visualization of the nanodiamond in cells allows comparison of the internalization for diamond nanoparticles in cancer A549 cell, non-cancer HFL-1, and Beas-2b cells. The dose-dependent and time-dependent behavior of nanodiamond uptake is observed in both cancer as well as non-cancer cells. The mechanism of nanodiamond uptake by cancer and non-cancer cells is analyzed by blocking different pathways. The uptake of nanodiamond in both cancer and non-cancer cells was found predominantly via clathrin-dependent endocytosis. In spite of observed similarity in the uptake mechanism for cancer and non-cancer cells, the nanodiamond uptake for cancer cell quantitatively exceeds the uptake for non-cancer cells, for the studied cell lines. The observed difference in internalization of nanodiamond by cancer and non-cancer cells is discussed

CD34+ cells cultured in stem cell factor and interleukin-2 generate CD56+ cells with antiproliferative effects on tumor cell lines

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Hensel Nancy

2005-04-01

Full Text Available Abstract In vitro stimulation of CD34+ cells with IL-2 induces NK cell differentiation. In order to define the stages of NK cell development, which influence their generation from CD34 cells, we cultured G-CSF mobilized peripheral blood CD34+ cells in the presence of stem cell factor and IL-2. After three weeks culture we found a diversity of CD56+ subsets which possessed granzyme A, but lacked the cytotoxic apparatus required for classical NK-like cytotoxicity. However, these CD56+ cells had the unusual property of inhibiting proliferation of K562 and P815 cell lines in a cell-contact dependent fashion.

Pluripotent stem cells and reprogrammed cells in farm animals.

Science.gov (United States)

Nowak-Imialek, Monika; Kues, Wilfried; Carnwath, Joseph W; Niemann, Heiner

2011-08-01

Pluripotent cells are unique because of their ability to differentiate into the cell lineages forming the entire organism. True pluripotent stem cells with germ line contribution have been reported for mice and rats. Human pluripotent cells share numerous features of pluripotentiality, but confirmation of their in vivo capacity for germ line contribution is impossible due to ethical and legal restrictions. Progress toward derivation of embryonic stem cells from domestic species has been made, but the derived cells were not able to produce germ line chimeras and thus are termed embryonic stem-like cells. However, domestic animals, in particular the domestic pig (Sus scrofa), are excellent large animals models, in which the clinical potential of stem cell therapies can be studied. Reprogramming technologies for somatic cells, including somatic cell nuclear transfer, cell fusion, in vitro culture in the presence of cell extracts, in vitro conversion of adult unipotent spermatogonial stem cells into germ line derived pluripotent stem cells, and transduction with reprogramming factors have been developed with the goal of obtaining pluripotent, germ line competent stem cells from domestic animals. This review summarizes the present state of the art in the derivation and maintenance of pluripotent stem cells in domestic animals.

Epithelial cell polarity, stem cells and cancer

DEFF Research Database (Denmark)

Martin-Belmonte, Fernando; Perez-Moreno, Mirna

2011-01-01

, deregulation of adhesion and polarity proteins can cause misoriented cell divisions and increased self-renewal of adult epithelial stem cells. In this Review, we highlight some advances in the understanding of how loss of epithelial cell polarity contributes to tumorigenesis.......After years of extensive scientific discovery much has been learned about the networks that regulate epithelial homeostasis. Loss of expression or functional activity of cell adhesion and cell polarity proteins (including the PAR, crumbs (CRB) and scribble (SCRIB) complexes) is intricately related...

Regulatory T cells and B cells: implication on autoimmune diseases

OpenAIRE

Wang, Ping; Zheng, Song Guo

2013-01-01

The regulatory T (Treg) cells play an important role in the maintenance of homeostasis and the prevention of autoimmune diseases. Although most studies are focusing on the role of Treg cells in T cells and T cells-mediated diseases, these cells also directly affect B cells and other non-T cells. This manuscript updates the role of Treg cells on the B cells and B cell-mediated diseases. In addition, the mechanisms whereby Treg cells suppress B cell responses have been discussed.

Sertoli cells maintain Leydig cell number and peritubular myoid cell activity in the adult mouse testis.

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Diane Rebourcet

Full Text Available The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.

Selection of radioresistant cells by vitamin A deficiency in a small cell lung cancer cell line

International Nuclear Information System (INIS)

Terasaki, Takeo; Shimosato, Yukio; Wada, Makio; Yokota, Jun; Terada, Masaaki

1990-01-01

Radiation sensitivity of a human small cell lung cancer cell line, Lu-134-B cells, cultured in serum-supplemented medium and of cells transferred to and cultured in delipidized serum-supplemented (vitamin A-deficient) medium was studied. The cells cultured in serum-supplemented medium showed the phenotype of classic small cell lung cancer sensitive to radiation, while cells transferred to delipidized serum-supplemented medium showed partial squamous cell differentiation and became resistant to radiation. These results suggest that some small cell lung cancer cells in vitro change their morphology and radiosensitivity depending on the culture conditions. The change in radiosensitivity was reproducible, and was not reversible by culture of the radioresistant cells in delipidized serum-supplemented medium with addition of retinoic acid (vitamin A-sufficient medium) for two months, although squamous cells disappeared. Acquisition of radioresistancy was considered to occur as the result of clonal selective growth in delipidized medium of a minor cell population in the original cell culture, based on a study of chromosome number. It was also found that there was no association of myc-family oncogenes with the changes of radiosensitivity in this cell line. (author)

Mast-Cell-Derived TNF Amplifies CD8+ Dendritic Cell Functionality and CD8+ T Cell Priming

Directory of Open Access Journals (Sweden)

Jan Dudeck

2015-10-01

Full Text Available Mast cells are critical promoters of adaptive immunity in the contact hypersensitivity model, but the mechanism of allergen sensitization is poorly understood. Using Mcpt5-CreTNFFL/FL mice, we show here that the absence of TNF exclusively in mast cells impaired the expansion of CD8+ T cells upon sensitization and the T-cell-driven adaptive immune response to elicitation. T cells primed in the absence of mast cell TNF exhibited a diminished efficiency to transfer sensitization to naive recipients. Specifically, mast cell TNF promotes CD8+ dendritic cell (DC maturation and migration to draining lymph nodes. The peripherally released mast cell TNF further critically boosts the CD8+ T-cell-priming efficiency of CD8+ DCs, thereby linking mast cell effects on T cells to DC modulation. Collectively, our findings identify the distinct potential of mast cell TNF to amplify CD8+ DC functionality and CD8+ T-cell-dominated adaptive immunity, which may be of great importance for immunotherapy and vaccination approaches.

The Emerging Cell Biology of Thyroid Stem Cells

Science.gov (United States)

Latif, Rauf; Minsky, Noga C.; Ma, Risheng

2011-01-01

Context: Stem cells are undifferentiated cells with the property of self-renewal and give rise to highly specialized cells under appropriate local conditions. The use of stem cells in regenerative medicine holds great promise for the treatment of many diseases, including those of the thyroid gland. Evidence Acquisition: This review focuses on the progress that has been made in thyroid stem cell research including an overview of cellular and molecular events (most of which were drawn from the period 1990–2011) and discusses the remaining problems encountered in their differentiation. Evidence Synthesis: Protocols for the in vitro differentiation of embryonic stem cells, based on normal developmental processes, have generated thyroid-like cells but without full thyrocyte function. However, agents have been identified, including activin A, insulin, and IGF-I, which are able to stimulate the generation of thyroid-like cells in vitro. In addition, thyroid stem/progenitor cells have been identified within the normal thyroid gland and within thyroid cancers. Conclusions: Advances in thyroid stem cell biology are providing not only insight into thyroid development but may offer therapeutic potential in thyroid cancer and future thyroid cell replacement therapy. PMID:21778219

Oral epithelial cells are susceptible to cell-free and cell-associated HIV-1 infection in vitro

International Nuclear Information System (INIS)

Moore, Jennifer S.; Rahemtulla, Firoz; Kent, Leigh W.; Hall, Stacy D.; Ikizler, Mine R.; Wright, Peter F.; Nguyen, Huan H.; Jackson, Susan

2003-01-01

Epithelial cells lining the oral cavity are exposed to HIV-1 through breast-feeding and oral-genital contact. Genital secretions and breast milk of HIV-1-infected subjects contain both cell-free and cell-associated virus. To determine if oral epithelial cells can be infected with HIV-1 we exposed gingival keratinocytes and adenoid epithelial cells to cell-free virus and HIV-1-infected peripheral blood mononuclear cells and monocytes. Using primary isolates we determined that gingival keratinocytes are susceptible to HIV-1 infection via cell-free CD4-independent infection only. R5 but not X4 viral strains were capable of infecting the keratinocytes. Further, infected cells were able to release infectious virus. In addition, primary epithelial cells isolated from adenoids were also susceptible to infection; both cell-free and cell-associated virus infected these cells. These data have potential implications in the transmission of HIV-1 in the oral cavity

Nonmalignant T cells stimulate growth of T-cell lymphoma cells in the presence of bacterial toxins

DEFF Research Database (Denmark)

Woetmann, Anders; Lovato, Paola; Eriksen, Karsten W

2007-01-01

Bacterial toxins including staphylococcal enterotoxins (SEs) have been implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCLs). Here, we investigate SE-mediated interactions between nonmalignant T cells and malignant T-cell lines established from skin and blood of CTCL patients....... The malignant CTCL cells express MHC class II molecules that are high-affinity receptors for SE. Although treatment with SE has no direct effect on the growth of the malignant CTCL cells, the SE-treated CTCL cells induce vigorous proliferation of the SE-responsive nonmalignant T cells. In turn, the nonmalignant...... T cells enhance proliferation of the malignant cells in an SE- and MHC class II-dependent manner. Furthermore, SE and, in addition, alloantigen presentation by malignant CTCL cells to irradiated nonmalignant CD4(+) T-cell lines also enhance proliferation of the malignant cells. The growth...

The CEA−/lo colorectal cancer cell population harbors cancer stem cells and metastatic cells

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Zhang, Bo; Mu, Lei; Huang, Kaiyu; Zhao, Hui; Ma, Chensen; Li, Xiaolan; Tao, Deding; Gong, Jianping; Qin, Jichao

2016-01-01

Serum carcinoembryonic antigen (CEA) is the most commonly used tumor marker in a variety of cancers including colorectal cancer (CRC) for tumor diagnosis and monitoring. Recent studies have shown that colonic crypt cells expressing little or no CEA may enrich for stem cells. Numerous studies have clearly shown that there exist CRC patients with normal serum CEA levels during tumor progression or even tumor relapse, although CEA itself is considered to promote metastasis and block cell differentiation. These seemingly contradictory observations prompted us to investigate, herein, the biological properties as well as tumorigenic and metastatic capacity of CRC cells that express high (CEA+) versus low CEA (CEA−/lo) levels of CEA. Our findings show that the abundance of CEA−/lo cells correlate with poor differentiation and poor prognosis, and moreover, CEA−/lo cells form more spheres in vitro, generate more tumors and exhibit a higher potential in developing liver and lung metastases than corresponding CEA+ cells. Applying RNAi-mediated approach, we found that IGF1R mediated tumorigenic and capacity of CEA−/lo cells but did not mediate those of CEA+ cells. Notably, our data demonstrated that CEA molecule was capable of protecting CEA−/lo cells from anoikis, implying that CEA+ cells, although themselves possessing less tumorigenic and metastatic capacity, may promote metastasis of CEA−/lo cells via secreting CEA molecule. Our observations suggest that, besides targeting CEA molecule, CEA−/lo cells may represent a critical source of tumor progression and metastasis, and should therefore be the target of future therapies. PMID:27813496

Basal cell carcinoma of the skin with areas of squamous cell carcinoma: a basosquamous cell carcinoma?

OpenAIRE

de Faria, J

1985-01-01

The diagnosis of basosquamous cell carcinoma is controversial. A review of cases of basal cell carcinoma showed 23 cases that had conspicuous areas of squamous cell carcinoma. This was distinguished from squamous differentiation and keratotic basal cell carcinoma by a comparative study of 40 cases of compact lobular and 40 cases of keratotic basal cell carcinoma. Areas of intermediate tumour differentiation between basal cell and squamous cell carcinoma were found. Basal cell carcinomas with ...

The cell biology of T-dependent B cell activation

DEFF Research Database (Denmark)

Owens, T; Zeine, R

1989-01-01

The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells...... includes multipoint intermolecular interactions that probably involve aggregation of both polymorphic and monomorphic T cell surface molecules. Such aggregations have been shown in vitro to markedly enhance and, in some cases, induce T cell activation. The production of T-derived lymphokines that have been...... implicated in B cell activation is dependent on the T cell receptor for antigen and its associated CD3 signalling complex. T-dependent help for B cell activation is therefore similarly MHC-restricted and involves T-B intercellular interaction. Recent reports that describe antigen-independent B cell...

Activation of glioma cells generates immune tolerant NKT cells.

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Tang, Bo; Wu, Wei; Wei, Xiaowei; Li, Yang; Ren, Gang; Fan, Wenhai

2014-12-12

Therapeutic outcomes of glioma are currently not encouraging. Tumor tolerance plays an important role in the pathogenesis of glioma. It is reported that micro RNAs (miR) are associated with tumor development. This study aims to investigate the role of miR-92a in the development of tolerant natural killer T (NKT) cells. In this study, U87 cells (a human glioma cell line) and primary glioma cells were prepared. The assessment of miR-92a was performed by real time RT-PCR. The expression of interleukin (IL)-10 and IL-6 in NKT cells was evaluated by flow cytometry. Results showed that abundant IL-6(+) IL-10(+) NKT cells were detected in glioma tissue. Cultures of glioma cells and NKT cells induced the expression of IL-6 and IL-10 in NKT cells. Glioma cells expressed miR-92a; the latter played a critical role in the induction of IL-6 and IL-10 expression in NKT cells. The expression of the antitumor molecules, including perforin, Fas ligand, and interferon-γ, was significantly attenuated compared with control NKT cells. The IL-6(+) IL-10(+) NKT cells showed less capability in the induction of apoptosis in glioma cells, but showed the immune suppressor functions on CD8(+) T cell activities. We conclude that glioma-derived miR-92a induces IL-6(+) IL-10(+) NKT cells; this fraction of NKT cells can suppress cytotoxic CD8(+) T cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Metformin inhibits cell cycle progression of B-cell chronic lymphocytic leukemia cells.

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Bruno, Silvia; Ledda, Bernardetta; Tenca, Claudya; Ravera, Silvia; Orengo, Anna Maria; Mazzarello, Andrea Nicola; Pesenti, Elisa; Casciaro, Salvatore; Racchi, Omar; Ghiotto, Fabio; Marini, Cecilia; Sambuceti, Gianmario; DeCensi, Andrea; Fais, Franco

2015-09-08

B-cell chronic lymphocytic leukemia (CLL) was believed to result from clonal accumulation of resting apoptosis-resistant malignant B lymphocytes. However, it became increasingly clear that CLL cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. Drugs interfering with CLL cell cycle entry would be greatly beneficial in the treatment of this disease. 1, 1-Dimethylbiguanide hydrochloride (metformin), the most widely prescribed oral hypoglycemic agent, inexpensive and well tolerated, has recently received increased attention for its potential antitumor activity. We wondered whether metformin has apoptotic and anti-proliferative activity on leukemic cells derived from CLL patients. Metformin was administered in vitro either to quiescent cells or during CLL cell activation stimuli, provided by classical co-culturing with CD40L-expressing fibroblasts. At doses that were totally ineffective on normal lymphocytes, metformin induced apoptosis of quiescent CLL cells and inhibition of cell cycle entry when CLL were stimulated by CD40-CD40L ligation. This cytostatic effect was accompanied by decreased expression of survival- and proliferation-associated proteins, inhibition of signaling pathways involved in CLL disease progression and decreased intracellular glucose available for glycolysis. In drug combination experiments, metformin lowered the apoptotic threshold and potentiated the cytotoxic effects of classical and novel antitumor molecules. Our results indicate that, while CLL cells after stimulation are in the process of building their full survival and cycling armamentarium, the presence of metformin affects this process.

Non-Small Cell Lung Cancer Cells Expressing CD44 Are Enriched for Stem Cell-Like Properties

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Leung, Elaine Lai-Han; Fiscus, Ronald R.; Tung, James W.; Tin, Vicky Pui-Chi; Cheng, Lik Cheung; Sihoe, Alan Dart-Loon; Fink, Louis M.; Ma, Yupo; Wong, Maria Pik

2010-01-01

Background The cancer stem cell theory hypothesizes that cancers are perpetuated by cancer stem cells (CSC) or tumor initiating cells (TIC) possessing self-renewal and other stem cell-like properties while differentiated non-stem/initiating cells have a finite life span. To investigate whether the hypothesis is applicable to lung cancer, identification of lung CSC and demonstration of these capacities is essential. Methodology/Principal Finding The expression profiles of five stem cell markers (CD34, CD44, CD133, BMI1 and OCT4) were screened by flow cytometry in 10 lung cancer cell lines. CD44 was further investigated by testing for in vitro and in vivo tumorigenecity. Formation of spheroid bodies and in vivo tumor initiation ability were demonstrated in CD44+ cells of 4 cell lines. Serial in vivo tumor transplantability in nude mice was demonstrated using H1299 cell line. The primary xenografts initiated from CD44+ cells consisted of mixed CD44+ and CD44− cells in similar ratio as the parental H1299 cell line, supporting in vivo differentiation. Semi-quantitative Real-Time PCR (RT-PCR) showed that both freshly sorted CD44+ and CD44+ cells derived from CD44+-initiated tumors expressed the pluripotency genes OCT4/POU5F1, NANOG, SOX2. These stemness markers were not expressed by CD44− cells. Furthermore, freshly sorted CD44+ cells were more resistant to cisplatin treatment with lower apoptosis levels than CD44− cells. Immunohistochemical analysis of 141 resected non-small cell lung cancers showed tumor cell expression of CD44 in 50.4% of tumors while no CD34, and CD133 expression was observed in tumor cells. CD44 expression was associated with squamous cell carcinoma but unexpectedly, a longer survival was observed in CD44-expressing adenocarcinomas. Conclusion/Significance Overall, our results demonstrated that stem cell-like properties are enriched in CD44-expressing subpopulations of some lung cancer cell lines. Further investigation is required to clarify

Satellite Cells and the Muscle Stem Cell Niche

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Yin, Hang; Price, Feodor

2013-01-01

Adult skeletal muscle in mammals is a stable tissue under normal circumstances but has remarkable ability to repair after injury. Skeletal muscle regeneration is a highly orchestrated process involving the activation of various cellular and molecular responses. As skeletal muscle stem cells, satellite cells play an indispensible role in this process. The self-renewing proliferation of satellite cells not only maintains the stem cell population but also provides numerous myogenic cells, which proliferate, differentiate, fuse, and lead to new myofiber formation and reconstitution of a functional contractile apparatus. The complex behavior of satellite cells during skeletal muscle regeneration is tightly regulated through the dynamic interplay between intrinsic factors within satellite cells and extrinsic factors constituting the muscle stem cell niche/microenvironment. For the last half century, the advance of molecular biology, cell biology, and genetics has greatly improved our understanding of skeletal muscle biology. Here, we review some recent advances, with focuses on functions of satellite cells and their niche during the process of skeletal muscle regeneration. PMID:23303905

TOPICAL REVIEW: Stem cells engineering for cell-based therapy

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Taupin, Philippe

2007-09-01

Stem cells carry the promise to cure a broad range of diseases and injuries, from diabetes, heart and muscular diseases, to neurological diseases, disorders and injuries. Significant progresses have been made in stem cell research over the past decade; the derivation of embryonic stem cells (ESCs) from human tissues, the development of cloning technology by somatic cell nuclear transfer (SCNT) and the confirmation that neurogenesis occurs in the adult mammalian brain and that neural stem cells (NSCs) reside in the adult central nervous system (CNS), including that of humans. Despite these advances, there may be decades before stem cell research will translate into therapy. Stem cell research is also subject to ethical and political debates, controversies and legislation, which slow its progress. Cell engineering has proven successful in bringing genetic research to therapy. In this review, I will review, in two examples, how investigators are applying cell engineering to stem cell biology to circumvent stem cells' ethical and political constraints and bolster stem cell research and therapy.

CellNet: Network Biology Applied to Stem Cell Engineering

Science.gov (United States)

Cahan, Patrick; Li, Hu; Morris, Samantha A.; da Rocha, Edroaldo Lummertz; Daley, George Q.; Collins, James J.

2014-01-01

SUMMARY Somatic cell reprogramming, directed differentiation of pluripotent stem cells, and direct conversions between differentiated cell lineages represent powerful approaches to engineer cells for research and regenerative medicine. We have developed CellNet, a network biology platform that more accurately assesses the fidelity of cellular engineering than existing methodologies and generates hypotheses for improving cell derivations. Analyzing expression data from 56 published reports, we found that cells derived via directed differentiation more closely resemble their in vivo counterparts than products of direct conversion, as reflected by the establishment of target cell-type gene regulatory networks (GRNs). Furthermore, we discovered that directly converted cells fail to adequately silence expression programs of the starting population, and that the establishment of unintended GRNs is common to virtually every cellular engineering paradigm. CellNet provides a platform for quantifying how closely engineered cell populations resemble their target cell type and a rational strategy to guide enhanced cellular engineering. PMID:25126793

CellNet: network biology applied to stem cell engineering.

Science.gov (United States)

Cahan, Patrick; Li, Hu; Morris, Samantha A; Lummertz da Rocha, Edroaldo; Daley, George Q; Collins, James J

2014-08-14

Somatic cell reprogramming, directed differentiation of pluripotent stem cells, and direct conversions between differentiated cell lineages represent powerful approaches to engineer cells for research and regenerative medicine. We have developed CellNet, a network biology platform that more accurately assesses the fidelity of cellular engineering than existing methodologies and generates hypotheses for improving cell derivations. Analyzing expression data from 56 published reports, we found that cells derived via directed differentiation more closely resemble their in vivo counterparts than products of direct conversion, as reflected by the establishment of target cell-type gene regulatory networks (GRNs). Furthermore, we discovered that directly converted cells fail to adequately silence expression programs of the starting population and that the establishment of unintended GRNs is common to virtually every cellular engineering paradigm. CellNet provides a platform for quantifying how closely engineered cell populations resemble their target cell type and a rational strategy to guide enhanced cellular engineering. Copyright © 2014 Elsevier Inc. All rights reserved.

Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

Directory of Open Access Journals (Sweden)

Tongfang Tang

Full Text Available BACKGROUND: Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD through alternation of liver innate immune response. AIMS: The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. METHODS: Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. RESULTS: High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4 expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. CONCLUSION: High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

Science.gov (United States)

Tang, Tongfang; Sui, Yongheng; Lian, Min; Li, Zhiping; Hua, Jing

2013-01-01

Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD) through alternation of liver innate immune response. The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4) expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

T cell-B cell interactions in primary immunodeficiencies.

Science.gov (United States)

Tangye, Stuart G; Deenick, Elissa K; Palendira, Umaimainthan; Ma, Cindy S

2012-02-01

Regulated interactions between cells of the immune system facilitate the generation of successful immune responses, thereby enabling efficient neutralization and clearance of pathogens and the establishment of both cell- and humoral-mediated immunological memory. The corollary of this is that impediments to efficient cell-cell interactions, normally necessary for differentiation and effector functions of immune cells, underly the clinical features and disease pathogenesis of primary immunodeficiencies. In affected individuals, these defects manifest as impaired long-term humoral immunity and susceptibility to infection by specific pathogens. In this review, we discuss the importance of, and requirements for, effective interactions between B cells and T cells during the formation of CD4(+) T follicular helper cells and the elicitation of cytotoxic function of virus-specific CD8(+) T cells, as well as how these processes are abrogated in primary immunodeficiencies due to loss-of-function mutations in defined genes. © 2012 New York Academy of Sciences.

Are cancer cells really softer than normal cells?

Science.gov (United States)

Alibert, Charlotte; Goud, Bruno; Manneville, Jean-Baptiste

2017-05-01

Solid tumours are often first diagnosed by palpation, suggesting that the tumour is more rigid than its surrounding environment. Paradoxically, individual cancer cells appear to be softer than their healthy counterparts. In this review, we first list the physiological reasons indicating that cancer cells may be more deformable than normal cells. Next, we describe the biophysical tools that have been developed in recent years to characterise and model cancer cell mechanics. By reviewing the experimental studies that compared the mechanics of individual normal and cancer cells, we argue that cancer cells can indeed be considered as softer than normal cells. We then focus on the intracellular elements that could be responsible for the softening of cancer cells. Finally, we ask whether the mechanical differences between normal and cancer cells can be used as diagnostic or prognostic markers of cancer progression. © 2017 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.

Exosome-Based Cell-Cell Communication in the Tumor Microenvironment

Directory of Open Access Journals (Sweden)

Joana Maia

2018-02-01

Full Text Available Tumors are not isolated entities, but complex systemic networks involving cell-cell communication between transformed and non-transformed cells. The milieu created by tumor-associated cells may either support or halt tumor progression. In addition to cell-cell contact, cells communicate through secreted factors via a highly complex system involving characteristics such as ligand concentration, receptor expression and integration of diverse signaling pathways. Of these, extracellular vesicles, such as exosomes, are emerging as novel cell-cell communication mediators in physiological and pathological scenarios. Exosomes, membrane vesicles of endocytic origin released by all cells (both healthy and diseased, ranging in size from 30 to 150 nm, transport all the main biomolecules, including lipids, proteins, DNAs, messenger RNAs and microRNA, and perform intercellular transfer of components, locally and systemically. By acting not only in tumor cells, but also in tumor-associated cells such as fibroblasts, endothelium, leukocytes and progenitor cells, tumor- and non-tumor cells-derived exosomes have emerged as new players in tumor growth and invasion, tumor-associated angiogenesis, tissue inflammation and immunologic remodeling. In addition, due to their property of carrying molecules from their cell of origin to the peripheral circulation, exosomes have been increasingly studied as sources of tumor biomarkers in liquid biopsies. Here we review the current literature on the participation of exosomes in the communication between tumor and tumor-associated cells, highlighting the role of this process in the setup of tumor microenvironments that modulate tumor initiation and metastasis.

Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

Directory of Open Access Journals (Sweden)

José R Sotelo

Full Text Available To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells at the site of injury to promote regeneration.

Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

Science.gov (United States)

Sotelo, José R; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José R; Xu, Lei; Wallrabe, Horst; Calliari, Aldo; Rosso, Gonzalo; Cal, Karina; Mercer, John A

2013-01-01

To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

Cell fusion induced by ionizing radiation in various cell lines

International Nuclear Information System (INIS)

Khair, M.B.

1994-07-01

Cell fusion induced by ionizing radiation has been studied in rat's hepatocytes in vivo and in different cell lines in vitro. These cell lines were: Hela cells, V-79 fibroblasts, human and rat lymphocytes. For irradiation, 0.85 MeV fission neutrons and 14 MeV fast neutrons were used. Cell analyses were performed by fluorescent dyes using immunofluorescent microscope and flow cytometre. Our results in vivo showed that, regardless the dose-rate, a dose of 1 Gy approximately was enough to induce a significant level of cell fusion depending on neutron energy and the age of rats. The level of cell fusion was also significant in Hela cells at a dose of 0.5 Gy. Similar effect, but to a lesser extent, was observed in V-79 cells. Whereas, in lymphocytes insignificant cell fusion was noticed. The varying levels of cell-fusion in different cell lines could be attributed to the type of cells and mutual contact between cells. Furthermore irradiation did not show any influence on cell division ability in both hepatocytes and Hela cells and that fused cells were also able to divide forming a new generation of cells. (author). 36 refs., 8 figs., 10 tabs

Cell proliferation alterations in Chlorella cells under stress conditions

International Nuclear Information System (INIS)

Rioboo, Carmen; O'Connor, Jose Enrique; Prado, Raquel; Herrero, Concepcion; Cid, Angeles

2009-01-01

Very little is known about growth and proliferation in relation to the cell cycle regulation of algae. The lack of knowledge is even greater when referring to the potential toxic effects of pollutants on microalgal cell division. To assess the effect of terbutryn, a triazine herbicide, on the proliferation of the freshwater microalga Chlorella vulgaris three flow cytometric approaches were used: (1) in vivo cell division using 5-,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining was measured, (2) the growth kinetics were determined by cytometric cell counting and (3) cell viability was evaluated with the membrane-impermeable double-stranded nucleic acid stain propidium iodide (PI). The results obtained in the growth kinetics study using CFSE to identify the microalgal cell progeny were consistent with those determined by cytometric cell counting. In all C. vulgaris cultures, each mother cell had undergone only one round of division through the 96 h of assay and the cell division occurred during the dark period. Cell division of the cultures exposed to the herbicide was asynchronous. Terbutryn altered the normal number of daughter cells (4 autospores) obtained from each mother cell. The number was only two in the cultures treated with 250 nM. The duration of the lag phase after the exposure to terbutryn could be dependent on the existence of a critical cell size to activate cytoplasmic division. Cell size, complexity and fluorescence of chlorophyll a of the microalgal cells presented a marked light/dark (day/night) cycle, except in the non-dividing 500 nM cultures, where terbutryn arrested cell division at the beginning of the cycle. Viability results showed that terbutryn has an algastatic effect in C. vulgaris cells at this concentration. The rapid and precise determination of cell proliferation by CFSE staining has allowed us to develop a model for assessing both the cell cycle of C. vulgaris and the in vivo effects of pollutants on growth and

Cell proliferation alterations in Chlorella cells under stress conditions

Energy Technology Data Exchange (ETDEWEB)

Rioboo, Carmen [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain); O' Connor, Jose Enrique [Laboratorio de Citomica, Unidad Mixta de Investigacion CIPF-UVEG, Centro de Investigacion Principe Felipe, Avda. Autopista del Saler, 16, 46013 Valencia (Spain); Prado, Raquel; Herrero, Concepcion [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain); Cid, Angeles, E-mail: cid@udc.es [Laboratorio de Microbiologia, Facultad de Ciencias, Universidad de A Coruna, Campus da Zapateira s/n, 15008 A Coruna (Spain)

2009-09-14

Very little is known about growth and proliferation in relation to the cell cycle regulation of algae. The lack of knowledge is even greater when referring to the potential toxic effects of pollutants on microalgal cell division. To assess the effect of terbutryn, a triazine herbicide, on the proliferation of the freshwater microalga Chlorella vulgaris three flow cytometric approaches were used: (1) in vivo cell division using 5-,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining was measured, (2) the growth kinetics were determined by cytometric cell counting and (3) cell viability was evaluated with the membrane-impermeable double-stranded nucleic acid stain propidium iodide (PI). The results obtained in the growth kinetics study using CFSE to identify the microalgal cell progeny were consistent with those determined by cytometric cell counting. In all C. vulgaris cultures, each mother cell had undergone only one round of division through the 96 h of assay and the cell division occurred during the dark period. Cell division of the cultures exposed to the herbicide was asynchronous. Terbutryn altered the normal number of daughter cells (4 autospores) obtained from each mother cell. The number was only two in the cultures treated with 250 nM. The duration of the lag phase after the exposure to terbutryn could be dependent on the existence of a critical cell size to activate cytoplasmic division. Cell size, complexity and fluorescence of chlorophyll a of the microalgal cells presented a marked light/dark (day/night) cycle, except in the non-dividing 500 nM cultures, where terbutryn arrested cell division at the beginning of the cycle. Viability results showed that terbutryn has an algastatic effect in C. vulgaris cells at this concentration. The rapid and precise determination of cell proliferation by CFSE staining has allowed us to develop a model for assessing both the cell cycle of C. vulgaris and the in vivo effects of pollutants on growth and

Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization.

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Hazim, Roni A; Karumbayaram, Saravanan; Jiang, Mei; Dimashkie, Anupama; Lopes, Vanda S; Li, Douran; Burgess, Barry L; Vijayaraj, Preethi; Alva-Ornelas, Jackelyn A; Zack, Jerome A; Kohn, Donald B; Gomperts, Brigitte N; Pyle, April D; Lowry, William E; Williams, David S

2017-10-02

Dysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation. We have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions. Using highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function. This report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by

Cell Cycle Regulation of Stem Cells by MicroRNAs.

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Mens, Michelle M J; Ghanbari, Mohsen

2018-06-01

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules involved in the regulation of gene expression. They are involved in the fine-tuning of fundamental biological processes such as proliferation, differentiation, survival and apoptosis in many cell types. Emerging evidence suggests that miRNAs regulate critical pathways involved in stem cell function. Several miRNAs have been suggested to target transcripts that directly or indirectly coordinate the cell cycle progression of stem cells. Moreover, previous studies have shown that altered expression levels of miRNAs can contribute to pathological conditions, such as cancer, due to the loss of cell cycle regulation. However, the precise mechanism underlying miRNA-mediated regulation of cell cycle in stem cells is still incompletely understood. In this review, we discuss current knowledge of miRNAs regulatory role in cell cycle progression of stem cells. We describe how specific miRNAs may control cell cycle associated molecules and checkpoints in embryonic, somatic and cancer stem cells. We further outline how these miRNAs could be regulated to influence cell cycle progression in stem cells as a potential clinical application.

Tetherin restricts productive HIV-1 cell-to-cell transmission.

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Nicoletta Casartelli

2010-06-01

Full Text Available The IFN-inducible antiviral protein tetherin (or BST-2/CD317/HM1.24 impairs release of mature HIV-1 particles from infected cells. HIV-1 Vpu antagonizes the effect of tetherin. The fate of virions trapped at the cell surface remains poorly understood. Here, we asked whether tetherin impairs HIV cell-to-cell transmission, a major means of viral spread. Tetherin-positive or -negative cells, infected with wild-type or DeltaVpu HIV, were used as donor cells and cocultivated with target lymphocytes. We show that tetherin inhibits productive cell-to-cell transmission of DeltaVpu to targets and impairs that of WT HIV. Tetherin accumulates with Gag at the contact zone between infected and target cells, but does not prevent the formation of virological synapses. In the presence of tetherin, viruses are then mostly transferred to targets as abnormally large patches. These viral aggregates do not efficiently promote infection after transfer, because they accumulate at the surface of target cells and are impaired in their fusion capacities. Tetherin, by imprinting virions in donor cells, is the first example of a surface restriction factor limiting viral cell-to-cell spread.

Ion channels involved in cell volume regulation: effects on migration, proliferation, and programmed cell death in non adherent EAT cells and adherent ELA cells.

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Hoffmann, Else Kay

2011-01-01

This mini review outlines studies of cell volume regulation in two closely related mammalian cell lines: nonadherent Ehrlich ascites tumour cells (EATC) and adherent Ehrlich Lettre ascites (ELA) cells. Focus is on the regulatory volume decrease (RVD) that occurs after cell swelling, the volume regulatory ion channels involved, and the mechanisms (cellular signalling pathways) that regulate these channels. Finally, I shall also briefly review current investigations in these two cell lines that focuses on how changes in cell volume can regulate cell functions such as cell migration, proliferation, and programmed cell death. Copyright © 2011 S. Karger AG, Basel.

Cholesterol inhibits entotic cell-in-cell formation and actomyosin contraction.

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Ruan, Banzhan; Zhang, Bo; Chen, Ang; Yuan, Long; Liang, Jianqing; Wang, Manna; Zhang, Zhengrong; Fan, Jie; Yu, Xiaochen; Zhang, Xin; Niu, Zubiao; Zheng, You; Gu, Songzhi; Liu, Xiaoqing; Du, Hongli; Wang, Jufang; Hu, Xianwen; Gao, Lihua; Chen, Zhaolie; Huang, Hongyan; Wang, Xiaoning; Sun, Qiang

2018-01-01

Cell-in-cell structure is prevalent in human cancer, and associated with several specific pathophysiological phenomena. Although cell membrane adhesion molecules were found critical for cell-in-cell formation, the roles of other membrane components, such as lipids, remain to be explored. In this study, we attempted to investigate the effects of cholesterol and phospholipids on the formation of cell-in-cell structures by utilizing liposome as a vector. We found that Lipofectamine-2000, the reagent commonly used for routine transfection, could significantly reduce entotic cell-in-cell formation in a cell-specific manner, which is correlated with suppressed actomyosin contraction as indicated by reduced β-actin expression and myosin light chain phosphorylation. The influence on cell-in-cell formation was likely dictated by specific liposome components as some liposomes affected cell-in-cell formation while some others didn't. Screening on a limited number of lipids, the major components of liposome, identified phosphatidylethanolamine (PE), stearamide (SA), lysophosphatidic acid (LPA) and cholesterol (CHOL) as the inhibitors of cell-in-cell formation. Importantly, cholesterol treatment significantly inhibited myosin light chain phosphorylation, which resembles the effect of Lipofectamine-2000, suggesting cholesterol might be partially responsible for liposomes' effects on cell-in-cell formation. Together, our findings supporting a role of membrane lipids and cholesterol in cell-in-cell formation probably via regulating actomyosin contraction. Copyright © 2017 Elsevier Inc. All rights reserved.

Selfish cells in altruistic cell society - a theoretical oncology.

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Chigira, M

1993-09-01

In multicellular organisms, internal evolution of individual cells is strictly forbidden and 'evolutional' DNA replication should be performed only by the sexual reproduction system. Wholistic negative control system called 'homeostasis' serves all service to germ line cells. All somatic cells are altruistic to the germ line cells. However, in malignant tumors, it seems that individual cells replicate and behave 'selfishly' and evolve against the internal microenvironment. Tumor cells only express the occult selfishness which is programmed in normal cells a priori. This phenomenon is based on the failure of identical DNA replication, and results in 'autonomy' and 'anomie' of cellular society as shown in tumor cells. Genetic programs of normal cells connote this cellular autonomy and anomie introduced by the deletion of regulators on structure genes. It is rather paradoxical that the somatic cells get their freedom from wholistic negative regulation programmed internally. However, this is not a true paradox, since multicellular organisms have clearly been evolved from 'monads' in which cells proliferate without wholistic regulation. Somatic cells revolt against germ cell DNA, called 'selfish replicator' by Dawkins. It is an inevitable destiny that the 'selfishness' coded in genome should be revenged by itself. Selfish replicator in germ cell line should be revolted by its selfishness in the expansion of somatic cells, since they have an orthogenesis to get more selfishness in order to increase their genome. Tumor heterogeneity and progression can be fully explained by this self-contradictory process which produces heterogeneous gene copies different from the original clone in the tumor, although 'selfish' gene replication is the final target of being. Furthermore, we have to discard the concept of clonality of tumor cells since genetic instability is a fundamental feature of tumors. Finally, tumor cells and proto-oncogenes can be considered as the ultimate parasite

The Antigen Presenting Cells Instruct Plasma Cell Differentiation

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Wei eXu

2014-01-01

Full Text Available The professional antigen presenting cells (APCs, including many subsets of dendritic cells and macrophages, not only mediate prompt but nonspecific response against microbes, but also bridge the antigen-specific adaptive immune response through antigen presentation. In the latter, typically activated B cells acquire cognate signals from T helper cells in the germinal center of lymphoid follicles to differentiate into plasma cells, which generate protective antibodies. Recent advances have revealed that many APC subsets provide not only signal 1 (the antigen, but also signal 2 to directly instruct the differentiation process of plasma cells in a T cell-independent manner. Herein, the different signals provided by these APC subsets to direct B cell proliferation, survival, class switching and terminal differentiation are discussed. We furthermore propose that the next generation of vaccines for boosting antibody response could be designed by targeting APCs.

Cell cycle variation in x-ray survival for cells from spheroids measured by volume cell sorting

International Nuclear Information System (INIS)

Freyer, J.P.; Wilder, M.E.; Raju, M.R.

1984-01-01

Considerable work has been done studying the variation in cell survival as a function of cell cycle position for monolayers or single cells exposed to radiation. Little is known about the effects of multicellular growth on the relative radiation sensitivity of cells in different cell cycle stages. The authors have developed a new technique for measuring the response of cells, using volume cell sorting, which is rapid, non-toxic, and does not require cell synchronization. By combining this technique with selective spheroid dissociation,they have measured the age response of cells located at various depths in EMT6 and Colon 26 spheroids. Although cells in the inner region had mostly G1-phase DNA contents, 15-20% had S- and G2-phase DNA contents. Analysis of these cells using BrdU labeling and flow cytometric analysis with a monoclonal antibody to BrdU indicated that the inner region cells were not synthesizing DNA. Thus, the authors were able to measure the radiation response of cells arrested in G1, S and G2 cell cycle phases. Comparison of inner and outer spheroid regions, and monolayer cultures, indicates that it is improper to extrapolate age response data in standard culture conditions to the situation in spheroids

Combination cell therapy with mesenchymal stem cells and neural stem cells for brain stroke in rats.

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Hosseini, Seyed Mojtaba; Farahmandnia, Mohammad; Razi, Zahra; Delavari, Somayeh; Shakibajahromi, Benafsheh; Sarvestani, Fatemeh Sabet; Kazemi, Sepehr; Semsar, Maryam

2015-05-01

Brain stroke is the second most important events that lead to disability and morbidity these days. Although, stroke is important, there is no treatment for curing this problem. Nowadays, cell therapy has opened a new window for treating central nervous system disease. In some previous studies the Mesenchymal stem cells and neural stem cells. In this study, we have designed an experiment to assess the combination cell therapy (Mesenchymal and Neural stem cells) effects on brain stroke. The Mesenchymal stem cells were isolated from adult rat bone marrow and the neural stem cells were isolated from ganglion eminence of rat embryo 14 days. The Mesenchymal stem cells were injected 1 day after middle cerebral artery occlusion (MCAO) and the neural stem cells transplanted 7 day after MCAO. After 28 days, the neurological outcomes and brain lesion volumes were evaluated. Also, the activity of Caspase 3 was assessed in different groups. The group which received combination cell therapy had better neurological examination and less brain lesion. Also the combination cell therapy group had the least Caspase 3 activity among the groups. The combination cell therapy is more effective than Mesenchymal stem cell therapy and neural stem cell therapy separately in treating the brain stroke in rats.

Tissue-resident natural killer (NK) cells are cell lineages distinct from thymic and conventional splenic NK cells

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Sojka, Dorothy K; Plougastel-Douglas, Beatrice; Yang, Liping; Pak-Wittel, Melissa A; Artyomov, Maxim N; Ivanova, Yulia; Zhong, Chao; Chase, Julie M; Rothman, Paul B; Yu, Jenny; Riley, Joan K; Zhu, Jinfang; Tian, Zhigang; Yokoyama, Wayne M

2014-01-01

Natural killer (NK) cells belong to the innate immune system; they can control virus infections and developing tumors by cytotoxicity and producing inflammatory cytokines. Most studies of mouse NK cells, however, have focused on conventional NK (cNK) cells in the spleen. Recently, we described two populations of liver NK cells, tissue-resident NK (trNK) cells and those resembling splenic cNK cells. However, their lineage relationship was unclear; trNK cells could be developing cNK cells, related to thymic NK cells, or a lineage distinct from both cNK and thymic NK cells. Herein we used detailed transcriptomic, flow cytometric, and functional analysis and transcription factor-deficient mice to determine that liver trNK cells form a distinct lineage from cNK and thymic NK cells. Taken together with analysis of trNK cells in other tissues, there are at least four distinct lineages of NK cells: cNK, thymic, liver (and skin) trNK, and uterine trNK cells. DOI: http://dx.doi.org/10.7554/eLife.01659.001 PMID:24714492

The recruitability and cell-cycle state of intestinal stem cells

International Nuclear Information System (INIS)

Potten, C.S.; Chadwick, C.; Ijiri, K.; Tsubouchi, S.; Hanson, W.R.

1984-01-01

Evidence is presented which suggests that the crypts of the small intestine contain at least two discrete but interdependent classes of stem cells, some with discrete cell kinetic properties and some with discrete radiation responses or radiosensitivities. Very low doses of X rays or gamma rays, or neutrons, kill a few cells in the stem cell regions of the crypt in a sensitive dose-dependent manner. Similar doses generate several different cell kinetic responses within either the clonogenic fraction or the cells at the stem cell position within the crypt. The cell kinetic responses range from apparent recruitment of G0 clonogenic cells into cycle, to a marked shortening of the average cell cycle of the cells at the stem cell position. It is suggested that the cell kinetic changes may be the consequence of the cell destruction

Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

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Chen Lei

2011-06-01

Full Text Available Abstract Background Cancer stem cells (CSCs are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44. Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

Th17 cell-mediated immune responses promote mast cell proliferation by triggering stem cell factor in keratinocytes

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Cho, Kyung-Ah; Park, Minhwa; Kim, Yu-Hee; Woo, So-Youn

2017-01-01

Although mast cells are traditionally thought to function as effector cells in allergic responses, they have increasingly been recognized as important regulators of various immune responses. Mast cells mature locally; thus, tissue-specific influences are important for promoting mast cell accumulation and survival in the skin and the gastrointestinal tract. In this study, we determined the effects of keratinocytes on mast cell accumulation during Th17-mediated skin inflammation. We observed increases in dermal mast cells in imiquimod-induced psoriatic dermatitis in mice accompanied by the expression of epidermal stem cell factor (SCF), a critical mast cell growth factor. Similar to mouse epidermal keratinocytes, SCF was highly expressed in the human HaCaT keratinocyte cell line following stimulation with IL−17. Further, keratinocytes promoted mast cell proliferation following stimulation with IL−17 in vitro. However, the effects of keratinocytes on mast cells were significantly diminished in the presence of anti−CD117 (stem cell factor receptor) blocking antibodies. Taken together, our results revealed that the Th17-mediated inflammatory environment promotes mast cell accumulation through keratinocyte-derived SCF. - Highlights: • Psoriasis-like skin inflammation increase dermal mast cells. • Keratinocyte produce stem cell factor in psoriasis-like skin inflammation. • Keratinocyte promote mast cell proliferation by stem cell factor dependent manner

NKp46+CD3+ cells - a novel non-conventional T-cell subset in cattle exhibiting both NK cell and T-cell features

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Connelley, Timothy K.; Longhi, Cassandra; Burrells, Alison; Degnan, Kathryn; Hope, Jayne; Allan, Alasdair; Hammond, John A.; Storset, Anne K.; Morrison, W. Ivan

2014-01-01

The NKp46 receptor demonstrates a high degree of lineage-specificity, being expressed almost exclusively in natural killer cells. Previous studies have demonstrated NKp46 expression by T-cells, but NKp46+CD3+ cells are rare and almost universally associated with NKp46 acquisition by T-cells following stimulation. In this study we demonstrate the existence of a population of NKp46+CD3+ cells resident in normal bovine PBMC which include cells of both the αβ TCR+ and γδ TCR+ lineages and is present at a frequency of 0.1-1.7%. NKp46+CD3+ cells express transcripts for a broad repertoire of both natural killer (NKR) and T-cell receptors (TCR) and also the CD3ζ, DAP10 and FcεR1γ but not DAP12 adaptor proteins. In vitro functional analysis of NKp46+CD3+ cells confirm that NKp46, CD16 and CD3 signalling pathways are all functionally competent and capable of mediating-re-direct cytolysis. However, only CD3 cross-ligation elicits IFN-γ release. NKp46+CD3+ cells exhibit cytotoxic activity against autologous Theileria parva infected cells in vitro and during in vivo challenge with this parasite an expansion of NKp46+CD3+ cells was observed in some animals, indicating the cells have the potential to act as an anti-pathogen effector population. The results presented herein identifies and describes a novel non-conventional NKp46+CD3+ T-cell subset that is phenotypically and functionally distinct from conventional NK and T-cells. The ability to exploit both NKR and TCR suggests these cells may fill a functional niche at the interface of innate and adaptive immune responses. PMID:24639352

Optimizing cell viability in droplet-based cell deposition

NARCIS (Netherlands)

Hendriks, Jan; Visser, C.W.; Henke, S.J.; Leijten, Jeroen Christianus Hermanus; Saris, Daniël B.F.; Sun, Chao; Lohse, Detlef; Karperien, Hermanus Bernardus Johannes

2015-01-01

Biofabrication commonly involves the use of liquid droplets to transport cells to the printed structure. However, the viability of the cells after impact is poorly controlled and understood, hampering applications including cell spraying, inkjet bioprinting, and laser-assisted cell transfer. Here,

Pituitary cell differentiation from stem cells and other cells: toward restorative therapy for hypopituitarism?

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Willems, Christophe; Vankelecom, Hugo

2014-01-01

The pituitary gland, key regulator of our endocrine system, produces multiple hormones that steer essential physiological processes. Hence, deficient pituitary function (hypopituitarism) leads to severe disorders. Hypopituitarism can be caused by defective embryonic development, or by damage through tumor growth/resection and traumatic brain injury. Lifelong hormone replacement is needed but associated with significant side effects. It would be more desirable to restore pituitary tissue and function. Recently, we showed that the adult (mouse) pituitary holds regenerative capacity in which local stem cells are involved. Repair of deficient pituitary may therefore be achieved by activating these resident stem cells. Alternatively, pituitary dysfunction may be mended by cell (replacement) therapy. The hormonal cells to be transplanted could be obtained by (trans-)differentiating various kinds of stem cells or other cells. Here, we summarize the studies on pituitary cell regeneration and on (trans-)differentiation toward hormonal cells, and speculate on restorative therapies for pituitary deficiency.

Single-Cell RNA Sequencing of Glioblastoma Cells.

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Sen, Rajeev; Dolgalev, Igor; Bayin, N Sumru; Heguy, Adriana; Tsirigos, Aris; Placantonakis, Dimitris G

2018-01-01

Single-cell RNA sequencing (sc-RNASeq) is a recently developed technique used to evaluate the transcriptome of individual cells. As opposed to conventional RNASeq in which entire populations are sequenced in bulk, sc-RNASeq can be beneficial when trying to better understand gene expression patterns in markedly heterogeneous populations of cells or when trying to identify transcriptional signatures of rare cells that may be underrepresented when using conventional bulk RNASeq. In this method, we describe the generation and analysis of cDNA libraries from single patient-derived glioblastoma cells using the C1 Fluidigm system. The protocol details the use of the C1 integrated fluidics circuit (IFC) for capturing, imaging and lysing cells; performing reverse transcription; and generating cDNA libraries that are ready for sequencing and analysis.

Relation Between the Cell Volume and the Cell Cycle Dynamics in Mammalian cell

International Nuclear Information System (INIS)

Magno, A.C.G.; Oliveira, I.L.; Hauck, J.V.S.

2016-01-01

The main goal of this work is to add and analyze an equation that represents the volume in a dynamical model of the mammalian cell cycle proposed by Gérard and Goldbeter (2011) [1]. The cell division occurs when the cyclinB/Cdkl complex is totally degraded (Tyson and Novak, 2011)[2] and it reaches a minimum value. At this point, the cell is divided into two newborn daughter cells and each one will contain the half of the cytoplasmic content of the mother cell. The equations of our base model are only valid if the cell volume, where the reactions occur, is constant. Whether the cell volume is not constant, that is, the rate of change of its volume with respect to time is explicitly taken into account in the mathematical model, then the equations of the original model are no longer valid. Therefore, every equations were modified from the mass conservation principle for considering a volume that changes with time. Through this approach, the cell volume affects all model variables. Two different dynamic simulation methods were accomplished: deterministic and stochastic. In the stochastic simulation, the volume affects every model's parameters which have molar unit, whereas in the deterministic one, it is incorporated into the differential equations. In deterministic simulation, the biochemical species may be in concentration units, while in stochastic simulation such species must be converted to number of molecules which are directly proportional to the cell volume. In an effort to understand the influence of the new equation a stability analysis was performed. This elucidates how the growth factor impacts the stability of the model's limit cycles. In conclusion, a more precise model, in comparison to the base model, was created for the cell cycle as it now takes into consideration the cell volume variation (paper)

Cell-substrate interaction with cell-membrane-stress dependent adhesion.

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Jiang, H; Yang, B

2012-01-10

Cell-substrate interaction is examined in a two-dimensional mechanics model. The cell and substrate are treated as a shell and an elastic solid, respectively. Their interaction through adhesion is treated using nonlinear springs. Compared to previous cell mechanics models, the present model introduces a cohesive force law that is dependent not only on cell-substrate distance but also on internal cell-membrane stress. It is postulated that a living cell would establish focal adhesion sites with density dependent on the cell-membrane stress. The formulated mechanics problem is numerically solved using coupled finite elements and boundary elements for the cell and the substrate, respectively. The nodes in the adhesion zone from either side are linked by the cohesive springs. The specific cases of a cell adhering to a homogeneous substrate and a heterogeneous bimaterial substrate are examined. The analyses show that the substrate stiffness affects the adhesion behavior significantly and regulates the direction of cell adhesion, in good agreement with the experimental results in the literature. By introducing a reactive parameter (i.e., cell-membrane stress) linking biological responses of a living cell to a mechanical environment, the present model offers a unified mechanistic vehicle for characterization and prediction of living cell responses to various kinds of mechanical stimuli including local extracellular matrix and neighboring cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

Dendritic cells fused with different pancreatic carcinoma cells induce different T-cell responses

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Andoh Y

2013-01-01

Full Text Available Yoshiaki Andoh,1,2 Naohiko Makino,2 Mitsunori Yamakawa11Department of Pathological Diagnostics, 2Department of Gastroenterology, Yamagata University School of Medicine, Yamagata, JapanBackground: It is unclear whether there are any differences in the induction of cytotoxic T lymphocytes (CTL and CD4+CD25high regulatory T-cells (Tregs among dendritic cells (DCs fused with different pancreatic carcinomas. The aim of this study was to compare the ability to induce cytotoxicity by human DCs fused with different human pancreatic carcinoma cell lines and to elucidate the causes of variable cytotoxicity among cell lines.Methods: Monocyte-derived DCs, which were generated from peripheral blood mononuclear cells (PBMCs, were fused with carcinoma cells such as Panc-1, KP-1NL, QGP-1, and KP-3L. The induction of CTL and Tregs, and cytokine profile of PBMCs stimulated by fused DCs were evaluated.Results: The cytotoxicity against tumor targets induced by PBMCs cocultured with DCs fused with QGP-1 (DC/QGP-1 was very low, even though PBMCs cocultured with DCs fused with other cell lines induced significant cytotoxicity against the respective tumor target. The factors causing this low cytotoxicity were subsequently investigated. DC/QGP-1 induced a significant expansion of Tregs in cocultured PBMCs compared with DC/KP-3L. The level of interleukin-10 secreted in the supernatants of PBMCs cocultured with DC/QGP-1 was increased significantly compared with that in DC/KP-3L. Downregulation of major histocompatibility complex class I expression and increased secretion of vascular endothelial growth factor were observed with QGP-1, as well as in the other cell lines.Conclusion: The present study demonstrated that the cytotoxicity induced by DCs fused with pancreatic cancer cell lines was different between each cell line, and that the reduced cytotoxicity of DC/QGP-1 might be related to the increased secretion of interleukin-10 and the extensive induction of Tregs

Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

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Kelly, Catriona, E-mail: catriona.kelly@qub.ac.uk [SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine (United Kingdom); Flatt, Peter R.; McClenaghan, Neville H. [SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine (United Kingdom)

2010-08-20

Research highlights: {yields} TGP52 cells display enhanced functionality in pseudoislet form. {yields} Somatostatin content was reduced, but secretion increased in high glucose conditions. {yields} Cellular interactions and environment alter the somatostatin status of TGP52 cells. -- Abstract: Introduction: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. Methods: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mM glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. Results: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. Conclusions: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.

Cell-to-cell communication and cellular environment alter the somatostatin status of delta cells

International Nuclear Information System (INIS)

Kelly, Catriona; Flatt, Peter R.; McClenaghan, Neville H.

2010-01-01

Research highlights: → TGP52 cells display enhanced functionality in pseudoislet form. → Somatostatin content was reduced, but secretion increased in high glucose conditions. → Cellular interactions and environment alter the somatostatin status of TGP52 cells. -- Abstract: Introduction: Somatostatin, released from pancreatic delta cells, is a potent paracrine inhibitor of insulin and glucagon secretion. Islet cellular interactions and glucose homeostasis are essential to maintain normal patterns of insulin secretion. However, the importance of cell-to-cell communication and cellular environment in the regulation of somatostatin release remains unclear. Methods: This study employed the somatostatin-secreting TGP52 cell line maintained in DMEM:F12 (17.5 mM glucose) or DMEM (25 mM glucose) culture media. The effect of pseudoislet formation and culture medium on somatostatin content and release in response to a variety of stimuli was measured by somatostatin EIA. In addition, the effect of pseudoislet formation on cellular viability (MTT and LDH assays) and proliferation (BrdU ELISA) was determined. Results: TGP52 cells readily formed pseudoislets and showed enhanced functionality in three-dimensional form with increased E-cadherin expression irrespective of the culture environment used. However, culture in DMEM decreased cellular somatostatin content (P < 0.01) and increased somatostatin secretion in response to a variety of stimuli including arginine, calcium and PMA (P < 0.001) when compared with cells grown in DMEM:F12. Configuration of TGP52 cells as pseudoislets reduced the proliferative rate and increased cellular cytotoxicity irrespective of culture medium used. Conclusions: Somatostatin secretion is greatly facilitated by cell-to-cell interactions and E-cadherin expression. Cellular environment and extracellular glucose also significantly influence the function of delta cells.

Microencapsulating and Banking Living Cells for Cell-Based Medicine

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Wujie Zhang

2011-01-01

Full Text Available A major challenge to the eventual success of the emerging cell-based medicine such as tissue engineering, regenerative medicine, and cell transplantation is the limited availability of the desired cell sources. This challenge can be addressed by cell microencapsulation to overcome the undesired immune response (i.e., to achieve immunoisolation so that non-autologous cells can be used to treat human diseases, and by cell/tissue preservation to bank living cells for wide distribution to end users so that they are readily available when needed in the future. This review summarizes the status quo of research in both cell microencapsulation and banking the microencapsulated cells. It is concluded with a brief outlook of future research directions in this important field.

Effect of cell-phone radiofrequency on angiogenesis and cell invasion in human head and neck cancer cells.

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Alahmad, Yaman M; Aljaber, Mohammed; Saleh, Alaaeldin I; Yalcin, Huseyin C; Aboulkassim, Tahar; Yasmeen, Amber; Batist, Gerald; Moustafa, Ala-Eddin Al

2018-05-13

Today, the cell phone is the most widespread technology globally. However, the outcome of cell-phone radiofrequency on head and neck cancer progression has not yet been explored. The chorioallantoic membrane (CAM) and human head and neck cancer cell lines, FaDu and SCC25, were used to explore the outcome of cell-phone radiofrequency on angiogenesis, cell invasion, and colony formation of head and neck cancer cells, respectively. Western blot analysis was used to investigate the impact of the cell phone on the regulation of E-cadherin and Erk1/Erk2 genes. Our data revealed that cell-phone radiofrequency promotes angiogenesis of the CAM. In addition, the cell phone enhances cell invasion and colony formation of human head and neck cancer cells; this is accompanied by a downregulation of E-cadherin expression. More significantly, we found that the cell phone can activate Erk1/Erk2 in our experimental models. Our investigation reveals that cell-phone radiofrequency could enhance head and neck cancer by stimulating angiogenesis and cell invasion via Erk1/Erk2 activation. © 2018 Wiley Periodicals, Inc.

Stabilization Of Apoptotic Cells: Generation Of Zombie Cells

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José A. Sánchez Alcázar

2015-08-01

Stabilization of apoptotic cells can be used for reliable detection and quantification of apoptosis in cultured cells and may allow a safer administration of apoptotic cells in clinical applications. Furthermore, it opens new avenues in the functional reconstruction of apoptotic cells for longer preservation.

Single cell transcriptome profiling of developing chick retinal cells.

Science.gov (United States)

Laboissonniere, Lauren A; Martin, Gregory M; Goetz, Jillian J; Bi, Ran; Pope, Brock; Weinand, Kallie; Ellson, Laura; Fru, Diane; Lee, Miranda; Wester, Andrea K; Liu, Peng; Trimarchi, Jeffrey M

2017-08-15

The vertebrate retina is a specialized photosensitive tissue comprised of six neuronal and one glial cell types, each of which develops in prescribed proportions at overlapping timepoints from a common progenitor pool. While each of these cells has a specific function contributing to proper vision in the mature animal, their differential representation in the retina as well as the presence of distinctive cellular subtypes makes identifying the transcriptomic signatures that lead to each retinal cell's fate determination and development challenging. We have analyzed transcriptomes from individual cells isolated from the chick retina throughout retinogenesis. While we focused our efforts on the retinal ganglion cells, our transcriptomes of developing chick cells also contained representation from multiple retinal cell types, including photoreceptors and interneurons at different stages of development. Most interesting was the identification of transcriptomes from individual mixed lineage progenitor cells in the chick as these cells offer a window into the cell fate decision-making process. Taken together, these data sets will enable us to uncover the most critical genes acting in the steps of cell fate determination and early differentiation of various retinal cell types. © 2017 Wiley Periodicals, Inc.

Mechanisms for Cell-to-Cell Transmission of HIV-1

Science.gov (United States)

Bracq, Lucie; Xie, Maorong; Benichou, Serge; Bouchet, Jérôme

2018-01-01

While HIV-1 infection of target cells with cell-free viral particles has been largely documented, intercellular transmission through direct cell-to-cell contact may be a predominant mode of propagation in host. To spread, HIV-1 infects cells of the immune system and takes advantage of their specific particularities and functions. Subversion of intercellular communication allows to improve HIV-1 replication through a multiplicity of intercellular structures and membrane protrusions, like tunneling nanotubes, filopodia, or lamellipodia-like structures involved in the formation of the virological synapse. Other features of immune cells, like the immunological synapse or the phagocytosis of infected cells are hijacked by HIV-1 and used as gateways to infect target cells. Finally, HIV-1 reuses its fusogenic capacity to provoke fusion between infected donor cells and target cells, and to form infected syncytia with high capacity of viral production and improved capacities of motility or survival. All these modes of cell-to-cell transfer are now considered as viral mechanisms to escape immune system and antiretroviral therapies, and could be involved in the establishment of persistent virus reservoirs in different host tissues. PMID:29515578

Cell origami: self-folding of three-dimensional cell-laden microstructures driven by cell traction force.

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Kaori Kuribayashi-Shigetomi

Full Text Available This paper describes a method of generating three-dimensional (3D cell-laden microstructures by applying the principle of origami folding technique and cell traction force (CTF. We harness the CTF as a biological driving force to fold the microstructures. Cells stretch and adhere across multiple microplates. Upon detaching the microplates from a substrate, CTF causes the plates to lift and fold according to a prescribed pattern. This self-folding technique using cells is highly biocompatible and does not involve special material requirements for the microplates and hinges to induce folding. We successfully produced various 3D cell-laden microstructures by just changing the geometry of the patterned 2D plates. We also achieved mass-production of the 3D cell-laden microstructures without causing damage to the cells. We believe that our methods will be useful for biotechnology applications that require analysis of cells in 3D configurations and for self-assembly of cell-based micro-medical devices.

Cell origami: self-folding of three-dimensional cell-laden microstructures driven by cell traction force.

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Kuribayashi-Shigetomi, Kaori; Onoe, Hiroaki; Takeuchi, Shoji

2012-01-01

This paper describes a method of generating three-dimensional (3D) cell-laden microstructures by applying the principle of origami folding technique and cell traction force (CTF). We harness the CTF as a biological driving force to fold the microstructures. Cells stretch and adhere across multiple microplates. Upon detaching the microplates from a substrate, CTF causes the plates to lift and fold according to a prescribed pattern. This self-folding technique using cells is highly biocompatible and does not involve special material requirements for the microplates and hinges to induce folding. We successfully produced various 3D cell-laden microstructures by just changing the geometry of the patterned 2D plates. We also achieved mass-production of the 3D cell-laden microstructures without causing damage to the cells. We believe that our methods will be useful for biotechnology applications that require analysis of cells in 3D configurations and for self-assembly of cell-based micro-medical devices.

Short-lived, transitory cell-cell interactions foster migration-dependent aggregation.

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Melissa D Pope

Full Text Available During embryonic development, motile cells aggregate into cohesive groups, which give rise to tissues and organs. The role of cell migration in regulating aggregation is unclear. The current paradigm for aggregation is based on an equilibrium model of differential cell adhesivity to neighboring cells versus the underlying substratum. In many biological contexts, however, dynamics is critical. Here, we provide evidence that multicellular aggregation dynamics involves both local adhesive interactions and transport by cell migration. Using time-lapse video microscopy, we quantified the duration of cell-cell contacts among migrating cells that collided and adhered to another cell. This lifetime of cell-cell interactions exhibited a monotonic decreasing dependence on substratum adhesivity. Parallel quantitative measurements of cell migration speed revealed that across the tested range of adhesive substrata, the mean time needed for cells to migrate and encounter another cell was greater than the mean adhesion lifetime, suggesting that aggregation dynamics may depend on cell motility instead of the local differential adhesivity of cells. Consistent with this hypothesis, aggregate size exhibited a biphasic dependence on substratum adhesivity, matching the trend we observed for cell migration speed. Our findings suggest a new role for cell motility, alongside differential adhesion, in regulating developmental aggregation events and motivate new design principles for tuning aggregation dynamics in tissue engineering applications.

Radiation-induced cell death in embryogenic cells of coniferous plants

International Nuclear Information System (INIS)

Watanabe, Yoshito; Homma-Takeda, Shino; Yukawa, Masae; Nishimura, Yoshikazu; Sasamoto, Hamako; Takahagi, Masahiko

2004-01-01

Reproductive processes are particularly radiosensitive in plant development, which was clearly illustrated in reduction of seed formation in native coniferous plants around Chernobyl after the nuclear accident. For the purpose to investigate the effects of ionizing radiation on embryonic formation in coniferous plants, we used an embryo-derived embryogenic cell culture of a Japanese native coniferous plant, Japanese cedar (Cryplomeria japonica). The embryogenic cells were so radiosensitive that most of the cells died by X-ray irradiation of 5 Gy. This indicated that the embryogenic cells are as radiosensitive as some mammalian cells including lymphocytes. We considered that this type of radiosensitive cell death in the embryogenic cells should be responsible for reproductive damages of coniferous plants by low dose of ionizing radiation. The cell death of the embryogenic cells was characteristic of nuclear DNA fragmentation, which is typically observed in radiation-induced programmed cell death, i.e. apoptosis, in mammalian cells. On the other hand, cell death with nuclear DNA fragmentation did not develop by X-ray irradiation in vegetative cells including meristematic cells of Japanese cedar. This suggests that an apoptosis-like programmed cell death should develop cell-specifically in embryogenic cells by ionizing radiation. The abortion of embryogenic cells may work to prevent transmission of radiation-induced genetic damages to the descendants. (author)

Stem Cell Lineages: Between Cell and Organism

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Melinda Bonnie Fagan

2017-01-01

Full Text Available Ontologies of living things are increasingly grounded on the concepts and practices of current life science. Biological development is a process, undergone by living things, which begins with a single cell and (in an important class of cases ends with formation of a multicellular organism. The process of development is thus prima facie central for ideas about biological individuality and organismality. However, recent accounts of these concepts do not engage developmental biology. This paper aims to fill the gap, proposing the lineage view of stem cells as an ontological framework for conceptualizing organismal development. This account is grounded on experimental practices of stem cell research, with emphasis on new techniques for generating biological organization in vitro. On the lineage view, a stem cell is the starting point of a cell lineage with a specific organismal source, time-interval of existence, and ‘tree topology’ of branch-points linking the stem to developmental termini. The concept of ‘enkapsis’ accommodates the cell-organism relation within the lineage view; this hierarchical notion is further explicated by considering the methods and results of stem cell experiments. Results of this examination include a (partial characterization of stem cells’ developmental versatility, and the context-dependence of developmental processes involving stem cells.

A millifluidic study of cell-to-cell heterogeneity in growth-rate and cell-division capability in populations of isogenic cells of Chlamydomonas reinhardtii.

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Shima P Damodaran

Full Text Available To address possible cell-to-cell heterogeneity in growth dynamics of isogenic cell populations of Chlamydomonas reinhardtii, we developed a millifluidic drop-based device that not only allows the analysis of populations grown from single cells over periods of a week, but is also able to sort and collect drops of interest, containing viable and healthy cells, which can be used for further experimentation. In this study, we used isogenic algal cells that were first synchronized in mixotrophic growth conditions. We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homogeneous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers and a significant subpopulation of slowly dividing cells (slow-growers. These observations suggest that algal cells from an isogenic population may be present in either of two states, a state of restricted division and a state of active division. When isogenic cells were allowed to propagate for about 1000 generations on solid agar plates, they displayed an increased heterogeneity in their growth dynamics. Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times. Moreover, populations dividing with the same growth-rate often reached different cell numbers in stationary phase, suggesting that the progenitor cells differed in the number of cell divisions they could undertake. We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes.

Stem cells in dentistry--part I: stem cell sources.

Science.gov (United States)

Egusa, Hiroshi; Sonoyama, Wataru; Nishimura, Masahiro; Atsuta, Ikiru; Akiyama, Kentaro

2012-07-01

Stem cells can self-renew and produce different cell types, thus providing new strategies to regenerate missing tissues and treat diseases. In the field of dentistry, adult mesenchymal stem/stromal cells (MSCs) have been identified in several oral and maxillofacial tissues, which suggests that the oral tissues are a rich source of stem cells, and oral stem and mucosal cells are expected to provide an ideal source for genetically reprogrammed cells such as induced pluripotent stem (iPS) cells. Furthermore, oral tissues are expected to be not only a source but also a therapeutic target for stem cells, as stem cell and tissue engineering therapies in dentistry continue to attract increasing clinical interest. Part I of this review outlines various types of intra- and extra-oral tissue-derived stem cells with regard to clinical availability and applications in dentistry. Additionally, appropriate sources of stem cells for regenerative dentistry are discussed with regard to differentiation capacity, accessibility and possible immunomodulatory properties. Copyright © 2012 Japan Prosthodontic Society. Published by Elsevier Ltd. All rights reserved.

T cell receptor-engineered T cells to treat solid tumors: T cell processing toward optimal T cell fitness

NARCIS (Netherlands)

C.H.J. Lamers (Cor); S. van Steenbergen-Langeveld (Sabine); M. van Brakel (Mandy); C.M. Groot-van Ruijven (Corrien); P.M.M.L. van Elzakker (Pascal); B.A. van Krimpen (Brigitte); S. Sleijfer (Stefan); J.E.M.A. Debets (Reno)

2014-01-01

textabstractTherapy with autologous T cells that have been gene-engineered to express chimeric antigen receptors (CAR) or T cell receptors (TCR) provides a feasible and broadly applicable treatment for cancer patients. In a clinical study in advanced renal cell carcinoma (RCC) patients with CAR T

Interaction of Stellate Cells with Pancreatic Carcinoma Cells

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Marco Siech

2010-09-01

Full Text Available Pancreatic cancer is characterized by its late detection, aggressive growth, intense infiltration into adjacent tissue, early metastasis, resistance to chemo- and radiotherapy and a strong “desmoplastic reaction”. The dense stroma surrounding carcinoma cells is composed of fibroblasts, activated stellate cells (myofibroblast-like cells, various inflammatory cells, proliferating vascular structures, collagens and fibronectin. In particular the cellular components of the stroma produce the tumor microenvironment, which plays a critical role in tumor growth, invasion, spreading, metastasis, angiogenesis, inhibition of anoikis, and chemoresistance. Fibroblasts, myofibroblasts and activated stellate cells produce the extracellular matrix components and are thought to interact actively with tumor cells, thereby promoting cancer progression. In this review, we discuss our current understanding of the role of pancreatic stellate cells (PSC in the desmoplastic response of pancreas cancer and the effects of PSC on tumor progression, metastasis and drug resistance. Finally we present some novel ideas for tumor therapy by interfering with the cancer cell-host interaction.

Bio optofluidics cell sorter: cell-BOCS concept and applications

Science.gov (United States)

Roth, Tue; Glückstad, Jesper

2012-03-01

The cell-BOCS is a novel microfluidics based cell-sorting instrument utilizing next generation optical trapping technology developed at the Technical University of Denmark. It is targeted emerging bio-medical research and diagnostics markets where it for certain applications offers a number of advantages over conventional fluorescence activated cell-sorting (FACSTM) technology. Advantages include gentle handling of cells, sterile sorting, easy operation, small footprint and lower cost allowing out-of-core-facility use. Application examples are found within sorting of fragile transfected cells, high value samples and primary cell lines, where traditional FACS technology has limited application due to it's droplet-based approach to cell-sorting. In the diagnostics field, in particular applying the cell-BOCS for isolating pure populations of circulating tumor cells is an area that has generated a lot of interest.

HIV Cell-to-Cell Spread Results in Earlier Onset of Viral Gene Expression by Multiple Infections per Cell.

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Mikaël Boullé

2016-11-01

Full Text Available Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more rapid than cell-free infection. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell line, as well as single cell staining for infection over time in primary cells. We compared cell-to-cell spread of HIV to cell-free infection, and limited both types of transmission to a two-hour window to minimize differences due to virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell line and by 35% in peripheral blood mononuclear cells relative to cell-free HIV infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses.

Primitive human hematopoietic cells give rise to differentially specified daughter cells upon their initial cell division.

NARCIS (Netherlands)

Giebel, B.; Zhang, T.; Beckmann, J.; Spanholtz, J.; Wernet, P.; Ho, A.; Punzel, M.

2006-01-01

It is often predicted that stem cells divide asymmetrically, creating a daughter cell that maintains the stem-cell capacity, and 1 daughter cell committed to differentiation. While asymmetric stem-cell divisions have been proven to occur in model organisms (eg, in Drosophila), it remains illusive

A Dominant-Negative PPARγ Mutant Promotes Cell Cycle Progression and Cell Growth in Vascular Smooth Muscle Cells

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Joey Z. Liu

2009-01-01

Full Text Available PPARγ ligands have been shown to have antiproliferative effects on many cell types. We herein report that a synthetic dominant-negative (DN PPARγ mutant functions like a growth factor to promote cell cycle progression and cell proliferation in human coronary artery smooth muscle cells (CASMCs. In quiescent CASMCs, adenovirus-expressed DN-PPARγ promoted G1→S cell cycle progression, enhanced BrdU incorporation, and increased cell proliferation. DN-PPARγ expression also markedly enhanced positive regulators of the cell cycle, increasing Rb and CDC2 phosphorylation and the expression of cyclin A, B1, D1, and MCM7. Conversely, overexpression of wild-type (WT or constitutively-active (CA PPARγ inhibited cell cycle progression and the activity and expression of positive regulators of the cell cycle. DN-PPARγ expression, however, did not up-regulate positive cell cycle regulators in PPARγ-deficient cells, strongly suggesting that DN-PPARγ effects on cell cycle result from blocking the function of endogenous wild-type PPARγ. DN-PPARγ expression enhanced phosphorylation of ERK MAPKs. Furthermore, the ERK specific-inhibitor PD98059 blocked DN-PPARγ-induced phosphorylation of Rb and expression of cyclin A and MCM7. Our data thus suggest that DN-PPARγ promotes cell cycle progression and cell growth in CASMCs by modulating fundamental cell cycle regulatory proteins and MAPK mitogenic signaling pathways in vascular smooth muscle cells (VSMCs.

MCF-10A-NeoST: A New Cell System for Studying Cell-ECM and Cell-Cell Interactions in Breast Cancer

Energy Technology Data Exchange (ETDEWEB)

Zantek, Nicole Dodge; Walker-Daniels, Jennifer; Stewart, Jane; Hansen, Rhonda K.; Robinson, Daniel; Miao, Hui; Wang, Bingcheng; Kung, Hsing-Jien; Bissell, Mina J.; Kinch, Michael S.

2001-08-22

There is a continuing need for genetically matched cell systems to model cellular behaviors that are frequently observed in aggressive breast cancers. We report here the isolation and initial characterization of a spontaneously arising variant of MCF-10A cells, NeoST, which provides a new model to study cell adhesion and signal transduction in breast cancer. NeoST cells recapitulate important biological and biochemical features of metastatic breast cancer, including anchorage-independent growth, invasiveness in threedimensional reconstituted membranes, loss of E-cadherin expression, and increased tyrosine kinase activity. A comprehensive analysis of tyrosine kinase expression revealed overexpression or functional activation of the Axl, FAK, and EphA2 tyrosine kinases in transformed MCF-10A cells. MCF-10A and these new derivatives provide a genetically matched model to study defects in cell adhesion and signaling that are relevant to cellular behaviors that often typify aggressive breast cancer cells.

Fabrication of corneal epithelial cell sheets maintaining colony-forming cells without feeder cells by oxygen-controlled method.

Science.gov (United States)

Nakajima, Ryota; Takeda, Shizu

2014-01-01

The use of murine 3T3 feeder cells needs to be avoided when fabricating corneal epithelial cell sheets for use in treating ocular surface diseases. However, the expression level of the epithelial stem/progenitor cell marker, p63, is down-regulated in feeder-free culture systems. In this study, in order to fabricate corneal epithelial cell sheets that maintain colony-forming cells without using any feeder cells, we investigated the use of an oxygen-controlled method that was developed previously to fabricate cell sheets efficiently. Rabbit limbal epithelial cells were cultured under hypoxia (1-10% O2) and under normoxia during stratification after reaching confluence. Multilayered corneal epithelial cell sheets were fabricated using an oxygen-controlled method, and immunofluorescence analysis showed that cytokeratin 3 and p63 was expressed in appropriate localization in the cell sheets. The colony-forming efficiency of the cell sheets fabricated by the oxygen-controlled method without feeder cells was significantly higher than that of cell sheets fabricated under 20% O2 without feeder cells. These results indicate that the oxygen-controlled method has the potential to achieve a feeder-free culture system for fabricating corneal epithelial cell sheets for corneal regeneration. Copyright © 2013 Elsevier Ltd. All rights reserved.

iNKT cells suppress the CD8+ T cell response to a murine Burkitt's-like B cell lymphoma.

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Ryan L Bjordahl

Full Text Available The T cell response to B cell lymphomas differs from the majority of solid tumors in that the malignant cells themselves are derived from B lymphocytes, key players in immune response. B cell lymphomas are therefore well situated to manipulate their surrounding microenvironment to enhance tumor growth and minimize anti-tumor T cell responses. We analyzed the effect of T cells on the growth of a transplantable B cell lymphoma and found that iNKT cells suppressed the anti-tumor CD8(+ T cell response. Lymphoma cells transplanted into syngeneic wild type (WT mice or Jalpha18(-/- mice that specifically lack iNKT cells grew initially at the same rate, but only the mice lacking iNKT cells were able to reject the lymphoma. This effect was due to the enhanced activity of tumor-specific CD8(+ T cells in the absence of iNKT cells, and could be partially reversed by reconstitution of iNKT cells in Jalpha 18(-/- mice. Treatment of tumor-bearing WT mice with alpha -galactosyl ceramide, an activating ligand for iNKT cells, reduced the number of tumor-specific CD8(+ T cells. In contrast, lymphoma growth in CD1d1(-/- mice that lack both iNKT and type II NKT cells was similar to that in WT mice, suggesting that type II NKT cells are required for full activation of the anti-tumor immune response. This study reveals a tumor-promoting role for iNKT cells and suggests their capacity to inhibit the CD8(+ T cell response to B cell lymphoma by opposing the effects of type II NKT cells.

Advances in reprogramming somatic cells to induced pluripotent stem cells.

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Patel, Minal; Yang, Shuying

2010-09-01

Traditionally, nuclear reprogramming of cells has been performed by transferring somatic cell nuclei into oocytes, by combining somatic and pluripotent cells together through cell fusion and through genetic integration of factors through somatic cell chromatin. All of these techniques changes gene expression which further leads to a change in cell fate. Here we discuss recent advances in generating induced pluripotent stem cells, different reprogramming methods and clinical applications of iPS cells. Viral vectors have been used to transfer transcription factors (Oct4, Sox2, c-myc, Klf4, and nanog) to induce reprogramming of mouse fibroblasts, neural stem cells, neural progenitor cells, keratinocytes, B lymphocytes and meningeal membrane cells towards pluripotency. Human fibroblasts, neural cells, blood and keratinocytes have also been reprogrammed towards pluripotency. In this review we have discussed the use of viral vectors for reprogramming both animal and human stem cells. Currently, many studies are also involved in finding alternatives to using viral vectors carrying transcription factors for reprogramming cells. These include using plasmid transfection, piggyback transposon system and piggyback transposon system combined with a non viral vector system. Applications of these techniques have been discussed in detail including its advantages and disadvantages. Finally, current clinical applications of induced pluripotent stem cells and its limitations have also been reviewed. Thus, this review is a summary of current research advances in reprogramming cells into induced pluripotent stem cells.

Tuft (caveolated) cells in two human colon carcinoma cell lines.

Science.gov (United States)

Barkla, D H; Whitehead, R H; Foster, H; Tutton, P J

1988-09-01

The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting from the apical surface. The microvilli are attached by a core of long microfilaments passing deep into the apical cytoplasm. Between the microvilli are parallel arrays of vesicles (caveoli) containing flocculent material. Two different but not mutually exclusive explanations for the presence of tuft cells are proposed. The first explanation is that tuft cells came from the resected tumour and have survived by mitotic division during subsequent passages. The second explanation suggests that tuft cells are the progeny of undifferentiated tumour cells. Descriptions of tuft cells in colon carcinomas are uncommon and possible reasons for this are presented. The morphology of tuft cells is consistent with that of a highly differentiated cell specialised for absorption, and these new models provide an opportunity to further investigate the structure and function of tuft cells.

Promotion of initiated cells by radiation-induced cell inactivation.

Science.gov (United States)

Heidenreich, W F; Paretzke, H G

2008-11-01

Cells on the way to carcinogenesis can have a growth advantage relative to normal cells. It has been hypothesized that a radiation-induced growth advantage of these initiated cells might be induced by an increased cell replacement probability of initiated cells after inactivation of neighboring cells by radiation. Here Monte Carlo simulations extend this hypothesis for larger clones: The effective clonal expansion rate decreases with clone size. This effect is stronger for the two-dimensional than for the three-dimensional situation. The clones are irregular, far from a circular shape. An exposure-rate dependence of the effective clonal expansion rate could come in part from a minimal recovery time of the initiated cells for symmetric cell division.

Cre-mediated cell ablation contests mast cell contribution in models of antibody- and T cell-mediated autoimmunity.

Science.gov (United States)

Feyerabend, Thorsten B; Weiser, Anne; Tietz, Annette; Stassen, Michael; Harris, Nicola; Kopf, Manfred; Radermacher, Peter; Möller, Peter; Benoist, Christophe; Mathis, Diane; Fehling, Hans Jörg; Rodewald, Hans-Reimer

2011-11-23

Immunological functions of mast cells remain poorly understood. Studies in Kit mutant mice suggest key roles for mast cells in certain antibody- and T cell-mediated autoimmune diseases. However, Kit mutations affect multiple cell types of both immune and nonimmune origin. Here, we show that targeted insertion of Cre-recombinase into the mast cell carboxypeptidase A3 locus deleted mast cells in connective and mucosal tissues by a genotoxic Trp53-dependent mechanism. Cre-mediated mast cell eradication (Cre-Master) mice had, with the exception of a lack of mast cells and reduced basophils, a normal immune system. Cre-Master mice were refractory to IgE-mediated anaphylaxis, and this defect was rescued by mast cell reconstitution. This mast cell-deficient strain was fully susceptible to antibody-induced autoimmune arthritis and to experimental autoimmune encephalomyelitis. Differences comparing Kit mutant mast cell deficiency models to selectively mast cell-deficient mice call for a systematic re-evaluation of immunological functions of mast cells beyond allergy. Copyright © 2011 Elsevier Inc. All rights reserved.

In vitro germ cell differentiation from cynomolgus monkey embryonic stem cells.

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Kaori Yamauchi

Full Text Available BACKGROUND: Mouse embryonic stem (ES cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro. METHODS AND FINDINGS: To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis. VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene. CONCLUSION: VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the

Mesenchymal stem cells: cell biology and potential use in therapy

DEFF Research Database (Denmark)

Kassem, Moustapha; Kristiansen, Malthe; Abdallah, Basem M

2004-01-01

Mesenchymal stem cells are clonogenic, non-haematopoietic stem cells present in the bone marrow and are able to differentiate into multiple mesoderm-type cell lineages e.g. osteoblasts, chondrocytes, endothelial-cells and also non-mesoderm-type lineages e.g. neuronal-like cells. Several methods...... are currently available for isolation of the mesenchymal stem cells based on their physical and immunological characteristics. Because of the ease of their isolation and their extensive differentiation potential, mesenchymal stem cells are among the first stem cell types to be introduced in the clinic. Recent...... studies have demonstrated that the life span of mesenchymal stem cells in vitro can be extended by increasing the levels of telomerase expression in the cells and thus allowing culture of large number of cells needed for therapy. In addition, it has been shown that it is possible to culture the cells...

Altered development of NKT cells, γδ T cells, CD8 T cells and NK cells in a PLZF deficient patient.

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Maggie Eidson

Full Text Available In mice, the transcription factor, PLZF, controls the development of effector functions in invariant NKT cells and a subset of NKT cell-like, γδ T cells. Here, we show that in human lymphocytes, in addition to invariant NKT cells, PLZF was also expressed in a large percentage of CD8+ and CD4+ T cells. Furthermore, PLZF was also found to be expressed in all γδ T cells and in all NK cells. Importantly, we show that in a donor lacking functional PLZF, all of these various lymphocyte populations were altered. Therefore, in contrast to mice, PLZF appears to control the development and/or function of a wide variety of human lymphocytes that represent more than 10% of the total PBMCs. Interestingly, the PLZF-expressing CD8+ T cell population was found to be expanded in the peripheral blood of patients with metastatic melanoma but was greatly diminished in patients with autoimmune disease.

Fuel Cell/Electrochemical Cell Voltage Monitor

Science.gov (United States)

Vasquez, Arturo

2012-01-01

A concept has been developed for a new fuel cell individual-cell-voltage monitor that can be directly connected to a multi-cell fuel cell stack for direct substack power provisioning. It can also provide voltage isolation for applications in high-voltage fuel cell stacks. The technology consists of basic modules, each with an 8- to 16-cell input electrical measurement connection port. For each basic module, a power input connection would be provided for direct connection to a sub-stack of fuel cells in series within the larger stack. This power connection would allow for module power to be available in the range of 9-15 volts DC. The relatively low voltage differences that the module would encounter from the input electrical measurement connection port, coupled with the fact that the module's operating power is supplied by the same substack voltage input (and so will be at similar voltage), provides for elimination of high-commonmode voltage issues within each module. Within each module, there would be options for analog-to-digital conversion and data transfer schemes. Each module would also include a data-output/communication port. Each of these ports would be required to be either non-electrical (e.g., optically isolated) or electrically isolated. This is necessary to account for the fact that the plurality of modules attached to the stack will normally be at a range of voltages approaching the full range of the fuel cell stack operating voltages. A communications/ data bus could interface with the several basic modules. Options have been identified for command inputs from the spacecraft vehicle controller, and for output-status/data feeds to the vehicle.

Differentiated cells are more efficient than adult stem cells for cloning by somatic cell nuclear transfer.

Science.gov (United States)

Sung, Li-Ying; Gao, Shaorong; Shen, Hongmei; Yu, Hui; Song, Yifang; Smith, Sadie L; Chang, Ching-Chien; Inoue, Kimiko; Kuo, Lynn; Lian, Jin; Li, Ao; Tian, X Cindy; Tuck, David P; Weissman, Sherman M; Yang, Xiangzhong; Cheng, Tao

2006-11-01

Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.

Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

Science.gov (United States)

Wang, Zhongde

2011-01-01

Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

Airway Basal Cell Heterogeneity and Lung Squamous Cell Carcinoma.

Science.gov (United States)

Hynds, Robert E; Janes, Sam M

2017-09-01

Basal cells are stem/progenitor cells that maintain airway homeostasis, enact repair following epithelial injury, and are a candidate cell-of-origin for lung squamous cell carcinoma. Heterogeneity of basal cells is recognized in terms of gene expression and differentiation capacity. In this Issue, Pagano and colleagues isolate a subset of immortalized basal cells that are characterized by high motility, suggesting that they might also be heterogeneous in their biophysical properties. Motility-selected cells displayed an increased ability to colonize the lung in vivo The possible implications of these findings are discussed in terms of basal cell heterogeneity, epithelial cell migration, and modeling of metastasis that occurs early in cancer evolution. Cancer Prev Res; 10(9); 491-3. ©2017 AACR See related article by Pagano et al., p. 514 . ©2017 American Association for Cancer Research.

Studies on ADCC (antibody-dependent cell-mediated cytotoxicity) using sheep red blood cells as target cells, 2

International Nuclear Information System (INIS)

Ichikawa, Yukinobu; Takaya, Masatoshi; Arimori, Shigeru

1979-01-01

A non-specific cytotoxic mediator from effector cells (human peripheral blood leukocytes) was investigated in the ADCC (antibody-dependent cell-mediated cytotoxicity) system using antibody-coated sheep red blood cells (SRBC) as target cells. 51 Cr-labelled homologous (sheep) or heterologous (human) red blood cells were used as adjacent cells. Either crude lymphocyte fraction, phagocyte depleted fraction or granulocyte rich fraction separated from human peripheral leukocytes showed moderate cytotoxic effect on homologous adjacent cells, however no cytotoxic activity on heterologous adjacent cells was demonstrated in any leukocyte fraction. This suggests that the cytotoxic effects on homologous adjacent cells were resulted from the translocation of antibody molecules to adjacent cells from antibody-coated target cells. We concluded that the cytotoxic mechanism in this ADCC system was not mediated by non-specific soluble factors released from either human peripheral lymphocytes, monocytes or granulocytes. (author)

Galvanic cells: setting up the Daniell cell.

OpenAIRE

Milla González, Miguel

2008-01-01

With the reagents (0.05M copper nitrate solution, 0.05M zinc nitrate solution) and material (glassware, potentiometer, electric wire) availabe in the laboratory, the user must set up the Daniell cell. Different configurations can be possible if the half cells are filled with either electrolyte solution. The cell connections to the measuring device can also be changed. In all instances, an explanation of the set up cell is obtained as well as of the measured potential difference.

Relative Contributions of B Cells and Dendritic Cells from Lupus-Prone Mice to CD4+ T Cell Polarization.

Science.gov (United States)

Choi, Seung-Chul; Xu, Zhiwei; Li, Wei; Yang, Hong; Roopenian, Derry C; Morse, Herbert C; Morel, Laurence

2018-05-01

Mouse models of lupus have shown that multiple immune cell types contribute to autoimmune disease. This study sought to investigate the involvement of B cells and dendritic cells in supporting the expansion of inflammatory and regulatory CD4 + T cells that are critical for lupus pathogenesis. We used lupus-prone B6.NZM2410.Sle1.Sle2.Sle3 (TC) and congenic C57BL/6J (B6) control mice to investigate how the genetic predisposition of these two cell types controls the activity of normal B6 T cells. Using an allogeneic in vitro assay, we showed that TC B1-a and conventional B cells expanded Th17 cells significantly more than their B6 counterparts. This expansion was dependent on CD86 and IL-6 expression and mapped to the Sle1 lupus-susceptibility locus. In vivo, TC B cells promoted greater differentiation of CD4 + T cells into Th1 and follicular helper T cells than did B6 B cells, but they limited the expansion of Foxp3 regulatory CD4 + T cells to a greater extent than did B6 B cells. Finally, when normal B6 CD4 + T cells were introduced into Rag1 -/- mice, TC myeloid/stromal cells caused their heightened activation, decreased Foxp3 regulatory CD4 + T cell differentiation, and increased renal infiltration of Th1 and Th17 cells in comparison with B6 myeloid/stromal cells. The results show that B cells from lupus mice amplify inflammatory CD4 + T cells in a nonredundant manner with myeloid/stromal cells. Copyright © 2018 by The American Association of Immunologists, Inc.

Electrorefining cell evaluation

Energy Technology Data Exchange (ETDEWEB)

Bronson, M.C.; Thomas, R.L. (ed.)

1989-04-14

Operational characteristics of the LANL electrorefining cell, a modified LANL electrorefining cell, and an advanced electrorefining cell (known as the CRAC cell) were determined. Average process yields achieved were: 75% for the LANL cell, 82% for the modified LANL cell, and 86% for the CRAC cell. All product metal from the LANL and modified LANL cells was within foundry specifications. Metal from one run in the CRAC cell exceeded foundry specifications for tantalum. The LANL and modified LANL cells were simple in design and operation, but product separation was more labor intensive than with the CRAC cell. The CRAC cell was more complicated in design but remained relatively simple in operation. A decision analysis concluded that the modified LANL cell was the preferred cell. It was recommended that the modified LANL cell be implemented by the Plutonium Recovery Project at Rocky Flats and that development of the CRAC cell continue. 8 refs., 22 figs., 12 tabs.

Canthin-6-one induces cell death, cell cycle arrest and differentiation in human myeloid leukemia cells.

Science.gov (United States)

Vieira Torquato, Heron F; Ribeiro-Filho, Antonio C; Buri, Marcus V; Araújo Júnior, Roberto T; Pimenta, Renata; de Oliveira, José Salvador R; Filho, Valdir C; Macho, Antonio; Paredes-Gamero, Edgar J; de Oliveira Martins, Domingos T

2017-04-01

Canthin-6-one is a natural product isolated from various plant genera and from fungi with potential antitumor activity. In the present study, we evaluate the antitumor effects of canthin-6-one in human myeloid leukemia lineages. Kasumi-1 lineage was used as a model for acute myeloid leukemia. Cells were treated with canthin-6-one and cell death, cell cycle and differentiation were evaluated in both total cells (Lin + ) and leukemia stem cell population (CD34 + CD38 - Lin -/low ). Among the human lineages tested, Kasumi-1 was the most sensitive to canthin-6-one. Canthin-6-one induced cell death with apoptotic (caspase activation, decrease of mitochondrial potential) and necrotic (lysosomal permeabilization, double labeling of annexin V/propidium iodide) characteristics. Moreover, canthin-6-one induced cell cycle arrest at G 0 /G 1 (7μM) and G 2 (45μM) evidenced by DNA content, BrdU incorporation and cyclin B1/histone 3 quantification. Canthin-6-one also promoted differentiation of Kasumi-1, evidenced by an increase in the expression of myeloid markers (CD11b and CD15) and the transcription factor PU.1. Furthermore, a reduction of the leukemic stem cell population and clonogenic capability of stem cells were observed. These results show that canthin-6-one can affect Kasumi-1 cells by promoting cell death, cell cycle arrest and cell differentiation depending on concentration used. Canthin-6-one presents an interesting cytotoxic activity against leukemic cells and represents a promising scaffold for the development of molecules for anti-leukemic applications, especially by its anti-leukemic stem cell activity. Copyright © 2017 Elsevier B.V. All rights reserved.

The antigen presenting cells instruct plasma cell differentiation.

Science.gov (United States)

Xu, Wei; Banchereau, Jacques

2014-01-06

The professional antigen presenting cells (APCs), including many subsets of dendritic cells and macrophages, not only mediate prompt but non-specific response against microbes, but also bridge the antigen-specific adaptive immune response through antigen presentation. In the latter, typically activated B cells acquire cognate signals from T helper cells in the germinal center of lymphoid follicles to differentiate into plasma cells (PCs), which generate protective antibodies. Recent advances have revealed that many APC subsets provide not only "signal 1" (the antigen), but also "signal 2" to directly instruct the differentiation process of PCs in a T-cell-independent manner. Herein, the different signals provided by these APC subsets to direct B cell proliferation, survival, class switching, and terminal differentiation are discussed. We furthermore propose that the next generation of vaccines for boosting antibody response could be designed by targeting APCs.

Stem cell biology and cell transplantation therapy in the retina.

Science.gov (United States)

Osakada, Fumitaka; Hirami, Yasuhiko; Takahashi, Masayo

2010-01-01

Embryonic stem (ES) cells, which are derived from the inner cell mass of mammalian blastocyst stage embryos, have the ability to differentiate into any cell type in the body and to grow indefinitely while maintaining pluripotency. During development, cells undergo progressive and irreversible differentiation into specialized adult cell types. Remarkably, in spite of this restriction in potential, adult somatic cells can be reprogrammed and returned to the naive state of pluripotency found in the early embryo simply by forcing expression of a defined set of transcription factors. These induced pluripotent stem (iPS) cells are molecularly and functionally equivalent to ES cells and provide powerful in vitro models for development, disease, and drug screening, as well as material for cell replacement therapy. Since functional impairment results from cell loss in most central nervous system (CNS) diseases, recovery of lost cells is an important treatment strategy. Although adult neurogenesis occurs in restricted regions, the CNS has poor potential for regeneration to compensate for cell loss. Thus, cell transplantation into damaged or diseased CNS tissues is a promising approach to treating various neurodegenerative disorders. Transplantation of photoreceptors or retinal pigment epithelium cells derived from human ES cells can restore some visual function. Patient-specific iPS cells may lead to customized cell therapy. However, regeneration of retinal function will require a detailed understanding of eye development, visual system circuitry, and retinal degeneration pathology. Here, we review the current progress in retinal regeneration, focusing on the therapeutic potential of pluripotent stem cells.

RNAi screen reveals host cell kinases specifically involved in Listeria monocytogenes spread from cell to cell.

Directory of Open Access Journals (Sweden)

Ryan Chong

Full Text Available Intracellular bacterial pathogens, such as Listeria monocytogenes and Rickettsia conorii display actin-based motility in the cytosol of infected cells and spread from cell to cell through the formation of membrane protrusions at the cell cortex. Whereas the mechanisms supporting cytosolic actin-based motility are fairly well understood, it is unclear whether specific host factors may be required for supporting the formation and resolution of membrane protrusions. To address this gap in knowledge, we have developed high-throughput fluorescence microscopy and computer-assisted image analysis procedures to quantify pathogen spread in human epithelial cells. We used the approach to screen a siRNA library covering the human kinome and identified 7 candidate kinases whose depletion led to severe spreading defects in cells infected with L. monocytogenes. We conducted systematic validation procedures with redundant silencing reagents and confirmed the involvement of the serine/threonine kinases, CSNK1A1 and CSNK2B. We conducted secondary assays showing that, in contrast with the situation observed in CSNK2B-depleted cells, L. monocytogenes formed wild-type cytosolic tails and displayed wild-type actin-based motility in the cytosol of CSNK1A1-depleted cells. Furthermore, we developed a protrusion formation assay and showed that the spreading defect observed in CSNK1A1-depleted cells correlated with the formation of protrusion that did not resolve into double-membrane vacuoles. Moreover, we developed sending and receiving cell-specific RNAi procedures and showed that CSNK1A was required in the sending cells, but was dispensable in the receiving cells, for protrusion resolution. Finally, we showed that the observed defects were specific to Listeria monocytogenes, as Rickettsia conorii displayed wild-type cell-to-cell spread in CSNK1A1- and CSNK2B-depleted cells. We conclude that, in addition to the specific host factors supporting cytosolic actin

β-Cell dedifferentiation, reduced duct cell plasticity, and impaired β-cell mass regeneration in middle-aged rats.

Science.gov (United States)

Téllez, Noèlia; Vilaseca, Marina; Martí, Yasmina; Pla, Arturo; Montanya, Eduard

2016-09-01

Limitations in β-cell regeneration potential in middle-aged animals could contribute to the increased risk to develop diabetes associated with aging. We investigated β-cell regeneration of middle-aged Wistar rats in response to two different regenerative stimuli: partial pancreatectomy (Px + V) and gastrin administration (Px + G). Pancreatic remnants were analyzed 3 and 14 days after surgery. β-Cell mass increased in young animals after Px and was further increased after gastrin treatment. In contrast, β-cell mass did not change after Px or after gastrin treatment in middle-aged rats. β-Cell replication and individual β-cell size were similarly increased after Px in young and middle-aged animals, and β-cell apoptosis was not modified. Nuclear immunolocalization of neurog3 or nkx6.1 in regenerative duct cells, markers of duct cell plasticity, was increased in young but not in middle-aged Px rats. The pancreatic progenitor-associated transcription factors neurog3 and sox9 were upregulated in islet β-cells of middle-aged rats and further increased after Px. The percentage of chromogranin A+/hormone islet cells was significantly increased in the pancreases of middle-aged Px rats. In summary, the potential for compensatory β-cell hyperplasia and hypertrophy was retained in middle-aged rats, but β-cell dedifferentiation and impaired duct cell plasticity limited β-cell regeneration. Copyright © 2016 the American Physiological Society.

Estimating cell capacity for multi-cell electrical energy system

Science.gov (United States)

Hashemi, Iman Ahari

A Multi-Cell Electrical Energy System is a set of batteries that are connected in series. The series batteries provide the required voltage necessary for the contraption. After using the energy that is provided by the batteries, some cells within the system tend to have a lower voltage than the other cells. Also, other factors, such as the number of times a battery has been charged or discharged, how long it has been within the system and many other factors, result in some cells having a lesser capacity compared to the other cells within the system. The outcome is that it lowers the required capacity that the electrical energy system is required to provide. By having an unknown cell capacity within the system, it is unknown how much of a charge can be provided to the system so that the cells are not overcharged or undercharged. Therefore, it is necessary to know the cells capacity within the system. Hence, if we were dealing with a single cell, the capacity could be obtained by a full charge and discharge of the cell. In a series system that contains multiple cells a full charging or discharging cannot happen as it might result in deteriorating the structure of some cells within the system. Hence, to find the capacity of a single cell within an electrical energy system it is required to obtain a method that can estimate the value of each cell within the electrical energy system. To approach this method an electrical energy system is required. The electrical energy system consists of rechargeable non-equal capacity batteries to provide the required energy to the system, a battery management system (BMS) board to monitor the cells voltages, an Arduino board that provides the required communication to BMS board, and the PC, and a software that is able to deliver the required data obtained from the Arduino board to the PC. The outcome, estimating the capacity of a cell within a multi-cell system, can be used in many battery related technologies to obtain unknown

Cross-talk between cd1d-restricted nkt cells and γδ cells in t regulatory cell response

Directory of Open Access Journals (Sweden)

Huber Sally A

2011-01-01

Full Text Available Abstract CD1d is a non-classical major histocompatibility class 1-like molecule which primarily presents either microbial or endogenous glycolipid antigens to T cells involved in innate immunity. Natural killer T (NKT cells and a subpopulation of γδ T cells expressing the Vγ4 T cell receptor (TCR recognize CD1d. NKT and Vγ4 T cells function in the innate immune response via rapid activation subsequent to infection and secrete large quantities of cytokines that both help control infection and modulate the developing adaptive immune response. T regulatory cells represent one cell population impacted by both NKT and Vγ4 T cells. This review discusses the evidence that NKT cells promote T regulatory cell activation both through direct interaction of NKT cell and dendritic cells and through NKT cell secretion of large amounts of TGFβ, IL-10 and IL-2. Recent studies have shown that CD1d-restricted Vγ4 T cells, in contrast to NKT cells, selectively kill T regulatory cells through a caspase-dependent mechanism. Vγ4 T cell elimination of the T regulatory cell population allows activation of autoimmune CD8+ effector cells leading to severe cardiac injury in a coxsackievirus B3 (CVB3 myocarditis model in mice. CD1d-restricted immunity can therefore lead to either immunosuppression or autoimmunity depending upon the type of innate effector dominating during the infection.

[gammadelta T cells stimulated by zoledronate kill osteosarcoma cells].

Science.gov (United States)

Jiang, Hui; Xu, Qiang; Yang, Chao; Cao, Zhen-Guo; Li, Zhao-Xu; Ye, Zhao-Ming

2010-12-01

To investigate the cytotoxicity of human γδT cells from PBMCs stimulated by zoledronate against osteosarcoma cell line HOS in vitro and in vivo and evaluate the relavent pathways. The peripheral blood mononuclear cells (PBMCs)of healthy donors were stimulated by single dose zoledronate and cultured in the present of IL-2 for two weeks, analysising the percentage of γδT cells on a FACSCalibur cytometer.Study the cytotoxicity of γδT cells against the osteosarcoma line HOS using LDH release assay kit. Pre-treatment of γδT cells with anti-human γδTCR antibody, anti-human NKG2D antibody and concanamycin A to bolck the relavent pathways for evaluating the mechenisms of its cytotoxicity. In vivo, BALB/c mice were inoculated subcutaneously osteosarcoma cell HOS for developing hypodermal tumors. And they were randomized into two groups: unteated group, γδT cell therapy group. Tumor volume and weight of the two groups were compared. After two weeks of culture, γδT cells from zoledronate-stimulated PBMCs could reach (95±3)%. When the E:T as 6:1, 12:1, 25:1, 50:1, the percentage of osteosarcoma cell HOS killed by γδT cells was 26.8%, 31.5%, 37.8%, 40.9%, respectively.When anti-huma γδTCR antibody, anti-human NKG2D antibody and concanamycin A blocked the relavent pathways, the percentage was 32.3%, 4.7%, 16.7% ( E:T as 25:1), respectively. In vivo, the tumor inhibition rate of the group of γδT cell therapy was 42.78%. γδT cells derived from PBMCs stimulated by zoledronate can acquired pure γδT cells. And they show strong cytoxicity against osteosarcoma cell line HOS in vitro and in vivo.

Tuft (caveolated) cells in two human colon carcinoma cell lines.

OpenAIRE

Barkla, D. H.; Whitehead, R. H.; Foster, H.; Tutton, P. J.

1988-01-01

The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting f...

Identification of Human Cutaneous Basal Cell Carcinoma Cancer Stem Cells.

Science.gov (United States)

Morgan, Huw; Olivero, Carlotta; Patel, Girish K

2018-04-20

The cancer stem cell model states that a subset of tumor cells, called "cancer stem cells," can initiate and propagate tumor growth through self-renewal, high proliferative capacity, and their ability to recreate tumor heterogeneity. In basal cell carcinoma (BCC), we have shown that tumor cells that express the cell surface protein CD200 fulfill the cancer stem cell hypothesis. CD200+ CD45- BCC cells represent 0.05-3.96% of all BCC cells and reside in small clusters at the tumor periphery. Using a novel, reproducible in vivo xenograft growth assay, we determined that tumor-initiating cell (TIC) frequencies are approximately 1 per 1.5 million unsorted BCC cells. The CD200+ CD45- BCC subpopulation recreated BCC tumor growth in vivo with typical histological architecture and expression of sonic hedgehog-regulated genes. Reproducible in vivo BCC growth was achieved with as few as 10,000 CD200+ CD45- cells, representing ~1500-fold enrichment. The methods used to identify and purify CD200+ CD45- BCC cells, as well as characterize gene expression, are described herein.

Radiation sensitivity of Merkel cell carcinoma cell lines

Energy Technology Data Exchange (ETDEWEB)

Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others

1995-07-30

Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

Radiation sensitivity of Merkell cell carcinoma cell lines

International Nuclear Information System (INIS)

Leonard, J. Helen; Ramsay, Jonathan R.; Kearsley, John H.; Birrell, Geoff W.

1995-01-01

Purpose: Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Methods and Materials: Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after γ irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. Results: We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to γ irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Conclusion: Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution

Hakai reduces cell-substratum adhesion and increases epithelial cell invasion

International Nuclear Information System (INIS)

Rodríguez-Rigueiro, Teresa; Valladares-Ayerbes, Manuel; Haz-Conde, Mar; Aparicio, Luis A; Figueroa, Angélica

2011-01-01

The dynamic regulation of cell-cell adhesions is crucial for developmental processes, including tissue formation, differentiation and motility. Adherens junctions are important components of the junctional complex between cells and are necessary for maintaining cell homeostasis and normal tissue architecture. E-cadherin is the prototype and best-characterized protein member of adherens junctions in mammalian epithelial cells. Regarded as a tumour suppressor, E-cadherin loss is associated with poor prognosis in carcinoma. The E3 ubiquitin-ligase Hakai was the first reported posttranslational regulator of the E-cadherin complex. Hakai specifically targetted E-cadherin for internalization and degradation and thereby lowered epithelial cell-cell contact. Hakai was also implicated in controlling proliferation, and promoted cancer-related gene expression by increasing the binding of RNA-binding protein PSF to RNAs encoding oncogenic proteins. We sought to investigate the possible implication of Hakai in cell-substratum adhesions and invasion in epithelial cells. Parental MDCK cells and MDCK cells stably overexpressing Hakai were used to analyse cell-substratum adhesion and invasion capabilities. Western blot and immunofluoresecence analyses were performed to assess the roles of Paxillin, FAK and Vinculin in cell-substratum adhesion. The role of the proteasome in controlling cell-substratum adhesion was studied using two proteasome inhibitors, lactacystin and MG132. To study the molecular mechanisms controlling Paxillin expression, MDCK cells expressing E-cadherin shRNA in a tetracycline-inducible manner was employed. Here, we present evidence that implicate Hakai in reducing cell-substratum adhesion and increasing epithelial cell invasion, two hallmark features of cancer progression and metastasis. Paxillin, an important protein component of the cell-matrix adhesion, was completely absent from focal adhesions and focal contacts in Hakai-overexpressing MDCK cells. The